Atmospheric-pressure plasma activation and surface characterization on polyethylene membrane separator

NASA Astrophysics Data System (ADS)

Tseng, Yu-Chien; Li, Hsiao-Ling; Huang, Chun

2017-01-01

The surface hydrophilic activation of a polyethylene membrane separator was achieved using an atmospheric-pressure plasma jet. The surface of the atmospheric-pressure-plasma-treated membrane separator was found to be highly hydrophilic realized by adjusting the plasma power input. The variations in membrane separator chemical structure were confirmed by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. Chemical analysis showed newly formed carbonyl-containing groups and high surface concentrations of oxygen-containing species on the atmospheric-pressure-plasma-treated polymeric separator surface. It also showed that surface hydrophilicity primarily increased from the polar component after atmospheric-pressure plasma treatment. The surface and pore structures of the polyethylene membrane separator were examined by scanning electron microscopy, revealing a slight alteration in the pore structure. As a result of the incorporation of polar functionalities by atmospheric-pressure plasma activation, the electrolyte uptake and electrochemical impedance of the atmospheric-pressure-plasma-treated membrane separator improved. The investigational results show that the separator surface can be controlled by atmospheric-pressure plasma surface treatment to tailor the hydrophilicity and enhance the electrochemical performance of lithium ion batteries.

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.

Plasma membrane isolation using immobilized concanavalin A magnetic beads.

PubMed

Lee, Yu-Chen; Srajer Gajdosik, Martina; Josic, Djuro; Lin, Sue-Hwa

2012-01-01

Isolation of highly purified plasma membranes is the key step in constructing the plasma membrane proteome. Traditional plasma membrane isolation method takes advantage of the differential density of organelles. While differential centrifugation methods are sufficient to enrich for plasma membranes, the procedure is lengthy and results in low recovery of the membrane fraction. Importantly, there is significant contamination of the plasma membranes with other organelles. The traditional agarose affinity matrix is suitable for isolating proteins but has limitation in separating organelles due to the density of agarose. Immobilization of affinity ligands to magnetic beads allows separation of affinity matrix from organelles through magnets and could be developed for the isolation of organelles. We have developed a simple method for isolating plasma membranes using lectin concanavalin A (ConA) magnetic beads. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. The ConA magnetic beads are used to bind glycosylated proteins present in the membranes. The bound membranes are solubilized from the magnetic beads with a detergent containing the competing sugar alpha methyl mannoside. In this study, we describe the procedure of isolating rat liver plasma membranes using sucrose density gradient centrifugation as described by Neville. We then further purify the membrane fraction by using ConA magnetic beads. After this purification step, main liver plasma membrane proteins, especially the highly glycosylated ones and proteins containing transmembrane domains could be identified by LC-ESI-MS/MS. While not described here, the magnetic bead method can also be used to isolate plasma membranes from cell lysates. This membrane purification method should expedite the cataloging of plasma membrane proteome.

Gas-liquid interfacial plasmas producing reactive species for cell membrane permeabilization

PubMed Central

Kaneko, Toshiro; Sasaki, Shota; Takashima, Keisuke; Kanzaki, Makoto

2017-01-01

Gas-liquid interfacial atmospheric-pressure plasma jets (GLI-APPJ) are used medically for plasma-induced cell-membrane permeabilization. In an attempt to identify the dominant factors induced by GLI-APPJ responsible for enhancing cell-membrane permeability, the concentration and distribution of plasma-produced reactive species in the gas and liquid phase regions are measured. These reactive species are classified in terms of their life-span: long-lived (e.g., H2O2), short-lived (e.g., O2•−), and extremely-short-lived (e.g., •OH). The concentration of plasma-produced •OHaq in the liquid phase region decreases with an increase in solution thickness (<1 mm), and plasma-induced cell-membrane permeabilization is found to decay markedly as the thickness of the solution increases. Furthermore, the horizontally center-localized distribution of •OHaq, resulting from the center-peaked distribution of •OH in the gas phase region, corresponds with the distribution of the permeabilized cells upon APPJ irradiation, whereas the overall plasma-produced oxidizing species such as H2O2aq in solution exhibit a doughnut-shaped horizontal distribution. These results suggest that •OHaq is likely one of the dominant factors responsible for plasma-induced cell-membrane permeabilization. PMID:28163376

Gas-liquid interfacial plasmas producing reactive species for cell membrane permeabilization.

PubMed

Kaneko, Toshiro; Sasaki, Shota; Takashima, Keisuke; Kanzaki, Makoto

2017-01-01

Gas-liquid interfacial atmospheric-pressure plasma jets (GLI-APPJ) are used medically for plasma-induced cell-membrane permeabilization. In an attempt to identify the dominant factors induced by GLI-APPJ responsible for enhancing cell-membrane permeability, the concentration and distribution of plasma-produced reactive species in the gas and liquid phase regions are measured. These reactive species are classified in terms of their life-span: long-lived (e.g., H 2 O 2 ), short-lived (e.g., O 2 •- ), and extremely-short-lived (e.g., • OH). The concentration of plasma-produced • OH aq in the liquid phase region decreases with an increase in solution thickness (<1 mm), and plasma-induced cell-membrane permeabilization is found to decay markedly as the thickness of the solution increases. Furthermore, the horizontally center-localized distribution of • OH aq , resulting from the center-peaked distribution of • OH in the gas phase region, corresponds with the distribution of the permeabilized cells upon APPJ irradiation, whereas the overall plasma-produced oxidizing species such as H 2 O 2aq in solution exhibit a doughnut-shaped horizontal distribution. These results suggest that • OH aq is likely one of the dominant factors responsible for plasma-induced cell-membrane permeabilization.

Plasma surface modification of nanofiltration (NF) thin-film composite (TFC) membranes to improve anti organic fouling

NASA Astrophysics Data System (ADS)

Kim, Eun-Sik; Yu, Qingsong; Deng, Baolin

2011-09-01

Commercial nanofiltration (NF) thin-film composite (TFC) membranes were treated by low-pressure NH3 plasma, and the effects of the plasma treatment were investigated in terms of the membrane hydrophilicity, pure water flux, salt rejection, protein adsorption, and humic acid fouling. Experimental results indicated that the membrane surface hydrophilicity was increased by the plasma treatment, and changes in the hydrophilicity as well as membrane performance including permeate flux and fouling varied with the original membrane characteristics (e.g., roughness and hydrophilicity). Water flux of plasma treated membranes was the highest with 10 min and 90 W of plasma treatment, and salt rejection was mainly affected by the intensity of the plasma power. Results of bovine serum albumin (BSA) adsorption demonstrated that the protein adsorption decreased with increasing plasma treatment time. The plasma treatment that resulted in more negatively charged surfaces could also better prevent Aldrich humic acid (AHA) attachment on the membrane surface.

Plasma membrane aquaporins mediates vesicle stability in broccoli

PubMed Central

Martínez-Ballesta, Maria del Carmen; García-Gomez, Pablo; Yepes-Molina, Lucía; Guarnizo, Angel L.; Teruel, José A.

2018-01-01

The use of in vitro membrane vesicles is attractive because of possible applications in therapies. Here we aimed to compare the stability and functionality of plasma membrane vesicles extracted from control and salt-treated broccoli. The impact of the amount of aquaporins was related to plasma membrane osmotic water permeability and the stability of protein secondary structure. Here, we describe for first time an increase in plant aquaporins acetylation under high salinity. Higher osmotic water permeability in NaCl vesicles has been related to higher acetylation, upregulation of aquaporins, and a more stable environment to thermal denaturation. Based on our findings, we propose that aquaporins play an important role in vesicle stability. PMID:29420651

Ras Diffusion Is Sensitive to Plasma Membrane Viscosity

PubMed Central

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

2005-01-01

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

Interaction between La(III) and proteins on the plasma membrane of horseradish

NASA Astrophysics Data System (ADS)

Yang, Guang-Mei; Chu, Yun-Xia; Lv, Xiao-Fen; Zhou, Qing; Huang, Xiao-Hua

2012-06-01

Lanthanum (La) is an important rare earth element in the ecological environment of plant. The proteins on the plasma membrane control the transport of molecules into and out of cell. It is very important to investigate the effect of La(III) on the proteins on the plasma membrane in the plant cell. In the present work, the interaction between La(III) and proteins on the plasma membrane of horseradish was investigated using optimization of the fluorescence microscopy and fluorescence spectroscopy. It is found that the fluorescence of the complex system of protoplasts and 1-aniline Kenai-8-sulfonic acid in horseradish treated with the low concentration of La(III) is increased compared with that of the control horseradish. The opposite effect is observed in horseradish treated with the high concentration of La(III). These results indicated that the low concentration of La(III) can interact with the proteins on the plasma membrane of horseradish, causing the improvement in the structure of proteins on the plasma membrane. The high concentration of La(III) can also interact with the proteins on the plasma membrane of horseradish, leading to the destruction of the structure of proteins on the plasma membrane. We demonstrate that the proteins on the plasma membrane are the targets of La(III) action on plant cell.

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

NASA Astrophysics Data System (ADS)

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

2018-02-01

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

Dynamics of HIV-1 RNA Near the Plasma Membrane during Virus Assembly.

PubMed

Sardo, Luca; Hatch, Steven C; Chen, Jianbo; Nikolaitchik, Olga; Burdick, Ryan C; Chen, De; Westlake, Christopher J; Lockett, Stephen; Pathak, Vinay K; Hu, Wei-Shau

2015-11-01

To increase our understanding of the events that lead to HIV-1 genome packaging, we examined the dynamics of viral RNA and Gag-RNA interactions near the plasma membrane by using total internal reflection fluorescence microscopy. We labeled HIV-1 RNA with a photoconvertible Eos protein via an RNA-binding protein that recognizes stem-loop sequences engineered into the viral genome. Near-UV light exposure causes an irreversible structural change in Eos and alters its emitted fluorescence from green to red. We studied the dynamics of HIV-1 RNA by photoconverting Eos near the plasma membrane, and we monitored the population of photoconverted red-Eos-labeled RNA signals over time. We found that in the absence of Gag, most of the HIV-1 RNAs stayed near the plasma membrane transiently, for a few minutes. The presence of Gag significantly increased the time that RNAs stayed near the plasma membrane: most of the RNAs were still detected after 30 min. We then quantified the proportion of HIV-1 RNAs near the plasma membrane that were packaged into assembling viral complexes. By tagging Gag with blue fluorescent protein, we observed that only a portion, ∼13 to 34%, of the HIV-1 RNAs that reached the membrane were recruited into assembling particles in an hour, and the frequency of HIV-1 RNA packaging varied with the Gag expression level. Our studies reveal the HIV-1 RNA dynamics on the plasma membrane and the efficiency of RNA recruitment and provide insights into the events leading to the generation of infectious HIV-1 virions. Nascent HIV-1 particles assemble on plasma membranes. During the assembly process, HIV-1 RNA genomes must be encapsidated into viral complexes to generate infectious particles. To gain insights into the RNA packaging and virus assembly mechanisms, we labeled and monitored the HIV-1 RNA signals near the plasma membrane. Our results showed that most of the HIV-1 RNAs stayed near the plasma membrane for only a few minutes in the absence of Gag, whereas

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.

Effect of hydrodynamic interactions on the diffusion of integral membrane proteins: diffusion in plasma membranes.

PubMed Central

Bussell, S J; Koch, D L; Hammer, D A

1995-01-01

Tracer diffusion coefficients of integral membrane proteins (IMPs) in intact plasma membranes are often much lower than those found in blebbed, organelle, and reconstituted membranes. We calculate the contribution of hydrodynamic interactions to the tracer, gradient, and rotational diffusion of IMPs in plasma membranes. Because of the presence of immobile IMPs, Brinkman's equation governs the hydrodynamics in plasma membranes. Solutions of Brinkman's equation enable the calculation of short-time diffusion coefficients of IMPs. There is a large reduction in particle mobilities when a fraction of them is immobile, and as the fraction increases, the mobilities of the mobile particles continue to decrease. Combination of the hydrodynamic mobilities with Monte Carlo simulation results, which incorporate excluded area effects, enable the calculation of long-time diffusion coefficients. We use our calculations to analyze results for tracer diffusivities in several different systems. In erythrocytes, we find that the hydrodynamic theory, when combined with excluded area effects, closes the gap between existing theory and experiment for the mobility of band 3, with the remaining discrepancy likely due to direct obstruction of band 3 lateral mobility by the spectrin network. In lymphocytes, the combined hydrodynamic-excluded area theory provides a plausible explanation for the reduced mobility of sIg molecules induced by binding concanavalin A-coated platelets. However, the theory does not explain all reported cases of "anchorage modulation" in all cell types in which receptor mobilities are reduced after binding by concanavalin A-coated platelets. The hydrodynamic theory provides an explanation of why protein lateral mobilities are restricted in plasma membranes and why, in many systems, deletion of the cytoplasmic tail of a receptor has little effect on diffusion rates. However, much more data are needed to test the theory definitively. We also predict that gradient and

Plasma membrane signaling in HIV-1 infection.

PubMed

Abbas, Wasim; Herbein, Georges

2014-04-01

Plasma membrane is a multifunctional structure that acts as the initial barrier against infection by intracellular pathogens. The productive HIV-1 infection depends upon the initial interaction of virus and host plasma membrane. Immune cells such as CD4+ T cells and macrophages contain essential cell surface receptors and molecules such as CD4, CXCR4, CCR5 and lipid raft components that facilitate HIV-1 entry. From plasma membrane HIV-1 activates signaling pathways that prepare the grounds for viral replication. Through viral proteins HIV-1 hijacks host plasma membrane receptors such as Fas, TNFRs and DR4/DR5, which results in immune evasion and apoptosis both in infected and uninfected bystander cells. These events are hallmark in HIV-1 pathogenesis that leads towards AIDS. The interplay between HIV-1 and plasma membrane signaling has much to offer in terms of viral fitness and pathogenicity, and a better understanding of this interplay may lead to development of new therapeutic approaches. This article is part of a Special Issue entitled: Viral Membrane Proteins - Channels for Cellular Networking. Copyright © 2013 Elsevier B.V. All rights reserved.

Caveolae as plasma membrane sensors, protectors and organizers.

PubMed

Parton, Robert G; del Pozo, Miguel A

2013-02-01

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

Plasma membrane changes during programmed cell deaths

PubMed Central

Zhang, Yingying; Chen, Xin; Gueydan, Cyril; Han, Jiahuai

2018-01-01

Ruptured and intact plasma membranes are classically considered as hallmarks of necrotic and apoptotic cell death, respectively. As such, apoptosis is usually considered a non-inflammatory process while necrosis triggers inflammation. Recent studies on necroptosis and pyroptosis, two types of programmed necrosis, revealed that plasma membrane rupture is mediated by MLKL channels during necroptosis but depends on non-selective gasdermin D (GSDMD) pores during pyroptosis. Importantly, the morphology of dying cells executed by MLKL channels can be distinguished from that executed by GSDMD pores. Interestingly, it was found recently that secondary necrosis of apoptotic cells, a previously believed non-regulated form of cell lysis that occurs after apoptosis, can be programmed and executed by plasma membrane pore formation like that of pyroptosis. In addition, pyroptosis is associated with pyroptotic bodies, which have some similarities to apoptotic bodies. Therefore, different cell death programs induce distinctive reshuffling processes of the plasma membrane. Given the fact that the nature of released intracellular contents plays a crucial role in dying/dead cell-induced immunogenicity, not only membrane rupture or integrity but also the nature of plasma membrane breakdown would determine the fate of a cell as well as its ability to elicit an immune response. In this review, we will discuss recent advances in the field of apoptosis, necroptosis and pyroptosis, with an emphasis on the mechanisms underlying plasma membrane changes observed on dying cells and their implication in cell death-elicited immunogenicity. PMID:29076500

Autophagosomal membranes assemble at ER-plasma membrane contact sites.

PubMed

Nascimbeni, Anna Chiara; Codogno, Patrice; Morel, Etienne

2017-01-01

The biogenesis of autophagosome, the double membrane bound organelle related to macro-autophagy, is a complex event requiring numerous key-proteins and membrane remodeling events. Our recent findings identify the extended synaptotagmins, crucial tethers of Endoplasmic Reticulum-plasma membrane contact sites, as key-regulators of this molecular sequence.

Terpenes increase the lipid dynamics in the Leishmania plasma membrane at concentrations similar to their IC50 values.

PubMed

Camargos, Heverton Silva; Moreira, Rodrigo Alves; Mendanha, Sebastião Antonio; Fernandes, Kelly Souza; Dorta, Miriam Leandro; Alonso, Antonio

2014-01-01

Although many terpenes have shown antitumor, antibacterial, antifungal, and antiparasitic activity, the mechanism of action is not well established. Electron paramagnetic resonance (EPR) spectroscopy of the spin-labeled 5-doxyl stearic acid revealed remarkable fluidity increases in the plasma membrane of terpene-treated Leishmania amazonensis promastigotes. For an antiproliferative activity assay using 5×10(6) parasites/mL, the sesquiterpene nerolidol and the monoterpenes (+)-limonene, α-terpineol and 1,8-cineole inhibited the growth of the parasites with IC50 values of 0.008, 0.549, 0.678 and 4.697 mM, respectively. The IC50 values of these terpenes increased as the parasite concentration used in the cytotoxicity assay increased, and this behavior was examined using a theoretical treatment of the experimental data. Cytotoxicity tests with the same parasite concentration as in the EPR experiments revealed a correlation between the IC50 values of the terpenes and the concentrations at which they altered the membrane fluidity. In addition, the terpenes induced small amounts of cell lysis (4-9%) at their respective IC50 values. For assays with high cell concentrations (2×10(9) parasites/mL), the incorporation of terpene into the cell membrane was very fast, and the IC50 values observed for 24 h and 5 min-incubation periods were not significantly different. Taken together, these results suggest that terpene cytotoxicity is associated with the attack on the plasma membrane of the parasite. The in vitro cytotoxicity of nerolidol was similar to that of miltefosine, and nerolidol has high hydrophobicity; thus, nerolidol might be used in drug delivery systems, such as lipid nanoparticles to treat leishmaniasis.

Terpenes Increase the Lipid Dynamics in the Leishmania Plasma Membrane at Concentrations Similar to Their IC50 Values

PubMed Central

Camargos, Heverton Silva; Moreira, Rodrigo Alves; Mendanha, Sebastião Antonio; Fernandes, Kelly Souza; Dorta, Miriam Leandro; Alonso, Antonio

2014-01-01

Although many terpenes have shown antitumor, antibacterial, antifungal, and antiparasitic activity, the mechanism of action is not well established. Electron paramagnetic resonance (EPR) spectroscopy of the spin-labeled 5-doxyl stearic acid revealed remarkable fluidity increases in the plasma membrane of terpene-treated Leishmania amazonensis promastigotes. For an antiproliferative activity assay using 5×106 parasites/mL, the sesquiterpene nerolidol and the monoterpenes (+)-limonene, α-terpineol and 1,8-cineole inhibited the growth of the parasites with IC50 values of 0.008, 0.549, 0.678 and 4.697 mM, respectively. The IC50 values of these terpenes increased as the parasite concentration used in the cytotoxicity assay increased, and this behavior was examined using a theoretical treatment of the experimental data. Cytotoxicity tests with the same parasite concentration as in the EPR experiments revealed a correlation between the IC50 values of the terpenes and the concentrations at which they altered the membrane fluidity. In addition, the terpenes induced small amounts of cell lysis (4–9%) at their respective IC50 values. For assays with high cell concentrations (2×109 parasites/mL), the incorporation of terpene into the cell membrane was very fast, and the IC50 values observed for 24 h and 5 min-incubation periods were not significantly different. Taken together, these results suggest that terpene cytotoxicity is associated with the attack on the plasma membrane of the parasite. The in vitro cytotoxicity of nerolidol was similar to that of miltefosine, and nerolidol has high hydrophobicity; thus, nerolidol might be used in drug delivery systems, such as lipid nanoparticles to treat leishmaniasis. PMID:25101672

Glucose rapidly decreases plasma membrane GLUT4 content in rat skeletal muscle.

PubMed

Marette, A; Dimitrakoudis, D; Shi, Q; Rodgers, C D; Klip, A; Vranic, M

1999-02-01

We have previously demonstrated that chronic hyperglycemia per se decreases GLUT4 glucose transporter expression and plasma membrane content in mildly streptozotocin- (STZ) diabetic rats (Biochem. J. 284, 341-348, 1992). In the present study, we investigated the effect of an acute rise in glycemia on muscle GLUT4 and GLUT1 protein contents in the plasma membrane, in the absence of insulin elevation. Four experimental groups of rats were analyzed in the postabsorptive state: 1. Control rats. 2. Hyperglycemic STZ-diabetic rats with moderately reduced fasting insulin levels. 3. STZ-diabetic rats made normoglycemic with phlorizin treatment. 4. Phlorizin-treated (normoglycemic) STZ-diabetic rats infused with glucose for 40 min. The uniqueness of the latter model is that glycemia can be rapidly raised without any concomitant increase in plasma insulin levels. Plasma membranes were isolated from hindlimb muscle and GLUT1 and GLUT4 proteins amounts determined by Western blot analysis. As predicted, STZ-diabetes caused a significant decrease in the abundance of GLUT4 in the isolated plasma membranes. Normalization of glycemia for 3 d with phlorizin treatment restored plasma membrane GLUT4 content in muscle of STZ-diabetic rats. A sudden rise in glycemia over a period of 40 min caused the GLUT4 levels in the plasma membrane fraction to decrease to those of nontreated STZ-diabetic rats. In contrast to the GLUT4 transporter, plasma membrane GLUT1 abundance was not changed by the acute glucose challenge. It is concluded that glucose can have regulatory effect by acutely reducing plasma membrane GLUT4 protein contents in rat skeletal muscle. We hypothesize that this glucose-induced downregulation of plasma membrane GLUT4 could represent a protective mechanism against excessive glucose uptake under hyperglycemic conditions accompanied by insulin resistance.

Plasma membrane disruption: repair, prevention, adaptation

NASA Technical Reports Server (NTRS)

McNeil, Paul L.; Steinhardt, Richard A.

2003-01-01

Many metazoan cells inhabit mechanically stressful environments and, consequently, their plasma membranes are frequently disrupted. Survival requires that the cell rapidly repair or reseal the disruption. Rapid resealing is an active and complex structural modification that employs endomembrane as its primary building block, and cytoskeletal and membrane fusion proteins as its catalysts. Endomembrane is delivered to the damaged plasma membrane through exocytosis, a ubiquitous Ca2+-triggered response to disruption. Tissue and cell level architecture prevent disruptions from occurring, either by shielding cells from damaging levels of force, or, when this is not possible, by promoting safe force transmission through the plasma membrane via protein-based cables and linkages. Prevention of disruption also can be a dynamic cell or tissue level adaptation triggered when a damaging level of mechanical stress is imposed. Disease results from failure of either the preventive or resealing mechanisms.

Enrichment of plasma membrane proteins using nanoparticle pellicles: comparison between silica and higher density nanoparticles

PubMed Central

Choksawangkarn, Waeowalee; Kim, Sung-Kyoung; Cannon, Joe R.; Edwards, Nathan J.; Lee, Sang Bok; Fenselau, Catherine

2013-01-01

Proteomic and other characterization of plasma membrane proteins is made difficult by their low abundance, hydrophobicity, frequent carboxylation and dynamic population. We and others have proposed that underrepresentation in LC-MS/MS analysis can be partially compensated by enriching the plasma membrane and its proteins using cationic nanoparticle pellicles. The nanoparticles increase the density of plasma membrane sheets and thus enhance separation by centrifugation from other lysed cellular components. Herein we test the hypothesis that the use of nanoparticles with increased densities can provide enhanced enrichment of plasma membrane proteins for proteomic analysis. Multiple myeloma cells were grown and coated in suspension with three different pellicles of three different densities and both pellicle coated and uncoated suspensions analyzed by high-throughput LC-MS/MS. Enrichment was evaluated by the total number and the spectral counts of identified plasma membrane proteins. PMID:23289353

Plasma treatment of polyethersulfone membrane for benzene removal from water by air gap membrane distillation.

PubMed

Pedram, Sara; Mortaheb, Hamid Reza; Arefi-Khonsari, Farzaneh

2018-01-01

In order to obtain a durable cost-effective membrane for membrane distillation (MD) process, flat sheet polyethersulfone (PES) membranes were modified by an atmospheric pressure nonequilibrium plasma generated using a dielectric barrier discharge in a mixture of argon and hexamethyldisiloxane as the organosilicon precursor. The surface properties of the plasma-modified membranes were characterized by water contact angle (CA), liquid entry pressure, X-ray photoelectron spectroscopy, scanning electron microscopy, and atomic force microscopy. The water CA of the membrane was increased from 64° to 104° by depositing a Si(CH 3 )-rich thin layer. While the pristine PES membrane was not applicable in the MD process, the modified PES membrane could be applied for the first time in an air gap membrane distillation setup for the removal of benzene as a volatile organic compound from water. The experimental design using central composite design and response surface methodology was applied to study the effects of feed temperature, concentration, and flow rate as well as their binary interactions on the overall permeate flux and separation factor. The separation factor and permeation flux of the modified PES membrane at optimum conditions were comparable with those of commercial polytetrafluoroethylene membrane.

Requirement for Coenzyme Q in Plasma Membrane Electron Transport

NASA Astrophysics Data System (ADS)

Sun, I. L.; Sun, E. E.; Crane, F. L.; Morre, D. J.; Lindgren, A.; Low, H.

1992-12-01

Coenzyme Q is required in the electron transport system of rat hepatocyte and human erythrocyte plasma membranes. Extraction of coenzyme Q from the membrane decreases NADH dehydrogenase and NADH:oxygen oxidoreductase activity. Addition of coenzyme Q to the extracted membrane restores the activity. Partial restoration of activity is also found with α-tocopherylquinone, but not with vitamin K_1. Analogs of coenzyme Q inhibit NADH dehydrogenase and oxidase activity and the inhibition is reversed by added coenzyme Q. Ferricyanide reduction by transmembrane electron transport from HeLa cells is inhibited by coenzyme Q analogs and restored with added coenzyme Q10. Reduction of external ferricyanide and diferric transferrin by HeLa cells is accompanied by proton release from the cells. Inhibition of the reduction by coenzyme Q analogs also inhibits the proton release, and coenzyme Q10 restores the proton release activity. Trans-plasma membrane electron transport stimulates growth of serum-deficient cells, and added coenzyme Q10 increases growth of HeLa (human adenocarcinoma) and BALB/3T3 (mouse fibroblast) cells. The evidence is consistent with a function for coenzyme Q in a trans-plasma membrane electron transport system which influences cell growth.

Na+/H+ exchange activity in the plasma membrane of Arabidopsis.

PubMed

Qiu, Quan-Sheng; Barkla, Bronwyn J; Vera-Estrella, Rosario; Zhu, Jian-Kang; Schumaker, Karen S

2003-06-01

In plants, Na+/H+ exchangers in the plasma membrane are critical for growth in high levels of salt, removing toxic Na+ from the cytoplasm by transport out of the cell. The molecular identity of a plasma membrane Na+/H+ exchanger in Arabidopsis (SOS1) has recently been determined. In this study, immunological analysis provided evidence that SOS1 localizes to the plasma membrane of leaves and roots. To characterize the transport activity of this protein, purified plasma membrane vesicles were isolated from leaves of Arabidopsis. Na+/H+ exchange activity, monitored as the ability of Na to dissipate an established pH gradient, was absent in plants grown without salt. However, exchange activity was induced when plants were grown in 250 mm NaCl and increased with prolonged salt exposure up to 8 d. H+-coupled exchange was specific for Na, because chloride salts of other monovalent cations did not dissipate the pH gradient. Na+/H+ exchange activity was dependent on Na (substrate) concentration, and kinetic analysis indicated that the affinity (apparent Km) of the transporter for Na+ is 22.8 mm. Data from two experimental approaches supports electroneutral exchange (one Na+ exchanged for one proton): (a) no change in membrane potential was measured during the exchange reaction, and (b) Na+/H+ exchange was unaffected by the presence or absence of a membrane potential. Results from this research provide a framework for future studies into the regulation of the plant plasma membrane Na+/H+ exchanger and its relative contribution to the maintenance of cellular Na+ homeostasis during plant growth in salt.

Plasma membranes modified by plasma treatment or deposition as solid electrolytes for potential application in solid alkaline fuel cells.

PubMed

Reinholdt, Marc; Ilie, Alina; Roualdès, Stéphanie; Frugier, Jérémy; Schieda, Mauricio; Coutanceau, Christophe; Martemianov, Serguei; Flaud, Valérie; Beche, Eric; Durand, Jean

2012-07-30

In the highly competitive market of fuel cells, solid alkaline fuel cells using liquid fuel (such as cheap, non-toxic and non-valorized glycerol) and not requiring noble metal as catalyst seem quite promising. One of the main hurdles for emergence of such a technology is the development of a hydroxide-conducting membrane characterized by both high conductivity and low fuel permeability. Plasma treatments can enable to positively tune the main fuel cell membrane requirements. In this work, commercial ADP-Morgane® fluorinated polymer membranes and a new brand of cross-linked poly(aryl-ether) polymer membranes, named AMELI-32®, both containing quaternary ammonium functionalities, have been modified by argon plasma treatment or triallylamine-based plasma deposit. Under the concomitant etching/cross-linking/oxidation effects inherent to the plasma modification, transport properties (ionic exchange capacity, water uptake, ionic conductivity and fuel retention) of membranes have been improved. Consequently, using plasma modified ADP-Morgane® membrane as electrolyte in a solid alkaline fuel cell operating with glycerol as fuel has allowed increasing the maximum power density by a factor 3 when compared to the untreated membrane.

Plasma Membranes Modified by Plasma Treatment or Deposition as Solid Electrolytes for Potential Application in Solid Alkaline Fuel Cells

PubMed Central

Reinholdt, Marc; Ilie, Alina; Roualdès, Stéphanie; Frugier, Jérémy; Schieda, Mauricio; Coutanceau, Christophe; Martemianov, Serguei; Flaud, Valérie; Beche, Eric; Durand, Jean

2012-01-01

In the highly competitive market of fuel cells, solid alkaline fuel cells using liquid fuel (such as cheap, non-toxic and non-valorized glycerol) and not requiring noble metal as catalyst seem quite promising. One of the main hurdles for emergence of such a technology is the development of a hydroxide-conducting membrane characterized by both high conductivity and low fuel permeability. Plasma treatments can enable to positively tune the main fuel cell membrane requirements. In this work, commercial ADP-Morgane® fluorinated polymer membranes and a new brand of cross-linked poly(aryl-ether) polymer membranes, named AMELI-32®, both containing quaternary ammonium functionalities, have been modified by argon plasma treatment or triallylamine-based plasma deposit. Under the concomitant etching/cross-linking/oxidation effects inherent to the plasma modification, transport properties (ionic exchange capacity, water uptake, ionic conductivity and fuel retention) of membranes have been improved. Consequently, using plasma modified ADP-Morgane® membrane as electrolyte in a solid alkaline fuel cell operating with glycerol as fuel has allowed increasing the maximum power density by a factor 3 when compared to the untreated membrane. PMID:24958295

Plasma membrane-associated platforms: dynamic scaffolds that organize membrane-associated events.

PubMed

Astro, Veronica; de Curtis, Ivan

2015-03-10

Specialized regions of the plasma membrane dedicated to diverse cellular processes, such as vesicle exocytosis, extracellular matrix remodeling, and cell migration, share a few cytosolic scaffold proteins that associate to form large plasma membrane-associated platforms (PMAPs). PMAPs organize signaling events and trafficking of membranes and molecules at specific membrane domains. On the basis of the intrinsic disorder of the proteins constituting the core of these PMAPs and of the dynamics of these structures at the periphery of motile cells, we propose a working model for the assembly and turnover of these platforms. Copyright © 2015, American Association for the Advancement of Science.

Purification and proteomic analysis of plant plasma membranes.

PubMed

Alexandersson, Erik; Gustavsson, Niklas; Bernfur, Katja; Karlsson, Adine; Kjellbom, Per; Larsson, Christer

2008-01-01

All techniques needed for proteomic analyses of plant plasma membranes are described in detail, from isolation of plasma membranes to protein identification by mass spectrometry (MS). Plasma membranes are isolated by aqueous two-phase partitioning yielding vesicles with a cytoplasmic side-in orientation and a purity of about 95%. These vesicles are turned inside-out by treatment with Brij 58, which removes soluble contaminating proteins enclosed in the vesicles as well as loosely attached proteins. The final plasma membrane preparation thus retains all integral proteins and many peripheral proteins. Proteins are separated by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and protein bands are excised and digested with trypsin. Peptides in tryptic digests are separated by nanoflow liquid chromatography and either fed directly into an ESI-MS or spotted onto matrix-assisted laser desorption ionization (MALDI) plates for analysis with MALDI-MS. Finally, data processing and database searching are used for protein identification to define a plasma membrane proteome.

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

PubMed

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

2015-01-01

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

Lateral mobility of plasma membrane lipids in dividing Xenopus eggs.

PubMed

Tetteroo, P A; Bluemink, J G; Dictus, W J; van Zoelen, E J; de Laat, S W

1984-07-01

The lateral mobility of plasma membrane lipids was analyzed during first cleavage of Xenopus laevis eggs by fluorescence photobleaching recovery (FPR) measurements, using the lipid analogs 5-(N-hexadecanoyl)aminofluorescein ("HEDAF") and 5-(N-tetradecanoyl)aminofluorescein ("TEDAF") as probes. The preexisting plasma membrane of the animal side showed an inhomogeneous, dotted fluorescence pattern after labeling and the lateral mobility of both probes used was below the detection limits of the FPR method (D much less than 10(-10) cm2/sec). In contrast, the preexisting plasma membrane of the vegetal side exhibited homogeneous fluorescence and the lateral diffusion coefficient of both probes used was relatively high (HEDAF, D = 2.8 X 10(-8) cm2/sec; TEDAF, D = 2.4 X 10(-8) cm2/sec). In the cleaving egg visible transfer of HEDAF or TEDAF from prelabeled plasma membrane to the new membrane in the furrow did not occur, even on the vegetal side. Upon labeling during cleavage, however, the new membrane was uniformly labeled and both probes were mobile, as in the vegetal preexisting plasma membrane. These data show that the membrane of the dividing Xenopus egg comprises three macrodomains: (i) the animal preexisting plasma membrane; (ii) the vegetal preexisting plasma membrane; (iii) the new furrow membrane.

Isolation of plasma membrane-associated membranes from rat liver.

PubMed

Suski, Jan M; Lebiedzinska, Magdalena; Wojtala, Aleksandra; Duszynski, Jerzy; Giorgi, Carlotta; Pinton, Paolo; Wieckowski, Mariusz R

2014-02-01

Dynamic interplay between intracellular organelles requires a particular functional apposition of membrane structures. The organelles involved come into close contact, but do not fuse, thereby giving rise to notable microdomains; these microdomains allow rapid communication between the organelles. Plasma membrane-associated membranes (PAMs), which are microdomains of the plasma membrane (PM) interacting with the endoplasmic reticulum (ER) and mitochondria, are dynamic structures that mediate transport of proteins, lipids, ions and metabolites. These structures have gained much interest lately owing to their roles in many crucial cellular processes. Here we provide an optimized protocol for the isolation of PAM, PM and ER fractions from rat liver that is based on a series of differential centrifugations, followed by the fractionation of crude PM on a discontinuous sucrose gradient. The procedure requires ∼8-10 h, and it can be easily modified and adapted to other tissues and cell types.

Response of plasma membrane H+-ATPase in rice (Oryza sativa) seedlings to simulated acid rain.

PubMed

Liang, Chanjuan; Ge, Yuqing; Su, Lei; Bu, Jinjin

2015-01-01

Understanding the adaptation of plants to acid rain is important to find feasible approaches to alleviate such damage to plants. We studied effects of acid rain on plasma membrane H(+)-ATPase activity and transcription, intracellular H(+), membrane permeability, photosynthetic efficiency, and relative growth rate during stress and recovery periods. Simulated acid rain at pH 5.5 did not affect plasma membrane H(+)-ATPase activity, intracellular H(+), membrane permeability, photosynthetic efficiency, and relative growth rate. Plasma membrane H(+)-ATPase activity and transcription in leaves treated with acid rain at pH 3.5 was increased to maintain ion homeostasis by transporting excessive H(+) out of cells. Then intracellular H(+) was close to the control after a 5-day recovery, alleviating damage on membrane and sustaining photosynthetic efficiency and growth. Simulated acid rain at pH 2.5 inhibited plasma membrane H(+)-ATPase activity by decreasing the expression of H(+)-ATPase at transcription level, resulting in membrane damage and abnormal intracellular H(+), and reduction in photosynthetic efficiency and relative growth rate. After a 5-day recovery, all parameters in leaves treated with pH 2.5 acid rain show alleviated damage, implying that the increased plasma membrane H(+)-ATPase activity and its high expression were involved in repairing process in acid rain-stressed plants. Our study suggests that plasma membrane H(+)-ATPase can play a role in adaptation to acid rain for rice seedlings.

Mammalian plasma membrane proteins as potential biomarkers and drug targets.

PubMed

Rucevic, Marijana; Hixson, Douglas; Josic, Djuro

2011-06-01

Defining the plasma membrane proteome is crucial to understand the role of plasma membrane in fundamental biological processes. Change in membrane proteins is one of the first events that take place under pathological conditions, making plasma membrane proteins a likely source of potential disease biomarkers with prognostic or diagnostic potential. Membrane proteins are also potential targets for monoclonal antibodies and other drugs that block receptors or inhibit enzymes essential to the disease progress. Despite several advanced methods recently developed for the analysis of hydrophobic proteins and proteins with posttranslational modifications, integral membrane proteins are still under-represented in plasma membrane proteome. Recent advances in proteomic investigation of plasma membrane proteins, defining their roles as diagnostic and prognostic disease biomarkers and as target molecules in disease treatment, are presented. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Fibrinogen Reduction During Selective Plasma Exchange due to Membrane Fouling.

PubMed

Ohkubo, Atsushi; Okado, Tomokazu; Miyamoto, Satoko; Hashimoto, Yurie; Komori, Shigeto; Yamamoto, Motoki; Maeda, Takuma; Itagaki, Ayako; Yamamoto, Hiroko; Seshima, Hiroshi; Kurashima, Naoki; Iimori, Soichiro; Naito, Shotaro; Sohara, Eisei; Uchida, Shinichi; Rai, Tatemitsu

2017-06-01

Fibrinogen is substantially reduced by most plasmapheresis modalities but retained in selective plasma exchange using Evacure EC-4A10 (EC-4A). Although EC-4A's fibrinogen sieving coefficient is 0, a session of selective plasma exchange reduced fibrinogen by approximately 19%. Here, we investigated sieving coefficient in five patients. When the mean processed plasma volume was 1.15 × plasma volume, the mean reduction of fibrinogen during selective plasma exchange was approximately 15%. Fibrinogen sieving coefficient was 0 when the processed plasma volume was 1.0 L, increasing to 0.07 when the processed plasma volume was 3.0 L, with a mean of 0.03 during selective plasma exchange. When fibrinogen sieving coefficient was 0, selective plasma exchange reduced fibrinogen by approximately 10%. Scanning electron microscopy images revealed internal fouling of EC-4A's hollow fiber membrane by substances such as fibrinogen fibrils. Thus, fibrinogen reduction by selective plasma exchange may be predominantly caused by membrane fouling rather than filtration. © 2017 International Society for Apheresis, Japanese Society for Apheresis, and Japanese Society for Dialysis Therapy.

Fluidity of pea root plasma membranes under altered gravity

NASA Astrophysics Data System (ADS)

Klymchuk, D. O.; Baranenko, V. V.; Vorobyova, T. V.; Dubovoy, V. D.

This investigation aims to determine whether clinorotation 2 rev min of pea Pisum sativum L seedlings induces the alterations in the physical-chemical properties of cellular membranes including the plasma membrane fluidity The last is an important regulator of functional activity of membrane enzymes The plasma membranes were isolated by aqueous two-phase partitioning from roots of 6-day old pea seedlings The membrane fluidity was examined by fluorescence spectroscopy using pyrene probe The plasma membrane vesicles with known protein concentration were added to the incubation buffer to a final concentration of 50 mu g of protein per ml A small amount by 1 mu l of pyrene solution in 2-propanol was added to the incubation mixture to a final probe concentration 5 mu M at constant mixing Fluorescence spectra were measured using a Perkin-Elmer LS-50 spectrofluorometer Perkin-Elmer England Pyrene was excited at 337 nm and fluorescence intensity of monomers I M and excimers I E were measured at 393 and 470 nm respectively The I E I M ratios were 0 081 pm 0 003 and 0 072 pm 0 004 in preparations obtained from clinorotated and the control seedlings respectively This fact indicates that rotation on the clinostat increases the membrane fluidity Compared with controls clinorotated seedlings have also showed a reduced growth and a higher level of total unsaturated fatty acids determined by gas chromatography The factors that influence on the fluidity of membrane lipids in bilayer appear to be the

Essentially All Excess Fibroblast Cholesterol Moves from Plasma Membranes to Intracellular Compartments

PubMed Central

Lange, Yvonne; Ye, Jin; Steck, Theodore L.

2014-01-01

It has been shown that modestly increasing plasma membrane cholesterol beyond its physiological set point greatly increases the endoplasmic reticulum and mitochondrial pools, thereby eliciting manifold feedback responses that return cell cholesterol to its resting state. The question arises whether this homeostatic mechanism reflects the targeting of cell surface cholesterol to specific intracellular sites or its general equilibration among the organelles. We now show that human fibroblast cholesterol can be increased as much as two-fold from 2-hydroxypropyl-β-cyclodextrin without changing the size of the cell surface pool. Rather, essentially all of the added cholesterol disperses rapidly among cytoplasmic membranes, increasing their overall cholesterol content by as much as five-fold. We conclude that the level of plasma membrane cholesterol is normally at capacity and that even small increments above this physiological set point redistribute essentially entirely to intracellular membranes, perhaps down their chemical activity gradients. PMID:25014655

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

PubMed

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

2015-01-01

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

Light-induced modification of plant plasma membrane ion transport.

PubMed

Marten, I; Deeken, R; Hedrich, R; Roelfsema, M R G

2010-09-01

Light is not only the driving force for electron and ion transport in the thylakoid membrane, but also regulates ion transport in various other membranes of plant cells. Light-dependent changes in ion transport at the plasma membrane and associated membrane potential changes have been studied intensively over the last century. These studies, with various species and cell types, revealed that apart from regulation by chloroplasts, plasma membrane transport can be controlled by phytochromes, phototropins or channel rhodopsins. In this review, we compare light-dependent plasma membrane responses of unicellular algae (Eremosphaera and Chlamydomonas), with those of a multicellular alga (Chara), liverworts (Conocephalum), mosses (Physcomitrella) and several angiosperm cell types. Light-dependent plasma membrane responses of Eremosphaera and Chara are characterised by the dominant role of K(+) channels during membrane potential changes. In most other species, the Ca(2+)-dependent activation of plasma membrane anion channels represents a general light-triggered event. Cell type-specific responses are likely to have evolved by modification of this general response or through the development of additional light-dependent signalling pathways. Future research to elucidate these light-activated signalling chains is likely to benefit from the recent identification of S-type anion channel genes and proteins capable of regulating these channels.

Taurine transport across hepatocyte plasma membranes: analysis in isolated rat liver sinusoidal plasma membrane vesicles.

PubMed

Inoue, M; Arias, I M

1988-07-01

To elucidate the mechanism of taurine transport across the hepatic plasma membranes, rat liver sinusoidal plasma membrane vesicles were isolated and the transport process was analyzed. In the presence of a sodium gradient across the membranes (vesicle inside less than vesicle outside), an overshooting uptake of taurine occurred. In the presence of other ion gradients (K+, Li+, and choline+), taurine uptake was very small and no such overshoot was observed. Sodium-dependent uptake of taurine occurred into an osmotically active intravesicular space. Taurine uptake was stimulated by preloading vesicles with unlabeled taurine (transstimulation) in the presence of NaCl, but not in the presence of KCl. Sodium-dependent transport followed saturation kinetics with respect to taurine concentration; double-reciprocal plots of uptake versus taurine concentration gave a straight line from which an apparent Km value of 0.38 mM and Vmax of 0.27 nmol/20 s x mg of protein were obtained. Valinomycin-induced K+-diffusion potential failed to enhance the rate of taurine uptake, suggesting that taurine transport does not depend on membrane potential. Taurine transport was inhibited by structurally related omega-amino acids, such as beta-alanine and gamma-aminobutyric acid, but not by glycine, epsilon-aminocaproic acid, or other alpha-amino acids, such as L-alanine. These results suggest that Na+-dependent uptake of taurine might occur across the hepatic sinusoidal plasma membranes via a transport system that is specific for omega-amino acids having 2-3 carbon chain length.

Na+/H+ Exchange Activity in the Plasma Membrane of Arabidopsis1

PubMed Central

Qiu, Quan-Sheng; Barkla, Bronwyn J.; Vera-Estrella, Rosario; Zhu, Jian-Kang; Schumaker, Karen S.

2003-01-01

In plants, Na+/H+ exchangers in the plasma membrane are critical for growth in high levels of salt, removing toxic Na+ from the cytoplasm by transport out of the cell. The molecular identity of a plasma membrane Na+/H+ exchanger in Arabidopsis (SOS1) has recently been determined. In this study, immunological analysis provided evidence that SOS1 localizes to the plasma membrane of leaves and roots. To characterize the transport activity of this protein, purified plasma membrane vesicles were isolated from leaves of Arabidopsis. Na+/H+ exchange activity, monitored as the ability of Na to dissipate an established pH gradient, was absent in plants grown without salt. However, exchange activity was induced when plants were grown in 250 mm NaCl and increased with prolonged salt exposure up to 8 d. H+-coupled exchange was specific for Na, because chloride salts of other monovalent cations did not dissipate the pH gradient. Na+/H+ exchange activity was dependent on Na (substrate) concentration, and kinetic analysis indicated that the affinity (apparent Km) of the transporter for Na+ is 22.8 mm. Data from two experimental approaches supports electroneutral exchange (one Na+ exchanged for one proton): (a) no change in membrane potential was measured during the exchange reaction, and (b) Na+/H+ exchange was unaffected by the presence or absence of a membrane potential. Results from this research provide a framework for future studies into the regulation of the plant plasma membrane Na+/H+ exchanger and its relative contribution to the maintenance of cellular Na+ homeostasis during plant growth in salt. PMID:12805632

Excess plasma membrane and effects of ionic amphipaths on mechanics of outer hair cell lateral wall.

PubMed

Morimoto, Noriko; Raphael, Robert M; Nygren, Anders; Brownell, William E

2002-05-01

The interaction between the outer hair cell (OHC) lateral wall plasma membrane and the underlying cortical lattice was examined by a morphometric analysis of cell images during cell deformation. Vesiculation of the plasma membrane was produced by micropipette aspiration in control cells and cells exposed to ionic amphipaths that alter membrane mechanics. An increase of total cell and vesicle surface area suggests that the plasma membrane possesses a membrane reservoir. Chlorpromazine (CPZ) decreased the pressure required for vesiculation, whereas salicylate (Sal) had no effect. The time required for vesiculation was decreased by CPZ, indicating that CPZ decreases the energy barrier required for vesiculation. An increase in total volume is observed during micropipette aspiration. A deformation-induced increase in hydraulic conductivity is also seen in response to micropipette-applied fluid jet deformation of the lateral wall. Application of CPZ and/or Sal decreased this strain-induced hydraulic conductivity. The impact of ionic amphipaths on OHC plasma membrane and lateral wall mechanics may contribute to their effects on OHC electromotility and hearing.

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

PubMed

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

1987-07-28

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

Whey protein hydrolysate increases translocation of GLUT-4 to the plasma membrane independent of insulin in wistar rats.

PubMed

Morato, Priscila Neder; Lollo, Pablo Christiano Barboza; Moura, Carolina Soares; Batista, Thiago Martins; Camargo, Rafael Ludemann; Carneiro, Everardo Magalhães; Amaya-Farfan, Jaime

2013-01-01

Whey protein (WP) and whey protein hydrolysate (WPH) have the recognized capacity to increase glycogen stores. The objective of this study was to verify if consuming WP and WPH could also increase the concentration of the glucose transporters GLUT-1 and GLUT-4 in the plasma membrane (PM) of the muscle cells of sedentary and exercised animals. Forty-eight Wistar rats were divided into 6 groups (n = 8 per group), were treated and fed with experimental diets for 9 days as follows: a) control casein (CAS); b) WP; c) WPH; d) CAS exercised; e) WP exercised; and f) WPH exercised. After the experimental period, the animals were sacrificed, muscle GLUT-1 and GLUT-4, p85, Akt and phosphorylated Akt were analyzed by western blotting, and the glycogen, blood amino acids, insulin levels and biochemical health indicators were analyzed using standard methods. Consumption of WPH significantly increased the concentrations of GLUT-4 in the PM and glycogen, whereas the GLUT-1 and insulin levels and the health indicators showed no alterations. The physical exercise associated with consumption of WPH had favorable effects on glucose transport into muscle. These results should encourage new studies dealing with the potential of both WP and WPH for the treatment or prevention of type II diabetes, a disease in which there is reduced translocation of GLUT-4 to the plasma membrane.

Direct chemical evidence for sphingolipid domains in the plasma membranes of fibroblasts [High-Resolution Chemical Imaging of Sphingolipid Distribution in the Plasma Membrane

DOE PAGES

Frisz, Jessica F.; Lou, Kaiyan; Klitzing, Haley A.; ...

2013-01-28

Sphingolipids play important roles in plasma membrane structure and cell signaling. Yet, their lateral distribution in the plasma membrane is poorly understood. Here we quantitatively analyzed the sphingolipid organization on the entire dorsal surface of intact cells by mapping the distribution of 15N-enriched ions from metabolically labeled 15N-sphingolipids in the plasma membrane using high-resolution imaging mass spectrometry. Many types of control experiments (internal, positive, negative, and fixation temperature), along with parallel experiments involving the imaging of fluorescent sphingolipids$-$both in living cells and during fixation of living cells$-$exclude potential artifacts. Micrometer-scale sphingolipid patches consisting of numerous 15Nsphingolipid microdomains with mean diametersmore » of ~200 nm are always present in the plasma membrane. Depletion of 30% of the cellular cholesterol did not eliminate the sphingolipid domains, but did reduce their abundance and long range organization in the plasma membrane. In contrast, disruption of the cytoskeleton eliminated the sphingolipid domains. These results indicate that these sphingolipid assemblages are not lipid rafts, and are instead a distinctly different type of sphingolipid-enriched plasma membrane domain that depends upon cortical actin.« less

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

PubMed

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

2013-12-13

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

The Role of the Plasma Membrane H+-ATPase in Plant Responses to Aluminum Toxicity.

PubMed

Zhang, Jiarong; Wei, Jian; Li, Dongxu; Kong, Xiangying; Rengel, Zed; Chen, Limei; Yang, Ye; Cui, Xiuming; Chen, Qi

2017-01-01

Aluminum (Al) toxicity is a key factor limiting plant growth and crop production on acid soils. Increasing the plant Al-detoxification capacity and/or breeding Al-resistant cultivars are a cost-effective strategy to support crop growth on acidic soils. The plasma membrane H + -ATPase plays a central role in all plant physiological processes. Changes in the activity of the plasma membrane H + -ATPase through regulating the expression and phosphorylation of this enzyme are also involved in many plant responses to Al toxicity. The plasma membrane H + -ATPase mediated H + influx may be associated with the maintenance of cytosolic pH and the plasma membrane gradients as well as Al-induced citrate efflux mediated by a H + -ATPase-coupled MATE co-transport system. In particular, modulating the activity of plasma membrane H + -ATPase through application of its activators (e.g., magnesium or IAA) or using transgenics has effectively enhanced plant resistance to Al stress in several species. In this review, we critically assess the available knowledge on the role of the plasma membrane H + -ATPase in plant responses to Al stress, incorporating physiological and molecular aspects.

Identification of new intrinsic proteins in Arabidopsis plasma membrane proteome.

PubMed

Marmagne, Anne; Rouet, Marie-Aude; Ferro, Myriam; Rolland, Norbert; Alcon, Carine; Joyard, Jacques; Garin, Jérome; Barbier-Brygoo, Hélène; Ephritikhine, Geneviève

2004-07-01

Identification and characterization of anion channel genes in plants represent a goal for a better understanding of their central role in cell signaling, osmoregulation, nutrition, and metabolism. Though channel activities have been well characterized in plasma membrane by electrophysiology, the corresponding molecular entities are little documented. Indeed, the hydrophobic protein equipment of plant plasma membrane still remains largely unknown, though several proteomic approaches have been reported. To identify new putative transport systems, we developed a new proteomic strategy based on mass spectrometry analyses of a plasma membrane fraction enriched in hydrophobic proteins. We produced from Arabidopsis cell suspensions a highly purified plasma membrane fraction and characterized it in detail by immunological and enzymatic tests. Using complementary methods for the extraction of hydrophobic proteins and mass spectrometry analyses on mono-dimensional gels, about 100 proteins have been identified, 95% of which had never been found in previous proteomic studies. The inventory of the plasma membrane proteome generated by this approach contains numerous plasma membrane integral proteins, one-third displaying at least four transmembrane segments. The plasma membrane localization was confirmed for several proteins, therefore validating such proteomic strategy. An in silico analysis shows a correlation between the putative functions of the identified proteins and the expected roles for plasma membrane in transport, signaling, cellular traffic, and metabolism. This analysis also reveals 10 proteins that display structural properties compatible with transport functions and will constitute interesting targets for further functional studies.

Interleaflet Coupling, Pinning, and Leaflet Asymmetry—Major Players in Plasma Membrane Nanodomain Formation

PubMed Central

Fujimoto, Toyoshi; Parmryd, Ingela

2017-01-01

The plasma membrane has a highly asymmetric distribution of lipids and contains dynamic nanodomains many of which are liquid entities surrounded by a second, slightly different, liquid environment. Contributing to the dynamics is a continuous repartitioning of components between the two types of liquids and transient links between lipids and proteins, both to extracellular matrix and cytoplasmic components, that temporarily pin membrane constituents. This make plasma membrane nanodomains exceptionally challenging to study and much of what is known about membrane domains has been deduced from studies on model membranes at equilibrium. However, living cells are by definition not at equilibrium and lipids are distributed asymmetrically with inositol phospholipids, phosphatidylethanolamines and phosphatidylserines confined mostly to the inner leaflet and glyco- and sphingolipids to the outer leaflet. Moreover, each phospholipid group encompasses a wealth of species with different acyl chain combinations whose lateral distribution is heterogeneous. It is becoming increasingly clear that asymmetry and pinning play important roles in plasma membrane nanodomain formation and coupling between the two lipid monolayers. How asymmetry, pinning, and interdigitation contribute to the plasma membrane organization is only beginning to be unraveled and here we discuss their roles and interdependence. PMID:28119914

Interleaflet Coupling, Pinning, and Leaflet Asymmetry-Major Players in Plasma Membrane Nanodomain Formation.

PubMed

Fujimoto, Toyoshi; Parmryd, Ingela

2016-01-01

The plasma membrane has a highly asymmetric distribution of lipids and contains dynamic nanodomains many of which are liquid entities surrounded by a second, slightly different, liquid environment. Contributing to the dynamics is a continuous repartitioning of components between the two types of liquids and transient links between lipids and proteins, both to extracellular matrix and cytoplasmic components, that temporarily pin membrane constituents. This make plasma membrane nanodomains exceptionally challenging to study and much of what is known about membrane domains has been deduced from studies on model membranes at equilibrium. However, living cells are by definition not at equilibrium and lipids are distributed asymmetrically with inositol phospholipids, phosphatidylethanolamines and phosphatidylserines confined mostly to the inner leaflet and glyco- and sphingolipids to the outer leaflet. Moreover, each phospholipid group encompasses a wealth of species with different acyl chain combinations whose lateral distribution is heterogeneous. It is becoming increasingly clear that asymmetry and pinning play important roles in plasma membrane nanodomain formation and coupling between the two lipid monolayers. How asymmetry, pinning, and interdigitation contribute to the plasma membrane organization is only beginning to be unraveled and here we discuss their roles and interdependence.

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

Giant plasma membrane vesicles: models for understanding membrane organization.

PubMed

Levental, Kandice R; Levental, Ilya

2015-01-01

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

Study on surface adhesion of Plasma modified Polytetrafluoroethylene hollow fiber membrane

NASA Astrophysics Data System (ADS)

Chen, Jiangrong; Zhang, Huifeng; Liu, Guochang; Guo, Chungang; Lv, Jinglie; Zhangb, Yushan

2018-01-01

Polytetrafluoroethylene (PTFE) is popular membrane material because of its excellent thermal stability, chemical stability and mechanical stability. However, the low surface energy and non-sticky property of PTFE present challenges for modification. In the present study, plasma treatment was performed to improve the surface adhesion of PTFE hollow fiber membrane. The effect of discharge voltage, treatment time on the adhesion of PTFE hollow fiber membrane was symmetrically evaluated. Results showed that the plasma treatment method contributed to improve the surface activity and roughness of PTFE hollow fiber membrane, and the adhesion strength depend significantly on discharge voltage, which was beneficial to seepage pressure of PTFE hollow fiber membrane module. The adhesion strength of PTFE membrane by plasma treated at 220V for 3min reached as high as 86.2 N, far surpassing the adhesion strength 12.7 N of pristine membrane. Furthermore, improvement of content of free radical and composition analysis changes of the plasma modified PTFE membrane were investigated. The seepage pressure of PTFE membrane by plasma treated at 220V for 3min was 0.375 MPa, which means that the plasma treatment is an effective technique to improve the adhesion strength of membrane.

Endoplasmic Reticulum-Plasma Membrane Contacts Regulate Cellular Excitability.

PubMed

Dickson, Eamonn J

2017-01-01

Cells that have intrinsic electrical excitability utilize changes in membrane potential to communicate with neighboring cells and initiate cellular cascades. Excitable cells like neurons and myocytes have evolved highly specialized subcellular architectures to translate these electrical signals into cellular events. One such structural specialization is sarco-/endoplasmic reticulum-plasma membrane contact sites. These membrane contact sites are positioned by specific membrane-membrane tethering proteins and contain an ever-expanding list of additional proteins that organize information transfer across the junctional space (~ 15-25 nm distance) to shape membrane identity and control cellular excitability. In this chapter we discuss how contacts between the sarco-/endoplasmic reticulum and plasma membrane are essential for regulated excitation-contraction coupling in striated muscle and control of lipid-dependent ion channels.

Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein is PAM involved in the capacitative calcium entry?

PubMed

Kozieł, Katarzyna; Lebiedzinska, Magdalena; Szabadkai, Gyorgy; Onopiuk, Marta; Brutkowski, Wojciech; Wierzbicka, Katarzyna; Wilczyński, Grzegorz; Pinton, Paolo; Duszyński, Jerzy; Zabłocki, Krzysztof; Wieckowski, Mariusz R

2009-12-01

A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca(2+) signalling and maintenance of Ca(2+) homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca(2+)-ATPase, Na(+), K(+)-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca(2+) ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca(2+) entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca(2+) entry, and their formation and rebuilding have an important regulatory role in cellular Ca(2+) homeostasis.

[Biocompatibility of poly-L-lactic acid/Bioglass-guided bone regeneration membranes processed with oxygen plasma].

PubMed

Fang, Wei; Zeng, Shu-Guang; Gao, Wen-Feng

2015-04-01

To prepare and characterize a nano-scale fibrous hydrophilic poly-L-lactic acid/ Bioglass (PLLA/BG) composite membrane and evaluate its biocompatibility as a composite membrane for guiding bone regeneration (GBR). PLLA/BG-guided bone regeneration membrane was treated by oxygen plasma to improved its hydrophilicity. The growth of MG-63 osteoblasts on the membrane was observed using Hoechst fluorescence staining, and the biocompatibility of the membrane was evaluated by calculating the cells adhesion rate and proliferation rate. Osteogenesis of MG-63 cells was assessed by detecting alkaline phosphatase (ALP), and the formation of calcified nodules and cell morphology changes were observed using scanning electron microscope (SEM). The cell adhesion rates of PLLA/BG-guided bone regeneration membrane treated with oxygen plasma were (30.570±0.96)%, (47.27±0.78)%, and (66.78±0.69)% at 1, 3, and 6 h, respectively, significantly higher than those on PLLA membrane and untreated PLLA/BG membrane (P<0.01). The cell proliferation rates on the 3 membranes increased with time, but highest on oxygen plasma-treated PLLA/BG membrane (P<0.01). Hoechst fluorescence staining revealed that oxygen plasma treatment of the PLLA/BG membrane promoted cell adhesion. The membranes with Bioglass promoted the matrix secretion of the osteoblasts. Under SEM, the formation of calcified nodules and spindle-shaped cell morphology were observed on oxygen plasma-treated PLLA/BG membrane. Oxygen plasma-treated PLLA/BG composite membrane has good biocompatibility and can promote adhesion, proliferation and osteogenesis of the osteoblasts.

The Chemical Potential of Plasma Membrane Cholesterol: Implications for Cell Biology.

PubMed

Ayuyan, Artem G; Cohen, Fredric S

2018-02-27

Cholesterol is abundant in plasma membranes and exhibits a variety of interactions throughout the membrane. Chemical potential accounts for thermodynamic consequences of molecular interactions, and quantifies the effective concentration (i.e., activity) of any substance participating in a process. We have developed, to our knowledge, the first method to measure cholesterol chemical potential in plasma membranes. This was accomplished by complexing methyl-β-cyclodextrin with cholesterol in an aqueous solution and equilibrating it with an organic solvent containing dissolved cholesterol. The chemical potential of cholesterol was thereby equalized in the two phases. Because cholesterol is dilute in the organic phase, here activity and concentration were equivalent. This equivalence allowed the amount of cholesterol bound to methyl-β-cyclodextrin to be converted to cholesterol chemical potential. Our method was used to determine the chemical potential of cholesterol in erythrocytes and in plasma membranes of nucleated cells in culture. For erythrocytes, the chemical potential did not vary when the concentration was below a critical value. Above this value, the chemical potential progressively increased with concentration. We used standard cancer lines to characterize cholesterol chemical potential in plasma membranes of nucleated cells. This chemical potential was significantly greater for highly metastatic breast cancer cells than for nonmetastatic breast cancer cells. Chemical potential depended on density of the cancer cells. A method to alter and fix the cholesterol chemical potential to any value (i.e., a cholesterol chemical potential clamp) was also developed. Cholesterol content did not change when cells were clamped for 24-48 h. It was found that the level of activation of the transcription factor STAT3 increased with increasing cholesterol chemical potential. The cholesterol chemical potential may regulate signaling pathways. Copyright © 2018. Published by

Reorganization of plasma membrane lipid domains during conidial germination.

PubMed

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

2017-02-01

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

Putting together a plasma membrane NADH oxidase: a tale of three laboratories.

PubMed

Löw, Hans; Crane, Frederick L; Morré, D James

2012-11-01

The observation that high cellular concentrations of NADH were associated with low adenylate cyclase activity led to a search for the mechanism of the effect. Since cyclase is in the plasma membrane, we considered the membrane might have a site for NADH action, and that NADH might be oxidized at that site. A test for NADH oxidase showed very low activity, which could be increased by adding growth factors. The plasma membrane oxidase was not inhibited by inhibitors of mitochondrial NADH oxidase such as cyanide, rotenone or antimycin. Stimulation of the plasma membrane oxidase by iso-proterenol or triiodothyronine was different from lack of stimulation in endoplasmic reticulum. After 25 years of research, three components of a trans membrane NADH oxidase have been discovered. Flavoprotein NADH coenzyme Q reductases (NADH cytochrome b reductase) on the inside, coenzyme Q in the middle, and a coenzyme Q oxidase on the outside as a terminal oxidase. The external oxidase segment is a copper protein with unique properties in timekeeping, protein disulfide isomerase and endogenous NADH oxidase activity, which affords a mechanism for control of cell growth by the overall NADH oxidase and the remarkable inhibition of oxidase activity and growth of cancer cells by a wide range of anti-tumor drugs. A second trans plasma membrane electron transport system has been found in voltage dependent anion channel (VDAC), which has NADH ferricyanide reductase activity. This activity must be considered in relation to ferricyanide stimulation of growth and increased VDAC antibodies in patients with autism. Copyright © 2012 Elsevier Ltd. All rights reserved.

An adhesion-based method for plasma membrane isolation: evaluating cholesterol extraction from cells and their membranes.

PubMed

Bezrukov, Ludmila; Blank, Paul S; Polozov, Ivan V; Zimmerberg, Joshua

2009-11-15

A method to isolate large quantities of directly accessible plasma membrane from attached cells is presented. The method is based on the adhesion of cells to an adsorbed layer of polylysine on glass plates, followed by hypotonic lysis with ice-cold distilled water and subsequent washing steps. Optimal conditions for coating glass plates and time for cell attachment were established. No additional chemical or mechanical treatments were used. Contamination of the isolated plasma membrane by cell organelles was less than 5%. The method uses inexpensive, commercially available polylysine and reusable glass plates. Plasma membrane preparations can be made in 15 min. Using this method, we determined that methyl-beta-cyclodextrin differentially extracts cholesterol from fibroblast cells and their plasma membranes and that these differences are temperature dependent. Determination of the cholesterol/phospholipid ratio from intact cells does not reflect methyl-beta-cyclodextrin plasma membrane extraction properties.

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

PubMed

Saarikangas, Juha; Zhao, Hongxia; Lappalainen, Pekka

2010-01-01

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

Composite plasma polymerized sulfonated polystyrene membrane for PEMFC

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

Nath, Bhabesh Kumar; Khan, Aziz; Chutia, Joyanti, E-mail: jchutiaiasst@gmail.com

2015-10-15

Highlights: • Methyl methane sulfonate (MMS) is used as the sulfonating agent. • The proton conductivity of the membrane is found to be 0.141 S cm{sup −1}. • Power density of fuel cell with styrene/MMS membrane is 0.5 W cm{sup −2}. • The membrane exhibits thermal stability up to 140 °C. - Abstract: This work presents the introduction of an organic compound methyl methane sulfonate (MMS) for the first time in fabrication of polystyrene based proton exchange membrane (PEM) by plasma polymerization process. The membrane is fabricated by co-polymerizing styrene and MMS in capacitively coupled continuous RF plasma. The chemicalmore » composition of the plasma polymerized polymer membrane is investigated using Fourier Transform Infrared Spectroscopy which reveals the formation of composite structure of styrene and MMS. The surface morphology studied using AFM and SEM depicts the effect of higher partial pressure of MMS on surface topography of the membrane. The proton transport property of the membrane studied using electrochemical impedance spectroscopy shows the achievement of maximum proton conductivity of 0.141 S cm{sup −1} which is comparable to Nafion 117 membrane. Fuel cell performance test of the synthesized membrane shows a maximum power density of 500 mW cm{sup −2} and current density of 0.62 A cm{sup −2} at 0.6 V.« less

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

PubMed Central

Kraft, Mary L.

2017-01-01

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

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

PubMed

Kraft, Mary L

2016-01-01

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

Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes.

PubMed

Csermely, P; Szamel, M; Resch, K; Somogyi, J

1988-05-15

In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested (Parker, P.J., Coussens, L., Totty, N., Rhee, L., Young, S., Chen, E., Stabel, S., Waterfield, M.D., and Ullrich, A. (1986) Science 233, 853-859). In the present report, we demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes, and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn2+, while Fe2+ and Mn2+ are only partially counteractive. Our results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca2+, phorbol ester, or antigen.

Protein diffusion in plant cell plasma membranes: the cell-wall corral.

PubMed

Martinière, Alexandre; Runions, John

2013-01-01

Studying protein diffusion informs us about how proteins interact with their environment. Work on protein diffusion over the last several decades has illustrated the complex nature of biological lipid bilayers. The plasma membrane contains an array of membrane-spanning proteins or proteins with peripheral membrane associations. Maintenance of plasma membrane microstructure can be via physical features that provide intrinsic ordering such as lipid microdomains, or from membrane-associated structures such as the cytoskeleton. Recent evidence indicates, that in the case of plant cells, the cell wall seems to be a major player in maintaining plasma membrane microstructure. This interconnection / interaction between cell-wall and plasma membrane proteins most likely plays an important role in signal transduction, cell growth, and cell physiological responses to the environment.

Necroptosis Execution Is Mediated by Plasma Membrane Nanopores Independent of Calcium.

PubMed

Ros, Uris; Peña-Blanco, Aida; Hänggi, Kay; Kunzendorf, Ulrich; Krautwald, Stefan; Wong, W Wei-Lynn; García-Sáez, Ana J

2017-04-04

Necroptosis is a form of regulated necrosis that results in cell death and content release after plasma membrane permeabilization. However, little is known about the molecular events responsible for the disruption of the plasma membrane. Here, we find that early increase in cytosolic calcium in TNF-induced necroptosis is mediated by treatment with a Smac mimetic via the TNF/RIP1/TAK1 survival pathway. This does not require the activation of the necrosome and is dispensable for necroptosis. Necroptosis induced by the activation of TLR3/4 pathways does not trigger early calcium flux. We also demonstrate that necroptotic plasma membrane rupture is mediated by osmotic forces and membrane pores around 4 nm in diameter. This late permeabilization step represents a hallmark in necroptosis execution that is cell and treatment independent and requires the RIP1/RIP3/MLKL core. In support of this, treatment with osmoprotectants reduces cell damage in an in vivo necroptosis model of ischemia-reperfusion injury. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Basolateral cholesterol depletion alters Aquaporin-2 post-translational modifications and disrupts apical plasma membrane targeting.

PubMed

Moeller, Hanne B; Fuglsang, Cecilia Hvitfeldt; Pedersen, Cecilie Nøhr; Fenton, Robert A

2018-01-01

Apical plasma membrane accumulation of the water channel Aquaporin-2 (AQP2) in kidney collecting duct principal cells is critical for body water homeostasis. Posttranslational modification (PTM) of AQP2 is important for regulating AQP2 trafficking. The aim of this study was to determine the role of cholesterol in regulation of AQP2 PTM and in apical plasma membrane targeting of AQP2. Cholesterol depletion from the basolateral plasma membrane of a collecting duct cell line (mpkCCD14) using methyl-beta-cyclodextrin (MBCD) increased AQP2 ubiquitylation. Forskolin, cAMP or dDAVP-mediated AQP2 phosphorylation at Ser269 (pS269-AQP2) was prevented by cholesterol depletion from the basolateral membrane. None of these effects on pS269-AQP2 were observed when cholesterol was depleted from the apical side of cells, or when MBCD was applied subsequent to dDAVP stimulation. Basolateral, but not apical, MBCD application prevented cAMP-induced apical plasma membrane accumulation of AQP2. These studies indicate that manipulation of the cholesterol content of the basolateral plasma membrane interferes with AQP2 PTM and subsequently regulated apical plasma membrane targeting of AQP2. Copyright © 2017 Elsevier Inc. All rights reserved.

Biochemical characterization and membrane fluidity of membranous vesicles isolated from boar seminal plasma.

PubMed

Piehl, Lidia L; Cisale, Humberto; Torres, Natalia; Capani, Francisco; Sterin-Speziale, Norma; Hager, Alfredo

2006-05-01

Mammalian seminal plasma contains membranous vesicles (MV), which differ in composition and origin. Among these particles, human prostasomes and equine prostasome-like MV have been the most studied. The aim of the present work is to characterize the biochemical composition and membrane fluidity of MV isolated from boar seminal plasma. The MV from boar seminal plasma were isolated by ultracentrifugation and further purification by gel filtration on Sephadex G-200. The MV were examined by electron microscopy (EM), amount of cholesterol, total phospholipid, protein content, and phospholipid composition were analyzed. Membrane fluidity of MV and spermatozoa were estimated from the electron spin resonance (ESR) spectra of the 5-doxilstearic acid incorporated into the vesicle membranes by the order parameter (S). The S parameter gives a measure of degree of structural order in the membrane and is defined as the ratio of the spectral anisotropy in the membranes to the maximum anisotropy obtained in a rigidly oriented system. The S parameter takes into consideration that S = 1 for a rapid spin-label motion of about only one axis and S = 0 for a rapid isotropic motion. Intermediate S values between S = 0 and S = 1 represents the consequence of decreased membrane fluidity. The EM revealed the presence of bilaminar and multilaminar electron-dense vesicles. Cholesterol to phospholipid molar ratio from the isolated MV was 1.8. Phospholipid composition showed a predominance of sphingomyelin. The S parameter for porcine MV and for boar spermatozoa was 0.73 +/- 0.02 and 0.644 +/- 0.008, respectively, with the S for MV being greater (p < 0.001) than the S for spermatozoa. The high order for S found for boar MV was in agreement with the greater cholesterol/phospholipids ratio and the lesser ratio for phosphatidylcholine/sphingomyelin. Results obtained in the present work indicate that MV isolated from boar semen share many biochemical and morphological characteristics with equine

Differences in Organizational Structure of Insulin Receptor on Rat Adipocyte and Liver Plasma Membranes: Role of Disulfide Bonds

NASA Astrophysics Data System (ADS)

Schweitzer, John B.; Smith, Robert M.; Jarett, Leonard

1980-08-01

Binding of 125I-labeled insulin to rat liver and adipocyte plasma membranes has been investigated after treatment of the membranes with agents that modify disulfide bonds or sulfhydryl groups. Dithiothreitol, a disulfide-reducing agent, produced a bimodal response in adipocyte plasma membranes with dose-dependent increases in binding occurring over the range of 0-1 mM dithiothreitol; 5 mM dithiothreitol produced decreased binding. Insulin binding reached its maximal increase at 1 mM and was 3 times control values. Scatchard analysis of the 1 mM dithiothreitol effect revealed a straight line plot indicative of one class of sites with a Ka of 1.0× 108 M-1 which is intermediate between the two Kas obtained from the curvilinear Scatchard plot of control membranes. There was a 20-fold increase in the number of intermediate-affinity receptors compared to high-affinity receptors. The increased 125I-labeled insulin binding after dithiothreitol treatment was reversed by oxidized glutathione in a dose-dependent manner. Interposition of treatment with N-ethylmaleimide, an alkylating agent, prevented oxidized glutathione from reversing the dithiothreitol effect. Reduced glutathione produced the same effect as dithiothreitol. Liver plasma membranes treated with up to 1 mM dithiothreitol exhibited a maximum increase in insulin binding of 20% compared to control. Dithiothreitol at 5 mM decreased insulin binding below that of control membranes. The results indicate that the dithiothreitol effect on insulin binding to adipocyte plasma membranes is due to disruption of disulfide bonds, and that the structural organization of the insulin receptor on the plasma membranes is different for liver and for adipose tissue. The data imply that the insulin receptors on the plasma membrane of adipocytes possess at least two functionally distinct subclasses of disulfide bond but liver insulin receptors do not.

Protein-centric N-glycoproteomics analysis of membrane and plasma membrane proteins.

PubMed

Sun, Bingyun; Hood, Leroy

2014-06-06

The advent of proteomics technology has transformed our understanding of biological membranes. The challenges for studying membrane proteins have inspired the development of many analytical and bioanalytical tools, and the techniques of glycoproteomics have emerged as an effective means to enrich and characterize membrane and plasma-membrane proteomes. This Review summarizes the development of various glycoproteomics techniques to overcome the hurdles formed by the unique structures and behaviors of membrane proteins with a focus on N-glycoproteomics. Example contributions of N-glycoproteomics to the understanding of membrane biology are provided, and the areas that require future technical breakthroughs are discussed.

Efficient adhesion-based plasma membrane isolation for cell surface N-glycan analysis.

PubMed

Mun, Ji-Young; Lee, Kyung Jin; Seo, Hoon; Sung, Min-Sun; Cho, Yee Sook; Lee, Seung-Goo; Kwon, Ohsuk; Oh, Doo-Byoung

2013-08-06

Glycans, which decorate cell surfaces, play crucial roles in various physiological events involving cell surface recognition. Despite the importance of surface glycans, most analyses have been performed using total cells or whole membranes rather than plasma membranes due to difficulties related to isolation. In the present study, we employed an adhesion-based method for plasma membrane isolation to analyze N-glycans on cell surfaces. Cells were attached to polylysine-coated glass plates and then ruptured by hypotonic pressure. After washing to remove intracellular organelles, only a plasma membrane fraction remained attached to the plates, as confirmed by fluorescence imaging using organelle-specific probes. The plate was directly treated with trypsin to digest and detach the glycoproteins from the plasma membrane. From the resulting glycopeptides, N-glycans were released and analyzed using MALDI-TOF mass spectrometry and HPLC. When N-glycan profiles obtained by this method were compared to those by other methods, the amount of high-mannose type glycans mainly contaminated from the endoplasmic reticulum was dramatically reduced, which enabled the efficient detection of complex type glycans present on the cell surface. Moreover, this method was successfully used to analyze the increase of high-mannose glycans on the surface as induced by a mannosidase inhibitor treatment.

The plasma membrane of Saccharomyces cerevisiae: structure, function, and biogenesis.

PubMed Central

van der Rest, M E; Kamminga, A H; Nakano, A; Anraku, Y; Poolman, B; Konings, W N

1995-01-01

The composition of phospholipids, sphingolipids, and sterols in the plasma membrane has a strong influence on the activity of the proteins associated or embedded in the lipid bilayer. Since most lipid-synthesizing enzymes in Saccharomyces cerevisiae are located in intracellular organelles, an extensive flux of lipids from these organelles to the plasma membrane is required. Although the pathway of protein traffic to the plasma membrane is similar to that of most of the lipids, the bulk flow of lipids is separate from vesicle-mediated protein transport. Recent advances in the analysis of membrane budding and membrane fusion indicate that the mechanisms of protein transport from the endoplasmic reticulum to the Golgi and from the Golgi to plasma membrane are similar. The majority of plasma membrane proteins transport solutes across the membrane. A number of ATP-dependent export systems have been detected that couple the hydrolysis of ATP to transport of molecules out of the cell. The hydrolysis of ATP by the plasma membrane H(+)-ATPase generates a proton motive force which is used to drive secondary transport processes. In S. cerevisiae, many substrates are transported by more than one system. Transport of monosaccharide is catalyzed by uniport systems, while transport of disaccharides, amino acids, and nucleosides is mediated by proton symport systems. Transport activity can be regulated at the level of transcription, e.g., induction and (catabolite) repression, but transport proteins can also be affected posttranslationally by a process termed catabolite inactivation. Catabolite inactivation is triggered by the addition of fermentable sugars, intracellular acidification, stress conditions, and/or nitrogen starvation. Phosphorylation and/or ubiquitination of the transport proteins has been proposed as an initial step in the controlled inactivation and degradation of the target enzyme. The use of artificial membranes, like secretory vesicles and plasma membranes

Cholesterol asymmetry in synaptic plasma membranes.

PubMed

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

2011-03-01

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

Mechanisms underlying anomalous diffusion in the plasma membrane.

PubMed

Krapf, Diego

2015-01-01

The plasma membrane is a complex fluid where lipids and proteins undergo diffusive motion critical to biochemical reactions. Through quantitative imaging analyses such as single-particle tracking, it is observed that diffusion in the cell membrane is usually anomalous in the sense that the mean squared displacement is not linear with time. This chapter describes the different models that are employed to describe anomalous diffusion, paying special attention to the experimental evidence that supports these models in the plasma membrane. We review models based on anticorrelated displacements, such as fractional Brownian motion and obstructed diffusion, and nonstationary models such as continuous time random walks. We also emphasize evidence for the formation of distinct compartments that transiently form on the cell surface. Finally, we overview heterogeneous diffusion processes in the plasma membrane, which have recently attracted considerable interest. Copyright © 2015. Published by Elsevier Inc.

Mammalian autophagy and the plasma membrane.

PubMed

Pavel, Mariana; Rubinsztein, David C

2017-03-01

Autophagy (literally 'self-eating') is an evolutionarily conserved degradation process where cytoplasmic components are engulfed by vesicles called autophagosomes, which are then delivered to lysosomes, where their contents are degraded. Under stress conditions, such as starvation or oxidative stress, autophagy is upregulated in order to degrade macromolecules and restore the nutrient balance. The source of membranes that participate in the initial formation of phagophores is still incompletely understood and many intracellular structures have been shown to act as lipid donors, including the endoplasmic reticulum, Golgi, nucleus, mitochondria and the plasma membrane. Here, we focus on the contributions of the plasma membrane to autophagosome biogenesis governed by ATG16L1 and ATG9A trafficking, and summarize the physiological and pathological implications of this macroautophagy route, from development and stem cell fate to neurodegeneration and cancer. © 2016 Federation of European Biochemical Societies.

Membranes produced by plasma enhanced chemical vapor deposition technique for low temperature fuel cell applications

NASA Astrophysics Data System (ADS)

Ennajdaoui, Aboubakr; Roualdes, Stéphanie; Brault, Pascal; Durand, Jean

A plasma polymerization process using a continuous glow discharge has been implemented for preparing proton conducting membranes from trifluoromethane sulfonic acid and styrene. The chemical and physical structure of plasma membranes has been investigated using FTIR and SEM. The films are homogeneous with a good adhesion on commercial gas diffusion layer (E-Tek ®). Their deposition rate can be increased with increasing flow rate and input power. The thermogravimetric analysis under air of plasma polymers has showed a thermal stability up to 140 °C. Compared to the pulsed glow discharge studied in a previous paper, the continuous glow discharge has enabled to enhance the proton conductivity of membranes by a factor 3 (up to 1.7 mS cm -1). Moreover, the low methanol permeability (methanol diffusion coefficient down to 5 × 10 -13 m 2 s -1) of membranes has been confirmed by this study. In an industrial context, a reactor prototype has been developed to manufacture by plasma processes all active layers of fuel cell cores to be integrated in original compact PEMFC or DMFC.

Plants and fungi in the era of heterogeneous plasma membranes.

PubMed

Opekarová, M; Malinsky, J; Tanner, W

2010-09-01

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

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.

The dynamics of plant plasma membrane proteins: PINs and beyond.

PubMed

Luschnig, Christian; Vert, Grégory

2014-08-01

Plants are permanently situated in a fixed location and thus are well adapted to sense and respond to environmental stimuli and developmental cues. At the cellular level, several of these responses require delicate adjustments that affect the activity and steady-state levels of plasma membrane proteins. These adjustments involve both vesicular transport to the plasma membrane and protein internalization via endocytic sorting. A substantial part of our current knowledge of plant plasma membrane protein sorting is based on studies of PIN-FORMED (PIN) auxin transport proteins, which are found at distinct plasma membrane domains and have been implicated in directional efflux of the plant hormone auxin. Here, we discuss the mechanisms involved in establishing such polar protein distributions, focusing on PINs and other key plant plasma membrane proteins, and we highlight the pathways that allow for dynamic adjustments in protein distribution and turnover, which together constitute a versatile framework that underlies the remarkable capabilities of plants to adjust growth and development in their ever-changing environment. © 2014. Published by The Company of Biologists Ltd.

Tools for phospho- and glycoproteomics of plasma membranes.

PubMed

Wiśniewski, Jacek R

2011-07-01

Analysis of plasma membrane proteins and their posttranslational modifications is considered as important for identification of disease markers and targets for drug treatment. Due to their insolubility in water, studying of plasma membrane proteins using mass spectrometry has been difficult for a long time. Recent technological developments in sample preparation together with important improvements in mass spectrometric analysis have facilitated analysis of these proteins and their posttranslational modifications. Now, large scale proteomic analyses allow identification of thousands of membrane proteins from minute amounts of sample. Optimized protocols for affinity enrichment of phosphorylated and glycosylated peptides have set new dimensions in the depth of characterization of these posttranslational modifications of plasma membrane proteins. Here, I summarize recent advances in proteomic technology for the characterization of the cell surface proteins and their modifications. In the focus are approaches allowing large scale mapping rather than analytical methods suitable for studying individual proteins or non-complex mixtures.

Anion channels in the sea urchin sperm plasma membrane.

PubMed

Morales, E; de la Torre, L; Moy, G W; Vacquier, V D; Darszon, A

1993-10-01

Ionic fluxes in sea urchin sperm plasma membrane regulate cell motility and the acrosome reaction (AR). Although cationic channels mediate some of the ionic movements, little is known about anion channels in these cells. The fusion of sperm plasma membranes into lipid bilayers allowed identification of a 150 pS anion channel. This anion channel was enriched from detergent-solubilized sperm plasma membranes using a wheat germ agglutinin Sepharose column. Vesicles formed from this preparation were fused into black lipid membranes (BLM), yielding single channel anion-selective activity with the properties of those found in the sperm membranes. The following anion selectivity sequence was found: NO3- > CNS- > Br- > Cl-. This anion channel has a high open probability at the holding potentials tested, it is partially blocked by 4,4'-diisothiocyano-2,2'-stilbendisulfonic acid (DIDS), and it often displays substates. The sperm AR was also inhibited by DIDS.

RNAi-mediated downregulation of poplar plasma membrane intrinsic proteins (PIPs) changes plasma membrane proteome composition and affects leaf physiology.

PubMed

Bi, Zhen; Merl-Pham, Juliane; Uehlein, Norbert; Zimmer, Ina; Mühlhans, Stefanie; Aichler, Michaela; Walch, Axel Karl; Kaldenhoff, Ralf; Palme, Klaus; Schnitzler, Jörg-Peter; Block, Katja

2015-10-14

Plasma membrane intrinsic proteins (PIPs) are one subfamily of aquaporins that mediate the transmembrane transport of water. To reveal their function in poplar, we generated transgenic poplar plants in which the translation of PIP genes was downregulated by RNA interference investigated these plants with a comprehensive leaf plasma membrane proteome and physiome analysis. First, inhibition of PIP synthesis strongly altered the leaf plasma membrane protein composition. Strikingly, several signaling components and transporters involved in the regulation of stomatal movement were differentially regulated in transgenic poplars. Furthermore, hormonal crosstalk related to abscisic acid, auxin and brassinosteroids was altered, in addition to cell wall biosynthesis/cutinization, the organization of cellular structures and membrane trafficking. A physiological analysis confirmed the proteomic results. The leaves had wider opened stomata and higher net CO2 assimilation and transpiration rates as well as greater mesophyll conductance for CO2 (gm) and leaf hydraulic conductance (Kleaf). Based on these results, we conclude that PIP proteins not only play essential roles in whole leaf water and CO2 flux but have important roles in the regulation of stomatal movement. Copyright © 2015. Published by Elsevier B.V.

AQP2 Plasma Membrane Diffusion Is Altered by the Degree of AQP2-S256 Phosphorylation

PubMed Central

Arnspang, Eva C.; Login, Frédéric H.; Koffman, Jennifer S.; Sengupta, Prabuddha; Nejsum, Lene N.

2016-01-01

Fine tuning of urine concentration occurs in the renal collecting duct in response to circulating levels of arginine vasopressin (AVP). AVP stimulates intracellular cAMP production, which mediates exocytosis of sub-apical vesicles containing the water channel aquaporin-2 (AQP2). Protein Kinase A (PKA) phosphorylates AQP2 on serine-256 (S256), which triggers plasma membrane accumulation of AQP2. This mediates insertion of AQP2 into the apical plasma membrane, increasing water permeability of the collecting duct. AQP2 is a homo-tetramer. When S256 on all four monomers is changed to the phosphomimic aspartic acid (S256D), AQP2-S256D localizes to the plasma membrane and internalization is decreased. In contrast, when S256 is mutated to alanine (S256A) to mimic non-phosphorylated AQP2, AQP2-S256A localizes to intracellular vesicles as well as the plasma membrane, with increased internalization from the plasma membrane. S256 phosphorylation is not necessary for exocytosis and dephosphorylation is not necessary for endocytosis, however, the degree of S256 phosphorylation is hypothesized to regulate the kinetics of AQP2 endocytosis and thus, retention time in the plasma membrane. Using k-space Image Correlation Spectroscopy (kICS), we determined how the number of phosphorylated to non-phosphorylated S256 monomers in the AQP2 tetramer affects diffusion speed of AQP2 in the plasma membrane. When all four monomers mimicked constitutive phosphorylation (AQP2-S256D), diffusion was faster than when all four were non-phosphorylated (AQP2-S256A). AQP2-WT diffused at a speed similar to that of AQP2-S256D. When an average of two or three monomers in the tetramer were constitutively phosphorylated, the average diffusion coefficients were not significantly different to that of AQP2-S256D. However, when only one monomer was phosphorylated, diffusion was slower and similar to AQP2-S256A. Thus, AQP2 with two to four phosphorylated monomers has faster plasma membrane kinetics, than the

AQP2 Plasma Membrane Diffusion Is Altered by the Degree of AQP2-S256 Phosphorylation.

PubMed

Arnspang, Eva C; Login, Frédéric H; Koffman, Jennifer S; Sengupta, Prabuddha; Nejsum, Lene N

2016-10-28

Fine tuning of urine concentration occurs in the renal collecting duct in response to circulating levels of arginine vasopressin (AVP). AVP stimulates intracellular cAMP production, which mediates exocytosis of sub-apical vesicles containing the water channel aquaporin-2 (AQP2). Protein Kinase A (PKA) phosphorylates AQP2 on serine-256 (S256), which triggers plasma membrane accumulation of AQP2. This mediates insertion of AQP2 into the apical plasma membrane, increasing water permeability of the collecting duct. AQP2 is a homo-tetramer. When S256 on all four monomers is changed to the phosphomimic aspartic acid (S256D), AQP2-S256D localizes to the plasma membrane and internalization is decreased. In contrast, when S256 is mutated to alanine (S256A) to mimic non-phosphorylated AQP2, AQP2-S256A localizes to intracellular vesicles as well as the plasma membrane, with increased internalization from the plasma membrane. S256 phosphorylation is not necessary for exocytosis and dephosphorylation is not necessary for endocytosis, however, the degree of S256 phosphorylation is hypothesized to regulate the kinetics of AQP2 endocytosis and thus, retention time in the plasma membrane. Using k-space Image Correlation Spectroscopy (kICS), we determined how the number of phosphorylated to non-phosphorylated S256 monomers in the AQP2 tetramer affects diffusion speed of AQP2 in the plasma membrane. When all four monomers mimicked constitutive phosphorylation (AQP2-S256D), diffusion was faster than when all four were non-phosphorylated (AQP2-S256A). AQP2-WT diffused at a speed similar to that of AQP2-S256D. When an average of two or three monomers in the tetramer were constitutively phosphorylated, the average diffusion coefficients were not significantly different to that of AQP2-S256D. However, when only one monomer was phosphorylated, diffusion was slower and similar to AQP2-S256A. Thus, AQP2 with two to four phosphorylated monomers has faster plasma membrane kinetics, than the

Purification of plant plasma membranes by two-phase partitioning and measurement of H+ pumping.

PubMed

Lund, Anette; Fuglsang, Anja Thoe

2012-01-01

Purification of plasma membranes by two-phase partitioning is based on the separation of microsomal membranes, dependent on their surface hydrophobicity. Here we explain the purification of plasma membranes from a relatively small amount of material (7-30 g). The fluorescent probe ACMA (9-amino-6-chloro-2-metoxyacridine) accumulates inside the vesicles upon protonation. Quenching of ACMA in the solution corresponds to the H(+) transport across the plasma membrane. Before running the assay, the plasma membranes are incubated with the detergent Brij-58 in order to create inside-out vesicles.Purification of plasma membranes by two-phase partitioning is based on the separation of microsomal membranes, dependent on their surface hydrophobicity. Here we explain the purification of plasma membranes from a relatively small amount of material (7-30 g). The fluorescent probe ACMA (9-amino-6-chloro-2-metoxyacridine) accumulates inside the vesicles upon protonation. Quenching of ACMA in the solution corresponds to the H(+) transport across the plasma membrane. Before running the assay, the plasma membranes are incubated with the detergent Brij-58 in order to create inside-out vesicles.

Layered plasma polymer composite membranes

DOEpatents

Babcock, Walter C.

1994-01-01

Layered plasma polymer composite fluid separation membranes are disclosed, which comprise alternating selective and permeable layers for a total of at least 2n layers, where n is .gtoreq.2 and is the number of selective layers.

LH-RH binding to purified pituitary plasma membranes: absence of adenylate cyclase activation.

PubMed

Clayton, R N; Shakespear, R A; Marshall, J C

1978-06-01

Purified bovine pituitary plasma membranes possess two specific LH-RH binding sites. The high affinity site (2.5 X 10(9) l/mol) has low capacity (9 X 10(-15) mol/mg membrane protein) while the low affinity site 6.1 X 10(5) l/mol) has a much higher capacity (1.1 X 10(-10) mol/mg). Specific LH-RH binding to plasma membranes is increased 8.5-fold during purification from homogenate whilst adenylate cyclase activity is enriched 7--8-fold. Distribution of specific LH-RH binding to sucrose density gradient interface fractions parallels that of adenylate cyclase activity. Mg2+ and Ca2+ inhibit specific [125I]LH-RH binding at micromolar concentrations. Synthetic LH-RH, up to 250 microgram/ml, failed to stimulate adenylase cyclase activity of the purified bovine membranes. Using a crude 10,800 g rat pituitary membrane preparation, LH-RH similarly failed to activate adenylate cyclase even in the presence of guanyl nucleotides. These data confirm the presence of LH-RH receptor sites on pituitary plasma membranes and suggest that LH-RH-induced gonadotrophin release may be mediated by mechanisms other than activation of adenylate cyclase.

Long-Time Plasma Membrane Imaging Based on a Two-Step Synergistic Cell Surface Modification Strategy.

PubMed

Jia, Hao-Ran; Wang, Hong-Yin; Yu, Zhi-Wu; Chen, Zhan; Wu, Fu-Gen

2016-03-16

Long-time stable plasma membrane imaging is difficult due to the fast cellular internalization of fluorescent dyes and the quick detachment of the dyes from the membrane. In this study, we developed a two-step synergistic cell surface modification and labeling strategy to realize long-time plasma membrane imaging. Initially, a multisite plasma membrane anchoring reagent, glycol chitosan-10% PEG2000 cholesterol-10% biotin (abbreviated as "GC-Chol-Biotin"), was incubated with cells to modify the plasma membranes with biotin groups with the assistance of the membrane anchoring ability of cholesterol moieties. Fluorescein isothiocyanate (FITC)-conjugated avidin was then introduced to achieve the fluorescence-labeled plasma membranes based on the supramolecular recognition between biotin and avidin. This strategy achieved stable plasma membrane imaging for up to 8 h without substantial internalization of the dyes, and avoided the quick fluorescence loss caused by the detachment of dyes from plasma membranes. We have also demonstrated that the imaging performance of our staining strategy far surpassed that of current commercial plasma membrane imaging reagents such as DiD and CellMask. Furthermore, the photodynamic damage of plasma membranes caused by a photosensitizer, Chlorin e6 (Ce6), was tracked in real time for 5 h during continuous laser irradiation. Plasma membrane behaviors including cell shrinkage, membrane blebbing, and plasma membrane vesiculation could be dynamically recorded. Therefore, the imaging strategy developed in this work may provide a novel platform to investigate plasma membrane behaviors over a relatively long time period.

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

PubMed

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

2017-05-01

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

Insights into plant plasma membrane aquaporin trafficking.

PubMed

Hachez, Charles; Besserer, Arnaud; Chevalier, Adrien S; Chaumont, François

2013-06-01

Plasma membrane intrinsic proteins (PIPs) are plant aquaporins that facilitate the diffusion of water and small uncharged solutes through the cell membrane. Deciphering the network of interacting proteins that modulate PIP trafficking to and activity in the plasma membrane is essential to improve our knowledge about PIP regulation and function. This review highlights the most recent advances related to PIP subcellular routing and dynamic redistribution, identifies some key molecular interacting proteins, and indicates exciting directions for future research in this field. A better understanding of the mechanisms by which plants optimize water movement might help in identifying new molecular players of agronomical relevance involved in the control of cellular water uptake and drought tolerance. Copyright © 2012 Elsevier Ltd. All rights reserved.

Trypanosoma cruzi subverts the sphingomyelinase-mediated plasma membrane repair pathway for cell invasion

PubMed Central

Fernandes, Maria Cecilia; Cortez, Mauro; Flannery, Andrew R.; Tam, Christina; Mortara, Renato A.

2011-01-01

Upon host cell contact, the protozoan parasite Trypanosoma cruzi triggers cytosolic Ca2+ transients that induce exocytosis of lysosomes, a process required for cell invasion. However, the exact mechanism by which lysosomal exocytosis mediates T. cruzi internalization remains unclear. We show that host cell entry by T. cruzi mimics a process of plasma membrane injury and repair that involves Ca2+-dependent exocytosis of lysosomes, delivery of acid sphingomyelinase (ASM) to the outer leaflet of the plasma membrane, and a rapid form of endocytosis that internalizes membrane lesions. Host cells incubated with T. cruzi trypomastigotes are transiently wounded, show increased levels of endocytosis, and become more susceptible to infection when injured with pore-forming toxins. Inhibition or depletion of lysosomal ASM, which blocks plasma membrane repair, markedly reduces the susceptibility of host cells to T. cruzi invasion. Notably, extracellular addition of sphingomyelinase stimulates host cell endocytosis, enhances T. cruzi invasion, and restores normal invasion levels in ASM-depleted cells. Ceramide, the product of sphingomyelin hydrolysis, is detected in newly formed parasitophorous vacuoles containing trypomastigotes but not in the few parasite-containing vacuoles formed in ASM-depleted cells. Thus, T. cruzi subverts the ASM-dependent ceramide-enriched endosomes that function in plasma membrane repair to infect host cells. PMID:21536739

Layered plasma polymer composite membranes

DOEpatents

Babcock, W.C.

1994-10-11

Layered plasma polymer composite fluid separation membranes are disclosed, which comprise alternating selective and permeable layers for a total of at least 2n layers, where n is [>=]2 and is the number of selective layers. 2 figs.

Role of STARD4 in sterol transport between the endocytic recycling compartment and the plasma membrane

PubMed Central

Iaea, David B.; Mao, Shu; Lund, Frederik W.; Maxfield, Frederick R.

2017-01-01

Cholesterol is an essential constituent of membranes in mammalian cells. The plasma membrane and the endocytic recycling compartment (ERC) are both highly enriched in cholesterol. The abundance and distribution of cholesterol among organelles are tightly controlled by a combination of mechanisms involving vesicular and nonvesicular sterol transport processes. Using the fluorescent cholesterol analogue dehydroergosterol, we examined sterol transport between the plasma membrane and the ERC using fluorescence recovery after photobleaching and a novel sterol efflux assay. We found that sterol transport between these organelles in a U2OS cell line has a t1/2 =12–15 min. Approximately 70% of sterol transport is ATP independent and therefore is nonvesicular. Increasing cellular cholesterol levels dramatically increases bidirectional transport rate constants, but decreases in cholesterol levels have only a modest effect. A soluble sterol transport protein, STARD4, accounts for ∼25% of total sterol transport and ∼33% of nonvesicular sterol transport between the plasma membrane and ERC. This study shows that nonvesicular sterol transport mechanisms and STARD4 in particular account for a large fraction of sterol transport between the plasma membrane and the ERC. PMID:28209730

Remodeling of the postsynaptic plasma membrane during neural development.

PubMed

Tulodziecka, Karolina; Diaz-Rohrer, Barbara B; Farley, Madeline M; Chan, Robin B; Di Paolo, Gilbert; Levental, Kandice R; Waxham, M Neal; Levental, Ilya

2016-11-07

Neuronal synapses are the fundamental units of neural signal transduction and must maintain exquisite signal fidelity while also accommodating the plasticity that underlies learning and development. To achieve these goals, the molecular composition and spatial organization of synaptic terminals must be tightly regulated; however, little is known about the regulation of lipid composition and organization in synaptic membranes. Here we quantify the comprehensive lipidome of rat synaptic membranes during postnatal development and observe dramatic developmental lipidomic remodeling during the first 60 postnatal days, including progressive accumulation of cholesterol, plasmalogens, and sphingolipids. Further analysis of membranes associated with isolated postsynaptic densities (PSDs) suggests the PSD-associated postsynaptic plasma membrane (PSD-PM) as one specific location of synaptic remodeling. We analyze the biophysical consequences of developmental remodeling in reconstituted synaptic membranes and observe remarkably stable microdomains, with the stability of domains increasing with developmental age. We rationalize the developmental accumulation of microdomain-forming lipids in synapses by proposing a mechanism by which palmitoylation of the immobilized scaffold protein PSD-95 nucleates domains at the postsynaptic plasma membrane. These results reveal developmental changes in lipid composition and palmitoylation that facilitate the formation of postsynaptic membrane microdomains, which may serve key roles in the function of the neuronal synapse. © 2016 Tulodziecka et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Rapid Preparation of a Plasma Membrane Fraction: Western Blot Detection of Translocated Glucose Transporter 4 from Plasma Membrane of Muscle and Adipose Cells and Tissues.

PubMed

Yamamoto, Norio; Yamashita, Yoko; Yoshioka, Yasukiyo; Nishiumi, Shin; Ashida, Hitoshi

2016-08-01

Membrane proteins account for 70% to 80% of all pharmaceutical targets, indicating their clinical relevance and underscoring the importance of identifying differentially expressed membrane proteins that reflect distinct disease properties. The translocation of proteins from the bulk of the cytosol to the plasma membrane is a critical step in the transfer of information from membrane-embedded receptors or transporters to the cell interior. To understand how membrane proteins work, it is important to separate the membrane fraction of cells. This unit provides a protocol for rapidly obtaining plasma membrane fractions for western blot analysis. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

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

PubMed

de Curtis, Ivan; Meldolesi, Jacopo

2012-10-01

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

Antifouling enhancement of polysulfone/TiO2 nanocomposite separation membrane by plasma etching

NASA Astrophysics Data System (ADS)

Chen, Z.; Yin, C.; Wang, S.; Ito, K.; Fu, Q. M.; Deng, Q. R.; Fu, P.; Lin, Z. D.; Zhang, Y.

2017-01-01

A polysulfone/TiO2 nanocomposite membrane was prepared via casting method, followed by the plasma etching of the membrane surface. Doppler broadened energy spectra vs. positron incident energy were employed to elucidate depth profiles of the nanostructure for the as-prepared and treated membranes. The results confirmed that the near-surface of the membrane was modified by the plasma treatment. The antifouling characteristics for the membranes, evaluated using the degradation of Rhodamin B, indicated that the plasma treatment enhances the photo catalytic ability of the membrane, suggesting that more TiO2 nanoparticles are exposed at the membrane surface after the plasma treatment as supported by the positron result.

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.

Strong Static Magnetic Fields Increase the Gel Signal in Partially Hydrated DPPC/DMPC Membranes.

PubMed

Tang, Jennifer; Alsop, Richard J; Schmalzl, Karin; Epand, Richard M; Rheinstädter, Maikel C

2015-09-29

NIt was recently reported that static magnetic fields increase lipid order in the hydrophobic membrane core of dehydrated native plant plasma membranes [Poinapen, Soft Matter 9:6804-6813, 2013]. As plasma membranes are multicomponent, highly complex structures, in order to elucidate the origin of this effect, we prepared model membranes consisting of a lipid species with low and high melting temperature. By controlling the temperature, bilayers coexisting of small gel and fluid domains were prepared as a basic model for the plasma membrane core. We studied molecular order in mixed lipid membranes made of dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) using neutron diffraction in the presence of strong static magnetic fields up to 3.5 T. The contribution of the hydrophobic membrane core was highlighted through deuterium labeling the lipid acyl chains. There was no observable effect on lipid organization in fluid or gel domains at high hydration of the membranes. However, lipid order was found to be enhanced at a reduced relative humidity of 43%: a magnetic field of 3.5 T led to an increase of the gel signal in the diffraction patterns of 5%. While all biological materials have weak diamagnetic properties, the corresponding energy is too small to compete against thermal disorder or viscous effects in the case of lipid molecules. We tentatively propose that the interaction between the fatty acid chains' electric moment and the external magnetic field is driving the lipid tails in the hydrophobic membrane core into a better ordered state.

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

PubMed Central

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

2015-01-01

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

Lipid Domain Structure of the Plasma Membrane Revealed by Patching of Membrane Components

PubMed Central

Harder, Thomas; Scheiffele, Peter; Verkade, Paul; Simons, Kai

1998-01-01

Lateral assemblies of glycolipids and cholesterol, “rafts,” have been implicated to play a role in cellular processes like membrane sorting, signal transduction, and cell adhesion. We studied the structure of raft domains in the plasma membrane of non-polarized cells. Overexpressed plasma membrane markers were evenly distributed in the plasma membrane. We compared the patching behavior of pairs of raft markers (defined by insolubility in Triton X-100) with pairs of raft/non-raft markers. For this purpose we cross-linked glycosyl-phosphatidylinositol (GPI)-anchored proteins placental alkaline phosphatase (PLAP), Thy-1, influenza virus hemagglutinin (HA), and the raft lipid ganglioside GM1 using antibodies and/or cholera toxin. The patches of these raft markers overlapped extensively in BHK cells as well as in Jurkat T–lymphoma cells. Importantly, patches of GPI-anchored PLAP accumulated src-like protein tyrosine kinase fyn, which is thought to be anchored in the cytoplasmic leaflet of raft domains. In contrast patched raft components and patches of transferrin receptor as a non-raft marker were sharply separated. Taken together, our data strongly suggest that coalescence of cross-linked raft elements is mediated by their common lipid environments, whereas separation of raft and non-raft patches is caused by the immiscibility of different lipid phases. This view is supported by the finding that cholesterol depletion abrogated segregation. Our results are consistent with the view that raft domains in the plasma membrane of non-polarized cells are normally small and highly dispersed but that raft size can be modulated by oligomerization of raft components. PMID:9585412

Hunting for low abundant redox proteins in plant plasma membranes.

PubMed

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

2009-04-13

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

Gravity Responsive NADH Oxidase of the Plasma Membrane

NASA Technical Reports Server (NTRS)

Morre, D. James (Inventor)

2002-01-01

A method and apparatus for sensing gravity using an NADH oxidase of the plasma membrane which has been found to respond to unit gravity and low centrifugal g forces. The oxidation rate of NADH supplied to the NADH oxidase is measured and translated to represent the relative gravitational force exerted on the protein. The NADH oxidase of the plasma membrane may be obtained from plant or animal sources or may be produced recombinantly.

Crystal structure of the plasma membrane proton pump.

PubMed

Pedersen, Bjørn P; Buch-Pedersen, Morten J; Morth, J Preben; Palmgren, Michael G; Nissen, Poul

2007-12-13

A prerequisite for life is the ability to maintain electrochemical imbalances across biomembranes. In all eukaryotes the plasma membrane potential and secondary transport systems are energized by the activity of P-type ATPase membrane proteins: H+-ATPase (the proton pump) in plants and fungi, and Na+,K+-ATPase (the sodium-potassium pump) in animals. The name P-type derives from the fact that these proteins exploit a phosphorylated reaction cycle intermediate of ATP hydrolysis. The plasma membrane proton pumps belong to the type III P-type ATPase subfamily, whereas Na+,K+-ATPase and Ca2+-ATPase are type II. Electron microscopy has revealed the overall shape of proton pumps, however, an atomic structure has been lacking. Here we present the first structure of a P-type proton pump determined by X-ray crystallography. Ten transmembrane helices and three cytoplasmic domains define the functional unit of ATP-coupled proton transport across the plasma membrane, and the structure is locked in a functional state not previously observed in P-type ATPases. The transmembrane domain reveals a large cavity, which is likely to be filled with water, located near the middle of the membrane plane where it is lined by conserved hydrophilic and charged residues. Proton transport against a high membrane potential is readily explained by this structural arrangement.

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

Multi-walled carbon nanotubes injure the plasma membrane of macrophages

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

Hirano, Seishiro; Kanno, Sanae; Furuyama, Akiko

2008-10-15

Carbon nanotubes (CNTs) are emerging nanotechnology materials which are likely to be mass-produced in the near future. However, prior to mass-production, certain health-related concerns should first be addressed. For example, when inhaled, the thin-fibrous shape and the biopersistent characteristics of CNTs may cause pulmonary diseases, in a manner similar to asbestos. In the present study, mouse macrophages (J774.1) were exposed to highly-purified multi-walled CNTs (MWCNTs, 67 nm) or to UICC crocidolite in order to evaluate the toxicity of these nano-size fibers. The cytotoxicity of MWCNTs was found to be higher than that of crocidolite. The toxic effect of MWCNTs wasmore » not affected by N-acetylcysteine, an antioxidant, or buthionine sulfoximine, a glutathione synthesis inhibitor. cDNA microarray analyses suggested that the cytotoxicity of MWCNTs could not be explained satisfactorily by either an increase or decrease of gene expression, although mRNA levels of some cytokines were slightly increased by MWCNTs. Moreover, MWCNTs did not significantly activate either MAP kinases such as ERK, JNK and p38, nor common apoptosis pathways such as caspase 3 and PARP. Electron microscopic studies indicated that MWCNTs associate with the plasma membrane of macrophages and disrupt the integrity of the membrane. Several proteins were found to adsorb onto MWCNTs when MWCNT-exposed macrophages were gently lysed. One of these proteins was macrophage receptor with collagenous structure (MARCO). MARCO-transfected CHO-K1 cells associated with MWCNTs more rapidly than mock-transfected cells. These results indicate that MWCNTs probably trigger cytotoxic effects in phagocytotic cells by reacting with MARCO on the plasma membrane and rupturing the plasma membrane.« less

Molecular dynamics study of lipid bilayers modeling the plasma membranes of mouse hepatocytes and hepatomas.

PubMed

Andoh, Yoshimichi; Aoki, Noriyuki; Okazaki, Susumu

2016-02-28

Molecular dynamics (MD) calculations of lipid bilayers modeling the plasma membranes of normal mouse hepatocytes and hepatomas in water have been performed under physiological isothermal-isobaric conditions (310.15 K and 1 atm). The changes in the membrane properties induced by hepatic canceration were investigated and were compared with previous MD calculations included in our previous study of the changes in membrane properties induced by murine thymic canceration. The calculated model membranes for normal hepatocytes and hepatomas comprised 23 and 24 kinds of lipids, respectively. These included phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. We referred to previously published experimental values for the mole fraction of the lipids adopted in the present calculations. The calculated structural and dynamic properties of the membranes such as lateral structure, order parameters, lateral self-diffusion constants, and rotational correlation times all showed that hepatic canceration causes plasma membranes to become more ordered laterally and less fluid. Interestingly, this finding contrasts with the less ordered structure and increased fluidity of plasma membranes induced by thymic canceration observed in our previous MD study.

Molecular dynamics study of lipid bilayers modeling the plasma membranes of mouse hepatocytes and hepatomas

NASA Astrophysics Data System (ADS)

Andoh, Yoshimichi; Aoki, Noriyuki; Okazaki, Susumu

2016-02-01

Molecular dynamics (MD) calculations of lipid bilayers modeling the plasma membranes of normal mouse hepatocytes and hepatomas in water have been performed under physiological isothermal-isobaric conditions (310.15 K and 1 atm). The changes in the membrane properties induced by hepatic canceration were investigated and were compared with previous MD calculations included in our previous study of the changes in membrane properties induced by murine thymic canceration. The calculated model membranes for normal hepatocytes and hepatomas comprised 23 and 24 kinds of lipids, respectively. These included phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. We referred to previously published experimental values for the mole fraction of the lipids adopted in the present calculations. The calculated structural and dynamic properties of the membranes such as lateral structure, order parameters, lateral self-diffusion constants, and rotational correlation times all showed that hepatic canceration causes plasma membranes to become more ordered laterally and less fluid. Interestingly, this finding contrasts with the less ordered structure and increased fluidity of plasma membranes induced by thymic canceration observed in our previous MD study.

Inhibition of vincristine binding to plasma membrane vesicles from daunorubicin-resistant Ehrlich ascites cells by multidrug resistance modulators.

PubMed Central

Sehested, M.; Jensen, P. B.; Skovsgaard, T.; Bindslev, N.; Demant, E. J.; Friche, E.; Vindeløv, L.

1989-01-01

The multidrug resistance (MDR) phenotype is presumed to be mostly dependent on changes in the resistant cell plasma membrane, notably the emergence of a 170 kDa glycoprotein called P-glycoprotein, which facilitate increased drug efflux. We have previously demonstrated that ATP-enhanced binding of vincristine (VCR) to plasma membrane vesicles is much greater in MDR than in wild type cells. The present study has shown that VCR binding to MDR Ehrlich ascites tumour cell plasma membrane vesicles is inhibited 50% most efficiently by quinidine (0.5 microM) followed by verapamil (4.1 microM) and trifluoperazine (23.2 microM). This is the reverse order of the effect on whole cells where a ranking of efficiency in terms of enhancement of VCR accumulation, inhibition of VCR efflux, DNA perturbation and modulation of resistance in a clonogenic assay, was trifluoperazine greater than or equal to verapamil much greater than quinidine. The detergent Tween 80 inhibited VCR binding to plasma membrane vesicles at 0.001% v/v which agreed with the level which modulated resistance and increased VCR accumulation in whole cells. No effect was observed on daunorubicin binding to MDR plasma membrane vesicles after incubation with either Tween 80 (up to 0.1% v/v) or verapamil (up to 25 microM). We conclude that the effect of a modulating drug in reversing resistance to VCR correlates with its ability to raise intracellular VCR levels but not with its capability to inhibit VCR binding to the plasma membrane. Thus, enhancement of VCR accumulation in MDR cells is hardly solely due to competition for a drug binding site on P-glycoprotein. Furthermore, the lack of a demonstrable effect on daunorubicin binding to the plasma membrane by modulators points to transport mechanisms which do not utilise specific drug binding to the plasma membrane. PMID:2605092

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.

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

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

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

2013-10-18

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

Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin

NASA Technical Reports Server (NTRS)

Hicks, G. R.; Rayle, D. L.; Jones, A. M.; Lomax, T. L.

1989-01-01

Plasma membrane vesicles were isolated from zucchini (Cucurbita pepo) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure (ca. 95%) plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-[7-3H]IAA ([3H]N3IAA), in a manner similar to the accumulation of [3H]IAA. The association of the [3H]N3IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of [3H]N3IAA to plasma membrane vesicles prior to exposure to UV light (15 sec; 300 nm) and detected by subsequent NaDodSO4/PAGE and fluorography. When the reaction temperature was lowered to -196 degrees C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Triton X-100 (0.1%) increased the specific activity of labeling and reduced the background, which suggests that the labeled polypeptides are intrinsic membrane proteins. The labeled polypeptides are of low abundance, as expected for auxin receptors. Further, the addition of IAA and auxin analogues to the photoaffinity reaction mixture resulted in reduced labeling that was qualitatively similar to their effects on the accumulation of radiolabeled IAA in membrane vesicles. Collectively, these results suggest that the radiolabeled polypeptides are auxin receptors. The covalent nature of the label should facilitate purification and further characterization of the receptors.

Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin.

PubMed

Hicks, G R; Rayle, D L; Jones, A M; Lomax, T L

1989-07-01

Plasma membrane vesicles were isolated from zucchini (Cucurbita pepo) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure (ca. 95%) plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-[7-3H]IAA ([3H]N3IAA), in a manner similar to the accumulation of [3H]IAA. The association of the [3H]N3IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of [3H]N3IAA to plasma membrane vesicles prior to exposure to UV light (15 sec; 300 nm) and detected by subsequent NaDodSO4/PAGE and fluorography. When the reaction temperature was lowered to -196 degrees C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Triton X-100 (0.1%) increased the specific activity of labeling and reduced the background, which suggests that the labeled polypeptides are intrinsic membrane proteins. The labeled polypeptides are of low abundance, as expected for auxin receptors. Further, the addition of IAA and auxin analogues to the photoaffinity reaction mixture resulted in reduced labeling that was qualitatively similar to their effects on the accumulation of radiolabeled IAA in membrane vesicles. Collectively, these results suggest that the radiolabeled polypeptides are auxin receptors. The covalent nature of the label should facilitate purification and further characterization of the receptors.

Exclusive photorelease of signalling lipids at the plasma membrane.

PubMed

Nadler, André; Yushchenko, Dmytro A; Müller, Rainer; Stein, Frank; Feng, Suihan; Mulle, Christophe; Carta, Mario; Schultz, Carsten

2015-12-21

Photoactivation of caged biomolecules has become a powerful approach to study cellular signalling events. Here we report a method for anchoring and uncaging biomolecules exclusively at the outer leaflet of the plasma membrane by employing a photocleavable, sulfonated coumarin derivative. The novel caging group allows quantifying the reaction progress and efficiency of uncaging reactions in a live-cell microscopy setup, thereby greatly improving the control of uncaging experiments. We synthesized arachidonic acid derivatives bearing the new negatively charged or a neutral, membrane-permeant coumarin caging group to locally induce signalling either at the plasma membrane or on internal membranes in β-cells and brain slices derived from C57B1/6 mice. Uncaging at the plasma membrane triggers a strong enhancement of calcium oscillations in β-cells and a pronounced potentiation of synaptic transmission while uncaging inside cells blocks calcium oscillations in β-cells and causes a more transient effect on neuronal transmission, respectively. The precise subcellular site of arachidonic acid release is therefore crucial for signalling outcome in two independent systems.

Plasma membrane damage to Candida albicans caused by chlorine dioxide (ClO2).

PubMed

Wei, M-K; Wu, Q-P; Huang, Q; Wu, J-L; Zhang, J-M

2008-08-01

To investigate the plasma membrane damage of chlorine dioxide (ClO(2)) to Candida albicans ATCC10231 at or below the minimal fungicidal concentration (MFC). ClO(2) at MFC or below was adopted to treat the cell suspensions of C. albicans ATCC10231. Using transmission electron microscopy, no visible physiological alteration of cell shape and plasma membrane occurred. Potassium (K(+)) leakages were significant; likewise, it showed time- and dose-dependent increases. However, adenosine triphosphate (ATP) leakages were very slight. Research shows that when 99% of the cells were inactivated, the leakage was measured at 0.04% of total ATP. Compared with the mortality-specific fluorescent dye of DiBAC(4)(3), majority of the inactivated cells were poorly stained by propidium iodide, another mortality-specific fluorescent dye which can be traced by flow cytometry. At or below MFC, ClO(2) damages the plasma membranes of C. albicans mainly by permeabilization, rather than by the disruption of their integrity. K(+) leakage and the concomitant depolarization of the cell membrane are some of the critical events. These insights into membrane damages are helpful in understanding the action mode of ClO(2).

A membrane-separator interface for mass-spectrometric analysis of blood plasma

NASA Astrophysics Data System (ADS)

Elizarov, A. Yu.; Gerasimov, D. G.

2014-09-01

We demonstrate the possibility of rapid mass-spectrometric determination of the content of anesthetic agents in blood plasma with the aid of a membrane-separator interface. The interface employs a hydrophobic selective membrane that is capable of separating various anesthetic drugs (including inhalation anesthetic sevofluran, noninhalation anesthetic thiopental, hypnotic propofol, and opioid analgesic fentanyl) from the blood plasma and introducing samples into a mass spectrometer. Analysis of the blood plasma was not accompanied by the memory effect and did not lead to membrane degradation. Results of clinical investigation of the concentration of anesthetics in the blood plasma of patients are presented.

Changes in plasma membrane lipids, aquaporins and proton pump of broccoli roots, as an adaptation mechanism to salinity.

PubMed

López-Pérez, Luis; Martínez-Ballesta, María Del Carmen; Maurel, Christophe; Carvajal, Micaela

2009-03-01

Salinity stress is known to modify the plasma membrane lipid and protein composition of plant cells. In this work, we determined the effects of salt stress on the lipid composition of broccoli root plasma membrane vesicles and investigated how these changes could affect water transport via aquaporins. Brassica oleracea L. var. Italica plants treated with different levels of NaCl (0, 40 or 80mM) showed significant differences in sterol and fatty acid levels. Salinity increased linoleic (18:2) and linolenic (18:3) acids and stigmasterol, but decreased palmitoleic (16:1) and oleic (18:1) acids and sitosterol. Also, the unsaturation index increased with salinity. Salinity increased the expression of aquaporins of the PIP1 and PIP2 subfamilies and the activity of the plasma membrane H(+)-ATPase. However, there was no effect of NaCl on water permeability (P(f)) values of root plasma membrane vesicles, as determined by stopped-flow light scattering. The counteracting changes in lipid composition and aquaporin expression observed in NaCl-treated plants could allow to maintain the membrane permeability to water and a higher H(+)-ATPase activity, thereby helping to reduce partially the Na(+) concentration in the cytoplasm of the cell while maintaining water uptake via cell-to-cell pathways. We propose that the modification of lipid composition could affect membrane stability and the abundance or activity of plasma membrane proteins such as aquaporins or H(+)-ATPase. This would provide a mechanism for controlling water permeability and for acclimation to salinity stress.

Reverse-osmosis membranes by plasma polymerization

NASA Technical Reports Server (NTRS)

Hollahan, J. R.; Wydeven, T.

1972-01-01

Thin allyl amine polymer films were developed using plasma polymerization. Resulting dry composite membranes effectively reject sodium chloride during reverse osmosis. Films are 98% sodium chloride rejective, and 46% urea rejective.

Increased ubiquitination and reduced plasma membrane trafficking of placental amino acid transporter SNAT-2 in human IUGR.

PubMed

Chen, Yi-Yung; Rosario, Fredrick J; Shehab, Majida Abu; Powell, Theresa L; Gupta, Madhulika B; Jansson, Thomas

2015-12-01

Placental amino acid transport is decreased in intrauterine growth restriction (IUGR); however, the underlying mechanisms remain largely unknown. We have shown that mechanistic target of rapamycin (mTOR) signalling regulates system A amino acid transport by modulating the ubiquitination and plasma membrane trafficking of sodium-coupled neutral amino acid transporter 2 (SNAT-2) in cultured primary human trophoblast cells. We hypothesize that IUGR is associated with (1) inhibition of placental mTORC1 and mTORC2 signalling pathways, (2) increased amino acid transporter ubiquitination in placental homogenates and (3) decreased protein expression of SNAT-2 in the syncytiotrophoblast microvillous plasma membrane (MVM). To test this hypothesis, we collected placental tissue and isolated MVM from women with pregnancies complicated by IUGR (n=25) and gestational age-matched women with appropriately grown control infants (n=19, birth weights between the twenty-fifth to seventy-fifth percentiles). The activity of mTORC1 and mTORC2 was decreased whereas the protein expression of the ubiquitin ligase NEDD4-2 (neural precursor cell expressed developmentally down-regulated protein 4-2; +72%, P<0.0001) and the ubiquitination of SNAT-2 (+180%, P<0.05) were increased in homogenates of IUGR placentas. Furthermore, IUGR was associated with decreased system A amino acid transport activity (-72%, P<0.0001) and SNAT-1 (-42%, P<0.05) and SNAT-2 (-31%, P<0.05) protein expression in MVM. In summary, these findings are consistent with the possibility that decreased placental mTOR activity causes down-regulation of placental system A activity by shifting SNAT-2 trafficking towards proteasomal degradation, thereby contributing to decreased fetal amino acid availability and restricted fetal growth in IUGR. © 2015 Authors; published by Portland Press Limited.

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

PubMed

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

2014-01-01

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

ABCA1, ABCG1, and ABCG4 Are Distributed to Distinct Membrane Meso-Domains and Disturb Detergent-Resistant Domains on the Plasma Membrane

PubMed Central

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

2014-01-01

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

Piracetam induces plasma membrane depolarization in rat brain synaptosomes.

PubMed

Fedorovich, Sergei V

2013-10-11

Piracetam is a cyclic derivative of γ-aminobutyric acid (GABA). It was the first nootropic drug approved for clinical use. However, mechanism of its action is still not clear. In present paper, I investigated effects of piracetam on neurotransmitter release, plasma membrane potential monitored by fluorescent dye DiSC3(5) and chloride transport monitored by fluorescent dye SPQ in rat brain synaptosomes. It was shown that piracetam (1 mM) induces slow weak plasma membrane depolarization. This effect was decreased on 43% and 58% by both AMPA/kainate receptor blockers NBQX (10 μM) and CNQX (100 μM), respectively, on 84% by GABA ionotropic receptor blocker picrotoxin (50 μM) and on 91% upon withdrawal of HCO(3-) ions from incubation medium. GABA (1 mM) and kainate (100 μM) were found not to produce changes of plasma membrane potential. Also, it was found that piracetam induces chloride efflux which seems to be the reason of depolarization. Thereby, piracetam induces depolarization of plasma membrane of isolated neuronal presynaptic endings by picrotoxin-sensitive way. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

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

PubMed

Luo, Yu; Russinova, Eugenia

2017-01-01

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

Boar seminal plasma exosomes maintain sperm function by infiltrating into the sperm membrane.

PubMed

Du, Jian; Shen, Jian; Wang, Yuanxian; Pan, Chuanying; Pang, Weijun; Diao, Hua; Dong, Wuzi

2016-09-13

Seminal plasma ingredients are important for maintenance of sperm viability. This study focuses on the effect of boar seminal plasma exosomes on sperm function during long-term liquid storage. Boar seminal plasma exosomes had typical nano-structure morphology as measured by scanning electron microscopy (SEM) and molecular markers such as AWN, CD9 and CD63 by western blot analysis. The effect on sperm parameters of adding different ratio of boar seminal plasma exosomes to boar sperm preparations was analyzed. Compared to the diluent without exosomes, the diluent with four times or sixteen times exosomes compared to original semen had higher sperm motility, prolonged effective survival time, improved sperm plasma membrane integrity (p < 0.05), increased total antioxidant capacity (T-AOC) activity and decreased malondialdehyde (MDA) content. The diluent containing four times concentration of exosomes compared to original semen was determined to inhibit premature capacitation, but not to influence capacitation induced in vitro. Inhibition of premature capacitation is likely related to the concentration of exosomes which had been demonstrated to transfer proteins including AWN and PSP-1 into sperm. In addition, using fluorescence microscopy and scanning electron microscopy analysis, it was demonstrated that exosomes in diluent were directly binding to the membrane of sperm head which could improve sperm plasma membrane integrity.

Boar seminal plasma exosomes maintain sperm function by infiltrating into the sperm membrane

PubMed Central

Du, Jian; Shen, Jian; Wang, Yuanxian; Pan, Chuanying; Pang, Weijun; Diao, Hua; Dong, Wuzi

2016-01-01

Seminal plasma ingredients are important for maintenance of sperm viability. This study focuses on the effect of boar seminal plasma exosomes on sperm function during long-term liquid storage. Boar seminal plasma exosomes had typical nano-structure morphology as measured by scanning electron microscopy (SEM) and molecular markers such as AWN, CD9 and CD63 by western blot analysis. The effect on sperm parameters of adding different ratio of boar seminal plasma exosomes to boar sperm preparations was analyzed. Compared to the diluent without exosomes, the diluent with four times or sixteen times exosomes compared to original semen had higher sperm motility, prolonged effective survival time, improved sperm plasma membrane integrity (p < 0.05), increased total antioxidant capacity (T-AOC) activity and decreased malondialdehyde (MDA) content. The diluent containing four times concentration of exosomes compared to original semen was determined to inhibit premature capacitation, but not to influence capacitation induced in vitro. Inhibition of premature capacitation is likely related to the concentration of exosomes which had been demonstrated to transfer proteins including AWN and PSP-1 into sperm. In addition, using fluorescence microscopy and scanning electron microscopy analysis, it was demonstrated that exosomes in diluent were directly binding to the membrane of sperm head which could improve sperm plasma membrane integrity. PMID:27542209

Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus

PubMed Central

Adu-Gyamfi, Emmanuel; Johnson, Kristen A.; Fraser, Mark E.; Scott, Jordan L.; Soni, Smita P.; Jones, Keaton R.; Digman, Michelle A.; Gratton, Enrico; Tessier, Charles R.

2015-01-01

ABSTRACT Lipid-enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Ebola virus, which buds from the plasma membrane of the host cell, causes viral hemorrhagic fever and has a high fatality rate. To date, little has been known about how budding and egress of Ebola virus are mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of Ebola virus matrix protein VP40. VP40 binds PS-containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress, whereas PS is typically found only on the inner leaflet. Taking the data together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus-like particles. IMPORTANCE The lipid-enveloped Ebola virus causes severe infection with a high mortality rate and currently lacks FDA-approved therapeutics or vaccines. Ebola virus harbors just seven genes in its genome, and there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell that it infects during the replication process. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This report, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry. PMID

Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus.

PubMed

Adu-Gyamfi, Emmanuel; Johnson, Kristen A; Fraser, Mark E; Scott, Jordan L; Soni, Smita P; Jones, Keaton R; Digman, Michelle A; Gratton, Enrico; Tessier, Charles R; Stahelin, Robert V

2015-09-01

Lipid-enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Ebola virus, which buds from the plasma membrane of the host cell, causes viral hemorrhagic fever and has a high fatality rate. To date, little has been known about how budding and egress of Ebola virus are mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of Ebola virus matrix protein VP40. VP40 binds PS-containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress, whereas PS is typically found only on the inner leaflet. Taking the data together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus-like particles. The lipid-enveloped Ebola virus causes severe infection with a high mortality rate and currently lacks FDA-approved therapeutics or vaccines. Ebola virus harbors just seven genes in its genome, and there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell that it infects during the replication process. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This report, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry. Copyright © 2015, American

Plant Defense Response to Fungal Pathogens (Activation of Host-Plasma Membrane H+-ATPase by Elicitor-Induced Enzyme Dephosphorylation).

PubMed Central

Vera-Estrella, R.; Barkla, B. J.; Higgins, V. J.; Blumwald, E.

1994-01-01

Elicitor preparations containing the avr5 gene products from race 4 of Cladosporium fulvum and tomato (Lycopersicon esculentum L.) cells near isogenic for the resistance gene Cf5 were used to investigate events following the treatment of host plasma membranes with elicitor. A 4-fold increase in H+-ATPase activity, coincident with the acidification of the extracellular medium, was detected immediately after elicitor treatment. The elicitor-induced stimulation of the plasma membrane H+-ATPase was inhibited by okadaic acid but not by staurosporine, suggesting that protein dephosphorylation was required for increased H+-ATPase activity. This observation was confirmed by [gamma]-32P labeling and immunodetection of the plasma membrane H+-ATPase. Effects of guanidine nucleotide analogs and mastoparan on the ATPase activity suggested the role of GTP-binding proteins in mediating the putative elicitor-receptor binding, resulting in activation of a phosphatase(s), which in turn stimulates the plasma membrane H+-ATPase by dephosphorylation. PMID:12232073

Plasma Modified Polypropylene Membranes as the Lithium-Ion Battery Separators

NASA Astrophysics Data System (ADS)

Wang, Zhengduo; Zhu, Huiqin; Yang, Lizhen; Wang, Xinwei; Liu, Zhongwei; Chen, Qiang

2016-04-01

To reduce the thermal shrinkage of the polymeric separators and improve the safety of the Li-ion batteries, plasma treatment and plasma enhanced vapor chemical deposition (PECVD) of SiOx-like are carried out on polypropylene (PP) separators, respectively. Critical parameters for separator properties, such as the thermal shrinkage rate, porosity, wettability, and mechanical strength, are evaluated on the plasma treated PP membranes. O2 plasma treatment is found to remarkably improve the wettability, porosity and electrolyte uptake. PECVD SiOx-like coatings are found to be able to effectively reduce the thermal shrinkage rate of the membranes and increase the ionic conductivity. The electrolyte-philicity of the SiOx-like coating surface can be tuned by the varying O2 content in the gas mixture during the deposition. Though still acceptable, the mechanical strength is reduced after PECVD, which is due to the plasma etching. supported by National Natural Science Foundation of China (Nos. 11175024, 11375031), the Beijing Institute of Graphic and Communication Key Project of China (No. 23190113051), the Shenzhen Science and Technology Innovation Committee of China (No. JCYJ20130329181509637), BJNSFC (No. KZ201510015014), and the State Key Laboratory of Electrical Insulation and Power Equipment of China (No. EIPE15208)

There Is No Simple Model of the Plasma Membrane Organization.

PubMed

Bernardino de la Serna, Jorge; Schütz, Gerhard J; Eggeling, Christian; Cebecauer, Marek

2016-01-01

Ever since technologies enabled the characterization of eukaryotic plasma membranes, heterogeneities in the distributions of its constituents were observed. Over the years this led to the proposal of various models describing the plasma membrane organization such as lipid shells, picket-and-fences, lipid rafts, or protein islands, as addressed in numerous publications and reviews. Instead of emphasizing on one model we in this review give a brief overview over current models and highlight how current experimental work in one or the other way do not support the existence of a single overarching model. Instead, we highlight the vast variety of membrane properties and components, their influences and impacts. We believe that highlighting such controversial discoveries will stimulate unbiased research on plasma membrane organization and functionality, leading to a better understanding of this essential cellular structure.

Detection of glycoproteins in the Acanthamoeba plasma membrane

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

Paatero, G.I.L.; Gahmberg, C.G.

1988-11-01

In the present study the authors have shown that glycoproteins are present in the plasma membrane of Acanthamoeba castellanii by utilizing different radioactive labeling techniques. Plasma membrane proteins in the amoeba were iodinated by {sup 125}I-lactoperoxidase labeling and the solubilized radiolabeled glycoproteins were separated by lectin-Sepharose affinity chromatography followed by polyacrylamide gel electrophoresis. The periodate/NaB{sup 3}H{sub 4} and galactose oxidase/NaB{sup 3}H{sub 4} labeling techniques were used for labeling of surface carbohydrates in the amoeba. Several surface-labeled glycoproteins were observed in addition to a diffusely labeled region with M{sub r} of 55,000-75,000 seen on electrophoresis, which could represent glycolipids. The presencemore » of glycoproteins in the plasma membrane of Acanthamoeba castellanii was confirmed by metabolic labeling with ({sup 35}S)methionine followed by lectin-Sepharose affinity chromatography and polyacrylamide gel electrophoresis.« less

Effect of Plasma Membrane Semipermeability in Making the Membrane Electric Double Layer Capacitances Significant.

PubMed

Sinha, Shayandev; Sachar, Harnoor Singh; Das, Siddhartha

2018-01-30

Electric double layers (or EDLs) formed at the membrane-electrolyte interface (MEI) and membrane-cytosol interface (MCI) of a charged lipid bilayer plasma membrane develop finitely large capacitances. However, these EDL capacitances are often much larger than the intrinsic capacitance of the membrane, and all of these capacitances are in series. Consequently, the effect of these EDL capacitances in dictating the overall membrane-EDL effective capacitance C eff becomes negligible. In this paper, we challenge this conventional notion pertaining to the membrane-EDL capacitances. We demonstrate that, on the basis of the system parameters, the EDL capacitance for both the permeable and semipermeable membranes can be small enough to influence C eff . For the semipermeable membranes, however, this lowering of the EDL capacitance can be much larger, ensuring a reduction of C eff by more than 20-25%. Furthermore, for the semipermeable membranes, the reduction in C eff is witnessed over a much larger range of system parameters. We attribute such an occurrence to the highly nonintuitive electrostatic potential distribution associated with the recently discovered phenomena of charge-inversion-like electrostatics and the attainment of a positive zeta potential at the MCI for charged semipermeable membranes. We anticipate that our findings will impact the quantification and the identification of a large number of biophysical phenomena that are probed by measuring the plasma membrane capacitance.

MAMP (microbe-associated molecular pattern)-induced changes in plasma membrane-associated proteins.

PubMed

Uhlíková, Hana; Solanský, Martin; Hrdinová, Vendula; Šedo, Ondrej; Kašparovský, Tomáš; Hejátko, Jan; Lochman, Jan

2017-03-01

Plant plasma membrane associated proteins play significant roles in Microbe-Associated Molecular Pattern (MAMP) mediated defence responses including signal transduction, membrane transport or energetic metabolism. To elucidate the dynamics of proteins associated with plasma membrane in response to cryptogein, a well-known MAMP of defence reaction secreted by the oomycete Phytophthora cryptogea, 2D-Blue Native/SDS gel electrophoresis of plasma membrane fractions was employed. This approach revealed 21 up- or down-regulated protein spots of which 15 were successfully identified as proteins related to transport through plasma membrane, vesicle trafficking, and metabolic enzymes including cytosolic NADP-malic enzyme and glutamine synthetase. Observed changes in proteins were also confirmed on transcriptional level by qRT-PCR analysis. In addition, a significantly decreased accumulation of transcripts observed after employment of a mutant variant of cryptogein Leu41Phe, exhibiting a conspicuous defect in induction of resistance, sustains the contribution of identified proteins in cryptogein-triggered cellular responses. Our data provide further evidence for dynamic MAMP-induced changes in plasma membrane associated proteins. Copyright © 2016 Elsevier GmbH. All rights reserved.

Switchable hydrophobic/hydrophilic surface of electrospun poly (l-lactide) membranes obtained by CF₄microwave plasma treatment

DOE PAGES

Yue, Mengyao; Zhou, Baoming; Jiao, Kunyan; ...

2014-11-29

A switchable surface that promotes either hydrophobic or hydrophilic wettability of poly (L-lactide) (PLLA) microfibrous membranes is obtained by CF₄ microwave plasma treatment in this paper. The results indicated that both etching and grafting process occurred during the CF₄ plasma treatment and these two factors synergistically affected the final surface wettability of PLLA membranes. When plasma treatment was taken under a relatively low power, the surface wettability of PLLA membranes turned from hydrophobic to hydrophilic. Especially when CF₄ plasma treatment was taken under 100 W for 10 min and 150 W for 5 min, the water contact angle sharply decreasedmore » from 116 ± 3.0° to ~0°. According to Field-emission scanning electron microscopy (FESEM) results, the PLLA fibers were notably etched by CF₄ plasma treatment. Combined with the X-ray photoelectron spectroscopy (XPS) measurements, only a few fluorine-containing groups were grafted onto the surface, so the etching effect directly affected the surface wettability of PLLA membranes in low plasma power condition. However, with the plasma power increasing to 200 W, the PLLA membrane surface turned to hydrophobic again. In contrast, the morphology changes of PLLA fiber surfaces were not obvious while a large number of fluorine-containing groups grafted onto the surface. So the grafting effect gradually became the major factor for the final surface wettability.« less

Cell cycle dependent changes in the plasma membrane organization of mammalian cells.

PubMed

Denz, Manuela; Chiantia, Salvatore; Herrmann, Andreas; Mueller, Peter; Korte, Thomas; Schwarzer, Roland

2017-03-01

Lipid membranes are major structural elements of all eukaryotic and prokaryotic organisms. Although many aspects of their biology have been studied extensively, their dynamics and lateral heterogeneity are still not fully understood. Recently, we observed a cell-to-cell variability in the plasma membrane organization of CHO-K1 cells (Schwarzer et al., 2014). We surmised that cell cycle dependent changes of the individual cells from our unsynchronized cell population account for this phenomenon. In the present study, this hypothesis was tested. To this aim, CHO-K1 cells were arrested in different cell cycle phases by chemical treatments, and the order of their plasma membranes was determined by various fluorescent lipid analogues using fluorescence lifetime imaging microscopy. Our experiments exhibit significant differences in the membrane order of cells arrested in the G2/M or S phase compared to control cells. Our single-cell analysis also enabled the specific selection of mitotic cells, which displayed a significant increase of the membrane order compared to the control. In addition, the lipid raft marker GPImYFP was used to study the lateral organization of cell cycle arrested cells as well as mitotic cells and freely cycling samples. Again, significant differences were found between control and arrested cells and even more pronounced between control and mitotic cells. Our data demonstrate a direct correlation between cell cycle progression and plasma membrane organization, underlining that cell-to-cell heterogeneities of membrane properties have to be taken into account in cellular studies especially at the single-cell level. Copyright © 2016 Elsevier B.V. All rights reserved.

Plasma membrane repair and cellular damage control: the annexin survival kit.

PubMed

Draeger, Annette; Monastyrskaya, Katia; Babiychuk, Eduard B

2011-03-15

Plasmalemmal injury is a frequent event in the life of a cell. Physical disruption of the plasma membrane is common in cells that operate under conditions of mechanical stress. The permeability barrier can also be breached by chemical means: pathogens gain access to host cells by secreting pore-forming toxins and phospholipases, and the host's own immune system employs pore-forming proteins to eliminate both pathogens and the pathogen-invaded cells. In all cases, the influx of extracellular Ca(2+) is being sensed and interpreted as an "immediate danger" signal. Various Ca(2+)-dependent mechanisms are employed to enable plasma membrane repair. Extensively damaged regions of the plasma membrane can be patched with internal membranes delivered to the cell surface by exocytosis. Nucleated cells are capable of resealing their injured plasmalemma by endocytosis of the permeabilized site. Likewise, the shedding of membrane microparticles is thought to be involved in the physical elimination of pores. Membrane blebbing is a further damage-control mechanism, which is triggered after initial attempts at plasmalemmal resealing have failed. The members of the annexin protein family are ubiquitously expressed and function as intracellular Ca(2+) sensors. Most cells contain multiple annexins, which interact with distinct plasma membrane regions promoting membrane segregation, membrane fusion and--in combination with their individual Ca(2+)-sensitivity--allow spatially confined, graded responses to membrane injury. Copyright © 2011 Elsevier Inc. All rights reserved.

Hyperpolarization of the plasma membrane potential provokes reorganization of the actin cytoskeleton and increases the stability of adherens junctions in bovine corneal endothelial cells in culture.

PubMed

Nin, Verónica; Hernández, Julio A; Chifflet, Silvia

2009-12-01

In previous works we showed that the depolarization of the plasma membrane potential (PMP) determines a reorganization of the cytoskeleton of diverse epithelia in culture, consisting mainly of a reallocation of peripheral actin toward the cell center, ultimately provoking intercellular disruption. In view of this evidence, we explored in this study the possible effects of membrane potential hyperpolarization on the cytoskeletal organization and adherens junction (AJ) morphology and the stability of confluent bovine corneal endothelial cells in culture. For this purpose, hyperpolarization was achieved by substitution of extracellular sodium by nondiffusible cations or via the incorporation of valinomycin to the control solution. Actin compactness at the cell periphery was assessed by quantitative analysis of fluorescence microscopy images. The stability of the AJ was challenged by calcium deprivation or temperature decrease. Our results showed that plasma membrane hyperpolarization provokes a compaction of AJ-associated actin filaments toward the plasma membrane and an increase in the stability of the AJs. We also observed that the hyperpolarizing procedures determined similar modifications in the actin cytoskeleton of endothelial cells in whole bovine corneas. Together with our previous work, the results of this study contribute to the idea that modifications in the PMP of nonexcitable cells participate in cellular adaptive responses involving reorganization of cytoskeletal components. (c) 2009 Wiley-Liss, Inc.

The isolation and subfractionation of plasma membrane from the cellular slime mould Dictyostelium discoideum

PubMed Central

Green, Anita A.; Newell, Peter C.

1974-01-01

A procedure for the isolation and separation of three different subfractions of plasma membrane from the cellular slime mould Dictyostelium discoideum is described. The cells were disrupted by freeze-thawing in liquid N2 and plasma membranes were purified by equilibrium centrifugation in a sucrose gradient. The cell surface was labelled with radioactive iodide by using the lactoperoxidase iodination method. Alkaline phosphatase was identified as a plasma-membrane marker by its co-distribution with [125I]iodide. 5′-Nucleotidase, which has been widely described as a plasma-membrane marker enzyme in mammalian tissues, was not localized to any marked extent in D. discoideum plasma membrane. The isolated plasma membranes showed a 24-fold enrichment of alkaline phosphatase specific activity relative to the homogenate and a yield of 50% of the total plasma membranes. Determination of succinate dehydrogenase and NADPH–cytochrome c reductase activities indicated that the preparation contained 2% of the total mitochondria and 3% of the endoplasmic reticulum. When the plasma-membrane preparation was further disrupted in a tight-fitting homogenizer, three plasma-membrane subfractions of different densities were obtained by isopycnic centrifugation. The enrichment of alkaline phosphatase was greatest in the subfraction with the lowest density. This fraction was enriched 36-fold relative to the homogenate and contained 19% of the total alkaline phosphatase activity but only 0.08% of the succinate dehydrogenase activity and 0.34% of the NADPH–cytochrome c reductase activity. Electron microscopy of this fraction showed it to consist of smooth membrane vesicles with no recognizable contaminants. ImagesPLATE 1 PMID:4156170

Phospholipase D1 regulates lymphocyte adhesion via upregulation of Rap1 at the plasma membrane.

PubMed

Mor, Adam; Wynne, Joseph P; Ahearn, Ian M; Dustin, Michael L; Du, Guangwei; Philips, Mark R

2009-06-01

Rap1 is a small GTPase that modulates adhesion of T cells by regulating inside-out signaling through LFA-1. The bulk of Rap1 is expressed in a GDP-bound state on intracellular vesicles. Exocytosis of these vesicles delivers Rap1 to the plasma membrane, where it becomes activated. We report here that phospholipase D1 (PLD1) is expressed on the same vesicular compartment in T cells as Rap1 and is translocated to the plasma membrane along with Rap1. Moreover, PLD activity is required for both translocation and activation of Rap1. Increased T-cell adhesion in response to stimulation of the antigen receptor depended on PLD1. C3G, a Rap1 guanine nucleotide exchange factor located in the cytosol of resting cells, translocated to the plasma membranes of stimulated T cells. Our data support a model whereby PLD1 regulates Rap1 activity by controlling exocytosis of a stored, vesicular pool of Rap1 that can be activated by C3G upon delivery to the plasma membrane.

There Is No Simple Model of the Plasma Membrane Organization

PubMed Central

Bernardino de la Serna, Jorge; Schütz, Gerhard J.; Eggeling, Christian; Cebecauer, Marek

2016-01-01

Ever since technologies enabled the characterization of eukaryotic plasma membranes, heterogeneities in the distributions of its constituents were observed. Over the years this led to the proposal of various models describing the plasma membrane organization such as lipid shells, picket-and-fences, lipid rafts, or protein islands, as addressed in numerous publications and reviews. Instead of emphasizing on one model we in this review give a brief overview over current models and highlight how current experimental work in one or the other way do not support the existence of a single overarching model. Instead, we highlight the vast variety of membrane properties and components, their influences and impacts. We believe that highlighting such controversial discoveries will stimulate unbiased research on plasma membrane organization and functionality, leading to a better understanding of this essential cellular structure. PMID:27747212

Shotgun proteomics of plant plasma membrane and microdomain proteins using nano-LC-MS/MS.

PubMed

Takahashi, Daisuke; Li, Bin; Nakayama, Takato; Kawamura, Yukio; Uemura, Matsuo

2014-01-01

Shotgun proteomics allows the comprehensive analysis of proteins extracted from plant cells, subcellular organelles, and membranes. Previously, two-dimensional gel electrophoresis-based proteomics was used for mass spectrometric analysis of plasma membrane proteins. In order to get comprehensive proteome profiles of the plasma membrane including highly hydrophobic proteins with a number of transmembrane domains, a mass spectrometry-based shotgun proteomics method using nano-LC-MS/MS for proteins from the plasma membrane proteins and plasma membrane microdomain fraction is described. The results obtained are easily applicable to label-free protein semiquantification.

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

PubMed

Sych, Taras; Mély, Yves; Römer, Winfried

2018-05-26

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

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

Tissue Factor Coagulant Activity is Regulated by the Plasma Membrane Microenvironment.

PubMed

Yu, Yuanjie; Böing, Anita N; Hau, Chi M; Hajji, Najat; Ruf, Wolfram; Sturk, Auguste; Nieuwland, Rienk

2018-06-01

 Tissue factor (TF) can be present in a non-coagulant and coagulant form. Whether the coagulant activity is affected by the plasma membrane microenvironment is unexplored.  This article studies the presence and coagulant activity of human TF in plasma membrane micro-domains.  Plasma membranes were isolated from human MIA PaCa2 cells, MDA-MB-231 cells and human vascular smooth muscle cells by Percoll gradient ultracentrifugation after cell disruption. Plasma membranes were fractionated by OptiPrep gradient ultracentrifugation, and the presence of TF, flotillin, caveolin, clathrin, protein disulphide isomerase (PDI), TF pathway inhibitor (TFPI) and phosphatidylserine (PS) were determined.  Plasma membranes contain two detergent-resistant membrane (DRM) compartments differing in density and biochemical composition. High-density DRMs (DRM-H) have a density ( ρ ) of 1.15 to 1.20 g/mL and contain clathrin, whereas low-density DRMs (DRM-L) have a density between 1.09 and 1.13 g/mL and do not contain clathrin. Both DRMs contain TF, flotillin and caveolin. PDI is detectable in DRM-H, TFPI is not detectable in either DMR-H or DRM-L and PS is detectable in DRM-L. The DRM-H-associated TF (> 95% of the TF antigen) lacks detectable coagulant activity, whereas the DRM-L-associated TF triggers coagulation. This coagulant activity is inhibited by lactadherin and thus PS-dependent, but seemed insensitive to 16F16, an inhibitor of PDI.  Non-coagulant and coagulant TF are present within different types of DRMs in the plasma membrane, and the composition of these DRMs may affect the TF coagulant activity. Schattauer GmbH Stuttgart.

Oxygen activation at the plasma membrane: relation between superoxide and hydroxyl radical production by isolated membranes.

PubMed

Heyno, Eiri; Mary, Véronique; Schopfer, Peter; Krieger-Liszkay, Anja

2011-07-01

Production of reactive oxygen species (hydroxyl radicals, superoxide radicals and hydrogen peroxide) was studied using EPR spin-trapping techniques and specific dyes in isolated plasma membranes from the growing and the non-growing zones of hypocotyls and roots of etiolated soybean seedlings as well as coleoptiles and roots of etiolated maize seedlings. NAD(P)H mediated the production of superoxide in all plasma membrane samples. Hydroxyl radicals were only produced by the membranes of the hypocotyl growing zone when a Fenton catalyst (FeEDTA) was present. By contrast, in membranes from other parts of the seedlings a low rate of spontaneous hydroxyl radical formation was observed due to the presence of small amounts of tightly bound peroxidase. It is concluded that apoplastic hydroxyl radical generation depends fully, or for the most part, on peroxidase localized in the cell wall. In soybean plasma membranes from the growing zone of the hypocotyl pharmacological tests showed that the superoxide production could potentially be attributed to the action of at least two enzymes, an NADPH oxidase and, in the presence of menadione, a quinone reductase.

Photostable bipolar fluorescent probe for video tracking plasma membranes related cellular processes.

PubMed

Zhang, Xinfu; Wang, Chao; Jin, Liji; Han, Zhuo; Xiao, Yi

2014-08-13

Plasma membranes can sense the stimulations and transmit the signals from extracellular environment and then make further responses through changes in locations, shapes or morphologies. Common fluorescent membrane markers are not well suited for long time tracking due to their shorter retention time inside plasma membranes and/or their lower photostability. To this end, we develop a new bipolar marker, Mem-SQAC, which can stably insert into plasma membranes of different cells and exhibits a long retention time over 30 min. Mem-SQAC also inherits excellent photostability from the BODIPY dye family. Large two-photon absorption cross sections and long wavelength fluorescence emissions further enhance the competitiveness of Mem-SQAC as a membrane marker. By using Mem-SQAC, significant morphological changes of plasma membranes have been monitored during heavy metal poisoning and drug induced apoptosis of MCF-7 cells; the change tendencies are so distinctly different from each other that they can be used as indicators to distinguish different cell injuries. Further on, the complete processes of endocytosis toward Staphylococcus aureus and Escherichia coli by RAW 264.7 cells have been dynamically tracked. It is discovered that plasma membranes take quite different actions in response to the two bacteria, information unavailable in previous research reports.

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

PubMed

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

2008-12-01

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

ESCRT-dependent degradation of ubiquitylated plasma membrane proteins in plants.

PubMed

Isono, Erika; Kalinowska, Kamila

2017-12-01

To control the abundance of plasma membrane receptors and transporters is crucial for proper perception and response to extracellular signals from surrounding cells and the environment. Posttranslational modification of plasma membrane proteins, especially ubiquitin conjugation or ubiquitylation, is key for the determination of stability for many transmembrane proteins localized on the cell surface. The targeted degradation is ensured by a complex network of proteins among which the endosomal sorting complex required for transport (ESCRT) plays a central role. This review focuses on progresses made in recent years on the understanding of the function of the ESCRT machinery in the degradation of ubiquitylated plasma membrane proteins in plants. Copyright © 2017 Elsevier Ltd. All rights reserved.

Plasma membrane proteome analysis identifies a role of barley membrane steroid binding protein in root architecture response to salinity.

PubMed

Witzel, Katja; Matros, Andrea; Møller, Anders L B; Ramireddy, Eswarayya; Finnie, Christine; Peukert, Manuela; Rutten, Twan; Herzog, Andreas; Kunze, Gotthard; Melzer, Michael; Kaspar-Schoenefeld, Stephanie; Schmülling, Thomas; Svensson, Birte; Mock, Hans-Peter

2018-06-01

Although the physiological consequences of plant growth under saline conditions have been well described, understanding the core mechanisms conferring plant salt adaptation has only started. We target the root plasma membrane proteomes of two barley varieties, cvs. Steptoe and Morex, with contrasting salinity tolerance. In total, 588 plasma membrane proteins were identified by mass spectrometry, of which 182 were either cultivar or salinity stress responsive. Three candidate proteins with increased abundance in the tolerant cv. Morex were involved either in sterol binding (a GTPase-activating protein for the adenosine diphosphate ribosylation factor [ZIGA2], and a membrane steroid binding protein [MSBP]) or in phospholipid synthesis (phosphoethanolamine methyltransferase [PEAMT]). Overexpression of barley MSBP conferred salinity tolerance to yeast cells, whereas the knock-out of the heterologous AtMSBP1 increased salt sensitivity in Arabidopsis. Atmsbp1 plants showed a reduced number of lateral roots under salinity, and root-tip-specific expression of barley MSBP in Atmsbp1 complemented this phenotype. In barley, an increased abundance of MSBP correlates with reduced root length and lateral root formation as well as increased levels of auxin under salinity being stronger in the tolerant cv. Morex. Hence, we concluded the involvement of MSBP in phytohormone-directed adaptation of root architecture in response to salinity. © 2018 John Wiley & Sons Ltd.

Intrinsic stability of Brassicaceae plasma membrane in relation to changes in proteins and lipids as a response to salinity.

PubMed

Chalbi, Najla; Martínez-Ballesta, Ma Carmen; Youssef, Nabil Ben; Carvajal, Micaela

2015-03-01

Changes in plasma membrane lipids, such as sterols and fatty acids, have been observed as a result of salt stress. These alterations, together with modification of the plasma membrane protein profile, confer changes in the physical properties of the membrane to be taken into account for biotechnological uses. In our experiments, the relationship between lipids and proteins in three different Brassicaceae species differing in salinity tolerance (Brassica oleracea, B. napus and Cakile maritima) and the final plasma membrane stability were studied. The observed changes in the sterol (mainly an increase in sitosterol) and fatty acid composition (increase in RUFA) in each species led to physical adaptation of the plasma membrane to salt stress. The in vitro vesicles stability was higher in the less tolerant (B. oleracea) plants together with low lipoxygenase activity. These results indicate that the proteins/lipids ratio and lipid composition is an important aspect to take into account for the use of natural vesicles in plant biotechnology. Copyright © 2014 Elsevier GmbH. All rights reserved.

NaCl regulation of plasma membrane H(+)-ATPase gene expression in a glycophyte and a halophyte.

PubMed

Niu, X; Narasimhan, M L; Salzman, R A; Bressan, R A; Hasegawa, P M

1993-11-01

NaCl regulation of plasma membrane H(+)-ATPase gene expression in the glycophyte tobacco (Nicotiana tabacum L. var Wisconsin 38) and the halophyte Atriplex nummularia L. was evaluated by comparison of organ-specific mRNA abundance using homologous cDNA probes encoding the ATPases of the respective plants. Accumulation of mRNA was induced by NaCl in fully expanded leaves and in roots but not in expanding leaves or stems. The NaCl responsiveness of the halophyte to accumulate plasma membrane H(+)-ATPase mRNA in roots was substantially greater than that of the glycophyte. Salt-induced transcript accumulation in A. nummularia roots was localized by in situ hybridization predominantly to the elongation zone, but mRNA levels also increased in the zone of differentiation. Increased message accumulation in A. nummularia roots could be detected within 8 h after NaCl (400 mM) treatment, and maximal levels were severalfold greater than in roots of untreated control plants. NaCl-induced plasma membrane H(+)-ATPase gene expression in expanded leaves and roots presumably indicates that these organs require increased H(+)-electrochemical potential gradients for the maintenance of plant ion homeostasis for salt adaptation. The greater capacity of the halophyte to induce plasma membrane H(+)-ATPase gene expression in response to NaCl may be a salt-tolerance determinant.

NaCl regulation of plasma membrane H(+)-ATPase gene expression in a glycophyte and a halophyte.

PubMed Central

Niu, X; Narasimhan, M L; Salzman, R A; Bressan, R A; Hasegawa, P M

1993-01-01

NaCl regulation of plasma membrane H(+)-ATPase gene expression in the glycophyte tobacco (Nicotiana tabacum L. var Wisconsin 38) and the halophyte Atriplex nummularia L. was evaluated by comparison of organ-specific mRNA abundance using homologous cDNA probes encoding the ATPases of the respective plants. Accumulation of mRNA was induced by NaCl in fully expanded leaves and in roots but not in expanding leaves or stems. The NaCl responsiveness of the halophyte to accumulate plasma membrane H(+)-ATPase mRNA in roots was substantially greater than that of the glycophyte. Salt-induced transcript accumulation in A. nummularia roots was localized by in situ hybridization predominantly to the elongation zone, but mRNA levels also increased in the zone of differentiation. Increased message accumulation in A. nummularia roots could be detected within 8 h after NaCl (400 mM) treatment, and maximal levels were severalfold greater than in roots of untreated control plants. NaCl-induced plasma membrane H(+)-ATPase gene expression in expanded leaves and roots presumably indicates that these organs require increased H(+)-electrochemical potential gradients for the maintenance of plant ion homeostasis for salt adaptation. The greater capacity of the halophyte to induce plasma membrane H(+)-ATPase gene expression in response to NaCl may be a salt-tolerance determinant. PMID:8022933

Nanoclustering as a dominant feature of plasma membrane organization.

PubMed

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

2014-12-01

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

Influence of Glucose Deprivation on Membrane Potentials of Plasma Membranes, Mitochondria and Synaptic Vesicles in Rat Brain Synaptosomes.

PubMed

Hrynevich, Sviatlana V; Pekun, Tatyana G; Waseem, Tatyana V; Fedorovich, Sergei V

2015-06-01

Hypoglycemia can cause neuronal cell death similar to that of glutamate-induced cell death. In the present paper, we investigated the effect of glucose removal from incubation medium on changes of mitochondrial and plasma membrane potentials in rat brain synaptosomes using the fluorescent dyes DiSC3(5) and JC-1. We also monitored pH gradients in synaptic vesicles and their recycling by the fluorescent dye acridine orange. Glucose deprivation was found to cause an inhibition of K(+)-induced Ca(2+)-dependent exocytosis and a shift of mitochondrial and plasma membrane potentials to more positive values. The sensitivity of these parameters to the energy deficit caused by the removal of glucose showed the following order: mitochondrial membrane potential > plasma membrane potential > pH gradient in synaptic vesicles. The latter was almost unaffected by deprivation compared with the control. The pH-dependent dye acridine orange was used to investigate synaptic vesicle recycling. However, the compound's fluorescence was shown to be enhanced also by the mixture of mitochondrial toxins rotenone (10 µM) and oligomycin (5 µg/mL). This means that acridine orange can presumably be partially distributed in the intermembrane space of mitochondria. Glucose removal from the incubation medium resulted in a 3.7-fold raise of acridine orange response to rotenone + oligomycin suggesting a dramatic increase in the mitochondrial pH gradient. Our results suggest that the biophysical characteristics of neuronal presynaptic endings do not favor excessive non-controlled neurotransmitter release in case of hypoglycemia. The inhibition of exocytosis and the increase of the mitochondrial pH gradient, while preserving the vesicular pH gradient, are proposed as compensatory mechanisms.

The Plasma Membrane Calcium Pump

NASA Technical Reports Server (NTRS)

Rasmussen, H.

1983-01-01

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

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

PubMed

Qiao, Zhenzhen; Libault, Marc

2017-10-03

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

Perforin Rapidly Induces Plasma Membrane Phospholipid Flip-Flop

PubMed Central

Metkar, Sunil S.; Wang, Baikun; Catalan, Elena; Anderluh, Gregor; Gilbert, Robert J. C.; Pardo, Julian; Froelich, Christopher J.

2011-01-01

The cytotoxic cell granule secretory pathway is essential for host defense. This pathway is fundamentally a form of intracellular protein delivery where granule proteases (granzymes) from cytotoxic lymphocytes are thought to diffuse through barrel stave pores generated in the plasma membrane of the target cell by the pore forming protein perforin (PFN) and mediate apoptotic as well as additional biological effects. While recent electron microscopy and structural analyses indicate that recombinant PFN oligomerizes to form pores containing 20 monomers (20 nm) when applied to liposomal membranes, these pores are not observed by propidium iodide uptake in target cells. Instead, concentrations of human PFN that encourage granzyme-mediated apoptosis are associated with pore structures that unexpectedly favor phosphatidylserine flip-flop measured by Annexin-V and Lactadherin. Efforts that reduce PFN mediated Ca influx in targets did not reduce Annexin-V reactivity. Antigen specific mouse CD8 cells initiate a similar rapid flip-flop in target cells. A lipid that augments plasma membrane curvature as well as cholesterol depletion in target cells enhance flip-flop. Annexin-V staining highly correlated with apoptosis after Granzyme B (GzmB) treatment. We propose the structures that PFN oligomers form in the membrane bilayer may include arcs previously observed by electron microscopy and that these unusual structures represent an incomplete mixture of plasma membrane lipid and PFN oligomers that may act as a flexible gateway for GzmB to translocate across the bilayer to the cytosolic leaflet of target cells. PMID:21931672

Plasma modified PLA electrospun membranes for actinorhodin production intensification in Streptomyces coelicolor immobilized-cell cultivations.

PubMed

Scaffaro, Roberto; Lopresti, Francesco; Sutera, Alberto; Botta, Luigi; Fontana, Rosa Maria; Gallo, Giuseppe

2017-09-01

Most of industrially relevant bioproducts are produced by submerged cultivations of actinomycetes. The immobilization of these Gram-positive filamentous bacteria on suitable porous supports may prevent mycelial cell-cell aggregation and pellet formation which usually negatively affect actinomycete submerged cultivations, thus, resulting in an improved biosynthetic capability. In this work, electrospun polylactic acid (PLA) membranes, subjected or not to O 2 -plasma treatment (PLA-plasma), were used as support for immobilized-cell submerged cultivations of Streptomyces coelicolor M145. This strain produces different bioactive compounds, including the blue-pigmented actinorhodin (ACT) and red-pigmented undecylprodigiosin (RED), and constitutes a model for the study of antibiotic-producing actinomycetes. Wet contact angles and X-ray photoelectron spectroscopy analysis confirmed the increased wettability of PLA-plasma due to the formation of polar functional groups such as carboxyl and hydroxyl moieties. Scanning electron microscope observations, carried out at different incubation times, revealed that S. coelicolor immobilized-cells created a dense "biofilm-like" mycelial network on both kinds of PLA membranes. Cultures of S. coelicolor immobilized-cells on PLA or PLA-plasma membranes produced higher biomass (between 1.5 and 2 fold) as well as higher levels of RED and ACT than planktonic cultures. In particular, cultures of immobilized-cells on PLA and PLA-plasma produced comparable levels of RED that were approximatively 4 and 5 fold higher than those produced by planktonic cultures, respectively. In contrast, levels of ACT produced by immobilized-cell cultures on PLA and PLA-plasma were different, being 5 and 10 fold higher than those of planktonic cultures, respectively. Therefore, this is study demonstrated the positive influence of PLA membrane on growth and secondary metabolite production in S. coelicolor and also revealed that O 2 -plasma treated PLA membranes

Plasma membrane calcium ATPases and related disorders.

PubMed

Giacomello, Marta; De Mario, Agnese; Scarlatti, Chiara; Primerano, Simona; Carafoli, Ernesto

2013-03-01

The plasma membrane Ca(2+) ATPases (PMCA pumps) cooperate with other transport systems in the plasma membrane and in the organelles in the regulation of cell Ca(2+). They have high Ca(2+) affinity and are thus the fine tuners of cytosolic Ca(2+). They belong to the superfamily of P-type ATPases: their four basic isoforms share the essential properties of the reaction cycle and the general membrane topography motif of 10 transmembrane domains and three large cytosolic units. However they also differ in other important properties, e.g., tissue distribution and regulatory mechanisms. Their chief regulator is calmodulin, that removes their C-terminal cytosolic tail from autoinhibitory binding sites next to the active site of the pump, restoring activity. The number of pump isoforms is increased to over 30 by alternative splicing of the transcripts at a N-terminal site (site A) and at site C within the C-terminal calmodulin binding domain: the splice variants are tissue specific and developmentally regulated. The importance of PMCAs in the maintenance of cellular Ca(2+) homeostasis is underlined by the disease phenotypes, genetic or acquired, caused by their malfunction. Non-genetic PMCA deficiencies have long been considered possible causative factors in disease conditions as important as cancer, hypertension, or neurodegeneration. Those of genetic origin are better characterized: some have now been discovered in humans as well. They concern all four PMCA isoforms, and range from cardiac dysfunctions, to deafness, to hypertension, to cerebellar ataxia. Copyright © 2012 Elsevier Ltd. All rights reserved.

Thymocyte plasma membrane of the rainbow trout, Salmo gairdneri: Associated immunoglobulin and heteroantigens

USGS Publications Warehouse

Warr, G.W.; DeLuca, D.; Anderson, D.P.

1983-01-01

1. Thymic lymphocytes of the rainbow trout, S. gairdneri were disrupted and a plasma membrane containing fraction isolated by differential and buoyant density centrifugation.2. Radioiodine introduced into the membrane by the lactoperoxidase catalyzed reaction and immunoglobulin (identified by radioimmunoassay with monoclonal antibody) both copurified in the plasma membrane fraction.3. Rabbit antibody raised to the plasma membrane fraction showed a strong reaction with trout lymphocytes in immunofluorescence, was mitogenic for trout lymphocytes, and recognized lymphocyte membrane heteroantigens of molecular weight > 70,000 in the thymus and 45,000–95,000 in the head kidney.

Increased ubiquitination and reduced plasma membrane trafficking of placental amino acid transporter SNAT-2 in human IUGR

PubMed Central

Rosario, Fredrick J.; Shehab, Majida Abu; Powell, Theresa L.; Gupta, Madhulika B.; Jansson, Thomas

2015-01-01

Placental amino acid transport is decreased in intrauterine growth restriction (IUGR); however, the underlying mechanisms remain largely unknown. We have shown that mechanistic target of rapamycin (mTOR) signalling regulates system A amino acid transport by modulating the ubiquitination and plasma membrane trafficking of sodium-coupled neutral amino acid transporter 2 (SNAT-2) in cultured primary human trophoblast cells. We hypothesize that IUGR is associated with (1) inhibition of placental mTORC1 and mTORC2 signalling pathways, (2) increased amino acid transporter ubiquitination in placental homogenates and (3) decreased protein expression of SNAT-2 in the syncytiotrophoblast microvillous plasma membrane (MVM). To test this hypothesis, we collected placental tissue and isolated MVM from women with pregnancies complicated by IUGR (n=25) and gestational age-matched women with appropriately grown control infants (n=19, birth weights between the twenty-fifth to seventy-fifth percentiles). The activity of mTORC1 and mTORC2 was decreased whereas the protein expression of the ubiquitin ligase NEDD4-2 (neural precursor cell expressed developmentally down-regulated protein 4-2; +72%, P<0.0001) and the ubiquitination of SNAT-2 (+180%, P<0.05) were increased in homogenates of IUGR placentas. Furthermore, IUGR was associated with decreased system A amino acid transport activity (–72%, P<0.0001) and SNAT-1 (–42%, P<0.05) and SNAT-2 (–31%, P<0.05) protein expression in MVM. In summary, these findings are consistent with the possibility that decreased placental mTOR activity causes down-regulation of placental system A activity by shifting SNAT-2 trafficking towards proteasomal degradation, thereby contributing to decreased fetal amino acid availability and restricted fetal growth in IUGR. PMID:26374858

The dynamics of plasma membrane PtdIns(4,5)P(2) at fertilization of mouse eggs.

PubMed

Halet, Guillaume; Tunwell, Richard; Balla, Tamas; Swann, Karl; Carroll, John

2002-05-15

A series of intracellular Ca2+ oscillations are responsible for triggering egg activation and cortical granule exocytosis at fertilization in mammals. These Ca2+ oscillations are generated by an increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)], which results from the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)]. Using confocal imaging to simultaneously monitor Ca2+ and plasma membrane PtdIns(4,5)P(2) in single living mouse eggs we have sought to establish the relationship between the kinetics of PtdIns(4,5)P(2) metabolism and the Ca2+ oscillations at fertilization. We report that there is no detectable net loss of plasma membrane PtdIns(4,5)P(2) either during the latent period or during the subsequent Ca2+ oscillations. When phosphatidylinositol 4-kinase is inhibited with micromolar wortmannin a limited decrease in plasma membrane PtdIns(4,5)P(2) is detected in half the eggs studied. Although we were unable to detect a widespread loss of PtdIns(4,5)P(2), we found that fertilization triggers a net increase in plasma membrane PtdIns(4,5)P(2) that is localized to the vegetal cortex. The fertilization-induced increase in PtdIns(4,5)P(2) follows the increase in Ca2+, is blocked by Ca2+ buffers and can be mimicked, albeit with slower kinetics, by photoreleasing Ins(1,4,5)P(3). Inhibition of Ca2+-dependent exocytosis of cortical granules, without interfering with Ca2+ transients, inhibits the PtdIns(4,5)P(2) increase. The increase appears to be due to de novo synthesis since it is inhibited by micromolar wortmannin. Finally, there is no increase in PtdIns(4,5)P(2) in immature oocytes that are not competent to extrude cortical granules. These studies suggest that fertilization does not deplete plasma membrane PtdIns(4,5)P(2) and that one of the pathways for increasing PtdIns(4,5)P(2) at fertilization is invoked by exocytosis of cortical granules.

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.

Cholesterol:phospholipid ratio is elevated in platelet plasma membrane in patients with hypertension.

PubMed

Benjamin, N; Robinson, B F; Graham, J G; Wilson, R B

1990-06-01

The cholesterol:phospholipid ratio was measured in platelet plasma membrane, red blood cell (RBC) membranes, low density lipoprotein (LDL) and whole plasma in patients with primary hypertension and in matched normal controls. The cholesterol:phospholipid ratio was raised in the platelet membrane from hypertensive patients compared with that from normal controls (0.65 +/- 0.03 vs 0.53 +/- 0.02: mean +/- SEM; P less than 0.01). The ratio observed in RBC membranes, LDL and whole blood was similar in the two groups. If this abnormality in the lipid composition of platelet plasma membrane is present in other cells it could account for some of the changes in cell membrane function that have been described in hypertension.

Transport proteins of the plant plasma membrane

NASA Technical Reports Server (NTRS)

Assmann, S. M.; Haubrick, L. L.; Evans, M. L. (Principal Investigator)

1996-01-01

Recently developed molecular and genetic approaches have enabled the identification and functional characterization of novel genes encoding ion channels, ion carriers, and water channels of the plant plasma membrane.

Cellulose microfibril deposition: coordinated activity at the plant plasma membrane.

PubMed

Lindeboom, J; Mulder, B M; Vos, J W; Ketelaar, T; Emons, A M C

2008-08-01

Plant cell wall production is a membrane-bound process. Cell walls are composed of cellulose microfibrils, embedded inside a matrix of other polysaccharides and glycoproteins. The cell wall matrix is extruded into the existing cell wall by exocytosis. This same process also inserts the cellulose synthase complexes into the plasma membrane. These complexes, the nanomachines that produce the cellulose microfibrils, move inside the plasma membrane leaving the cellulose microfibrils in their wake. Cellulose microfibril angle is an important determinant of cell development and of tissue properties and as such relevant for the industrial use of plant material. Here, we provide an integrated view of the events taking place in the not more than 100 nm deep area in and around the plasma membrane, correlating recent results provided by the distinct field of plant cell biology. We discuss the coordinated activities of exocytosis, endocytosis, and movement of cellulose synthase complexes while producing cellulose microfibrils and the link of these processes to the cortical microtubules.

Heterogeneity of Arabinogalactan-Proteins on the Plasma Membrane of Rose Cells.

PubMed Central

Serpe, M. D.; Nothnagel, E. A.

1996-01-01

Arabinogalactan-proteins (AGPs) have been purified from the plasma membrane of suspension-cultured Paul's Scarlet rose (Rosa sp.) cells. The two most abundant and homogeneous plasma membrane AGP fractions were named plasma membrane AGP1 (PM-AGP1) and plasma membrane AGP2 (PM-AGP2) and had apparent molecular masses of 140 and 217 kD, respectively. Both PM-AGP1 and PM-AGP2 had [beta]-(1-3)-, [beta]-(1,6)-, and [beta]-(1,3,6)-galactopyranosyl residues, predominantly terminal [alpha]-arabinofuranosyl residues, and (1,4)- and terminal glucuronopyranosyl residues. The protein moieties of PM-AGP1 and PM-AGP2 were both rich in hydroxyproline, alanine, and serine, but differed in the abundance of hydroxyproline, which was 1.6 times higher in PM-AGP2 than in PM-AGP1. Another difference was the overall protein content, which was 3.7% (w/w) in PM-AGP1 and 15% in PM-AGP2. As judged by their behavior on reverse-phase chromatography, PM-AGP1 and PM-AGP2 were not more hydrophobic than AGPs from the cell wall or culture medium. In contrast, a minor plasma membrane AGP fraction eluted later on reverse-phase chromatography and was more negatively charged at pH 5 than either PM-AGP1 or PM-AGP2. The more negatively charged fraction contained molecules with a glycosyl composition characteristic of AGPs and included at least two different macromolecules. The results of this investigation indicate that Rosa plasma membrane contains at least four distinct AGPs or AGP-like molecules. These molecules differed from each other in size, charge, hydrophobicity, amino-acyl composition, and/or protein content. PMID:12226444

Surface monofunctionalized polymethyl pentene hollow fiber membranes by plasma treatment and hemocompatibility modification for membrane oxygenators

NASA Astrophysics Data System (ADS)

Huang, Xin; Wang, Weiping; Zheng, Zhi; Fan, Wenling; Mao, Chun; Shi, Jialiang; Li, Lei

2016-01-01

The hemocompatibility of polymethyl pentene (PMP) hollow fiber membranes (HFMs) was improved through surface modification for membrane oxygenator applications. The modification was performed stepwise with the following: (1) oxygen plasma treatment, (2) functionalization of monosort hydroxyl groups through NaBH4 reduction, and (3) grafting 2-methacryloyloxyethyl phosphorylcholine (MPC) or heparin. SEM, ATR-FTIR, and XPS analyses were conducted to confirm successful grafting during the modification. The hemocompatibility of PMP HFMs was analyzed and compared through protein adsorption, platelet adhesion, and coagulation tests. Pure CO2 and O2 permeation rates, as well as in vitro gas exchange rates, were determined to evaluate the mass transfer properties of PMP HFMs. SEM results showed that different nanofibril topographies were introduced on the HFM surface. ATR-FTIR and XPS spectra indicated the presence of functionalization of monosort hydroxyl group and the grafting of MPC and heparin. Hemocompatibility evaluation results showed that the modified PMP HFMs presented optimal hemocompatibility compared with pristine HFMs. Gas permeation results revealed that gas permeation flux increased in the modified HFMs because of dense surface etching during the plasma treatment. The results of in vitro gas exchange rates showed that all modified PMP HFMs presented decreased gas exchange rates because of potential surface fluid wetting. The proposed strategy exhibits a potential for fabricating membrane oxygenators for biomedical applications to prevent coagulation formation and alter plasma-induced surface topology and composition.

Role of STARD4 in sterol transport between the endocytic recycling compartment and the plasma membrane.

PubMed

Iaea, David B; Mao, Shu; Lund, Frederik W; Maxfield, Frederick R

2017-04-15

Cholesterol is an essential constituent of membranes in mammalian cells. The plasma membrane and the endocytic recycling compartment (ERC) are both highly enriched in cholesterol. The abundance and distribution of cholesterol among organelles are tightly controlled by a combination of mechanisms involving vesicular and nonvesicular sterol transport processes. Using the fluorescent cholesterol analogue dehydroergosterol, we examined sterol transport between the plasma membrane and the ERC using fluorescence recovery after photobleaching and a novel sterol efflux assay. We found that sterol transport between these organelles in a U2OS cell line has a t 1/2 =12-15 min. Approximately 70% of sterol transport is ATP independent and therefore is nonvesicular. Increasing cellular cholesterol levels dramatically increases bidirectional transport rate constants, but decreases in cholesterol levels have only a modest effect. A soluble sterol transport protein, STARD4, accounts for ∼25% of total sterol transport and ∼33% of nonvesicular sterol transport between the plasma membrane and ERC. This study shows that nonvesicular sterol transport mechanisms and STARD4 in particular account for a large fraction of sterol transport between the plasma membrane and the ERC. © 2017 Iaea et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

The structure of the yeast plasma membrane SNARE complex reveals destabilizing water-filled cavities.

PubMed

Strop, Pavel; Kaiser, Stephen E; Vrljic, Marija; Brunger, Axel T

2008-01-11

SNARE proteins form a complex that leads to membrane fusion between vesicles, organelles, and plasma membrane in all eukaryotic cells. We report the 1.7A resolution structure of the SNARE complex that mediates exocytosis at the plasma membrane in the yeast Saccharomyces cerevisiae. Similar to its neuronal and endosomal homologues, the S. cerevisiae SNARE complex forms a parallel four-helix bundle in the center of which is an ionic layer. The S. cerevisiae SNARE complex exhibits increased helix bending near the ionic layer, contains water-filled cavities in the complex core, and exhibits reduced thermal stability relative to mammalian SNARE complexes. Mutagenesis experiments suggest that the water-filled cavities contribute to the lower stability of the S. cerevisiae complex.

Binding and Fusion of Extracellular Vesicles to the Plasma Membrane of Their Cell Targets.

PubMed

Prada, Ilaria; Meldolesi, Jacopo

2016-08-09

Exosomes and ectosomes, extracellular vesicles of two types generated by all cells at multivesicular bodies and the plasma membrane, respectively, play critical roles in physiology and pathology. A key mechanism of their function, analogous for both types of vesicles, is the fusion of their membrane to the plasma membrane of specific target cells, followed by discharge to the cytoplasm of their luminal cargo containing proteins, RNAs, and DNA. Here we summarize the present knowledge about the interactions, binding and fusions of vesicles with the cell plasma membrane. The sequence initiates with dynamic interactions, during which vesicles roll over the plasma membrane, followed by the binding of specific membrane proteins to their cell receptors. Membrane binding is then converted rapidly into fusion by mechanisms analogous to those of retroviruses. Specifically, proteins of the extracellular vesicle membranes are structurally rearranged, and their hydrophobic sequences insert into the target cell plasma membrane which undergoes lipid reorganization, protein restructuring and membrane dimpling. Single fusions are not the only process of vesicle/cell interactions. Upon intracellular reassembly of their luminal cargoes, vesicles can be regenerated, released and fused horizontally to other target cells. Fusions of extracellular vesicles are relevant also for specific therapy processes, now intensely investigated.

Effects of seminal plasma concentration on sperm motility and plasma and acrosome membrane integrity in chilled canine spermatozoa.

PubMed

Pan, C; Wu, Y; Yang, Q; Ye, J

2018-03-01

Depending on the mammal species, the use of seminal plasma during semen processing for cryopreservation has been found to have both beneficial and detrimental effects. This study was designed to determine the effects of seminal plasma concentration on the motility, sperm movement characteristics, and plasma and acrosome membrane integrity of chilled canine spermatozoa. After pooling the semen from seven dogs, samples for each assay were preserved at 4oC for 96h in extenders containing different seminal plasma concentrations (0, 25, 50, 75 and 100% (v/v) seminal plasma). After 96h cold storage, group 25% (v/v) seminal plasma showed significantly higher percentages of sperm cells with motility [46.4 ± 1.65% (p<0.05)], intact plasma membrane [46.5 ± 3.11% (p<0.05)] and intact acrosome[58.5 ± 1.86 % (p<0.05)] than other groups. In conclusion, supplementing semen extender with an appropriate seminal plasma concentration (25% (v/v) seminal plasma) is able to adequately preserve the sperm motility, integrity of the plasma and acrosome membrane in canine spermatozoa chilled at 4oC. Copyright© by the Polish Academy of Sciences.

NEU3 Sialidase Protein Interactors in the Plasma Membrane and in the Endosomes*

PubMed Central

Cirillo, Federica; Ghiroldi, Andrea; Fania, Chiara; Piccoli, Marco; Torretta, Enrica; Tettamanti, Guido; Gelfi, Cecilia; Anastasia, Luigi

2016-01-01

NEU3 sialidase has been shown to be a key player in many physio- and pathological processes, including cell differentiation, cellular response to hypoxic stress, and carcinogenesis. The enzyme, peculiarly localized on the outer leaflet of the plasma membrane, has been shown to be able to remove sialic acid residues from the gangliosides present on adjacent cells, thus creating cell to cell interactions. Nonetheless, herein we report that the enzyme localization is dynamically regulated between the plasma membrane and the endosomes, where a substantial amount of NEU3 is stored with low enzymatic activity. However, under opportune stimuli, NEU3 is shifted from the endosomes to the plasma membrane, where it greatly increases the sialidase activity. Finally, we found that NEU3 possesses also the ability to interact with specific proteins, many of which are different in each cell compartment. They were identified by mass spectrometry, and some selected ones were also confirmed by cross-immunoprecipitation with the enzyme, supporting NEU3 involvement in the cell stress response, protein folding, and intracellular trafficking. PMID:26987901

High-protein-PUFA supplementation, red blood cell membranes, and plasma antioxidant activity in volleyball athletes.

PubMed

Malaguti, Marco; Baldini, Marta; Angeloni, Cristina; Biagi, Pierluigi; Hrelia, Silvana

2008-06-01

The authors evaluated the role of a high-protein, low-calorie, polyunsaturated fatty-acid (PUFA) -supplemented diet on anthropometric parameters, erythrocyte-membrane fatty-acid composition, and plasma antioxidant defenses of nonprofessional volleyball athletes. The athletes were divided in two groups: One (n = 5) followed the Mediterranean diet, and the other (n = 6) followed a high-protein, low-calorie diet with a 3-g/day fish-oil supplementation. All the athletes had anthropometric measurements taken, both at the beginning and at the end of the study, which lasted for 2 months. Body-mass index and total body fat were significantly diminished in the second group, while they remained unchanged in the first. Plasma total antioxidant activity (TAA) was significantly increased in the plasma of both groups, with no differences between the groups, suggesting that physical activity, not the different diets, is the main contributor to the increase of plasma TAA. The second group showed a significant increase in erythrocyte-membrane PUFA content and in the unsaturation index value (UI) because of the fish-oil supplementation.A high-protein, low-carbohydrate, fish-oil-supplemented diet seems to be useful only when the aim of the diet is to obtain weight loss in a short-term period. The significant increase in the UI of erythrocyte membranes indicates the potential for harm, because a high intake of PUFA might increase susceptibility to lipid peroxidation not counterbalanced by a higher increase in TAA. Adherence to the Mediterranean diet seems to be the better choice.

Cold-induced ultrastructural changes in bull and boar sperm plasma membranes.

PubMed

De Leeuw, F E; Chen, H C; Colenbrander, B; Verkleij, A J

1990-04-01

The effect of low temperatures on the ultrastructure of the plasma membrane of bull and boar spermatozoa was investigated. Cold-induced changes in the organization of sperm plasma membrane components were demonstrated by the use of fast-freezing combined with freeze-fracture electron microscopy. This preparation technique ensures fixation without artifacts. At 38 degrees C bull and boar spermatozoa exhibited a random distribution of intramembranous particles over the plasma membrane of both head and tail. Exposure to 0 degree C resulted in redistribution of the intramembranous particles: on the head and principal piece of bull spermatozoa and on the principal piece of boar spermatozoa, particle-free areas were observed, whereas on the boar sperm head, particle aggregates were present. The original particle distribution was restored upon rewarming of bull and boar spermatozoa to 38 degrees C, as well as after freezing and thawing of bull spermatozoa. Dilution of bull and boar semen into Tris-dilution buffer and Beltsville Thaw Solution-dilution buffer, respectively, could not prevent cold-induced redistribution of intramembranous particles. The observed particle reorganization upon cooling was interpreted as the result of lateral phase separation in the plasma membrane. Species-dependent differences in cold-induced ultrastructural changes were considered to be determined by lipid composition and asymmetry of the plasma membrane, and might be related to differences in cold resistance between species.

Biomimetic carriers mimicking leukocyte plasma membrane to increase tumor vasculature permeability

NASA Astrophysics Data System (ADS)

Palomba, R.; Parodi, A.; Evangelopoulos, M.; Acciardo, S.; Corbo, C.; De Rosa, E.; Yazdi, I. K.; Scaria, S.; Molinaro, R.; Furman, N. E. Toledano; You, J.; Ferrari, M.; Salvatore, F.; Tasciotti, E.

2016-10-01

Recent advances in the field of nanomedicine have demonstrated that biomimicry can further improve targeting properties of current nanotechnologies while simultaneously enable carriers with a biological identity to better interact with the biological environment. Immune cells for example employ membrane proteins to target inflamed vasculature, locally increase vascular permeability, and extravasate across inflamed endothelium. Inspired by the physiology of immune cells, we recently developed a procedure to transfer leukocyte membranes onto nanoporous silicon particles (NPS), yielding Leukolike Vectors (LLV). LLV are composed of a surface coating containing multiple receptors that are critical in the cross-talk with the endothelium, mediating cellular accumulation in the tumor microenvironment while decreasing vascular barrier function. We previously demonstrated that lymphocyte function-associated antigen (LFA-1) transferred onto LLV was able to trigger the clustering of intercellular adhesion molecule 1 (ICAM-1) on endothelial cells. Herein, we provide a more comprehensive analysis of the working mechanism of LLV in vitro in activating this pathway and in vivo in enhancing vascular permeability. Our results suggest the biological activity of the leukocyte membrane can be retained upon transplant onto NPS and is critical in providing the particles with complex biological functions towards tumor vasculature.

Plasma membrane changes during the liquid storage of boar spermatozoa: a comparison of methods.

PubMed

Gaczarzewicz, Dariusz; Piasecka, Małgorzata; Udała, Jan; Błaszczyk, Barbara; Stankiewicz, Tomasz; Laszczyńska, Maria

2010-03-01

Studies were performed on boar semen routinely used at the local artificial insemination (AI) centre. The semen was stored in a Safe Cell Plus commercial extender at 17 degrees C for nine days. The aim of our research was focused on changes in sperm plasma membrane integrity. The integrity of the sperm plasma membrane and acrosome as well as sperm motility decreased after dilution and during storage of the semen. The highest percentage of live sperm was identified by the eosin-nigrosin method, a lower percentage by the SYBR-14/PI test, and the lowest percentage of live cells was discovered by the hypoosmotic swelling (HOS) test (P < 0.01). There were significant differences between the results of staining methods and sperm motility (P < 0.01). No significant differences were found between the HOS test results and sperm motility. The plasma membrane integrity parameters positively correlated (P < 0.001) with each other and with sperm motility but negatively with aspartate aminotransferase activity. Our findings confirmed that the boar sperm aging changes, which increased during liquid semen preservation, were connected with the loss of function and integrity of the sperm plasma membrane. The employed complementary tests are comprehensive indicators of sperm membrane integrity during long-term semen preservation, and they can help establish the actual number of 'healthy' cells. The assays may be used in AI laboratories and should be incorporated into the routine of semen analysis.

Agonist-activated Ca2+ influx occurs at stable plasma membrane and endoplasmic reticulum junctions

PubMed Central

Treves, Susan; Vukcevic, Mirko; Griesser, Johanna; Armstrong, Clara-Franzini; Zhu, Michael X.; Zorzato, Fancesco

2010-01-01

Junctate is a 33 kDa integral protein of sarco(endo)plasmic reticulum membranes that forms a macromolecular complex with inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptors and TRPC3 channels. TIRF microscopy shows that junctate enhances the number of fluorescent puncta on the plasma membrane. The size and distribution of these puncta are not affected by the addition of agonists that mobilize Ca2+ from Ins(1,4,5)P3-sensitive stores. Puncta are associated with a significantly larger number of peripheral junctions between endoplasmic reticulum and plasma membrane, which are further enhanced upon stable co-expression of junctate and TRPC3. The gap between the membranes of peripheral junctions is bridged by regularly spaced electron-dense structures of 10 nm. Ins(1,4,5)P3 inhibits the interaction of the cytoplasmic N-terminus of junctate with the ligand-binding domain of the Ins(1,4,5)P3 receptor. Furthermore, Ca2+ influx evoked by activation of Ins(1,4,5)P3 receptors is increased where puncta are located. We conclude that stable peripheral junctions between the plasma membrane and endoplasmic reticulum are the anatomical sites of agonist-activated Ca2+ entry. PMID:21062895

Insulin and adenosine regulate the phosphatidylcholine concentration in isolated rat adipocyte plasma membranes.

PubMed

Kiechle, F L; Sykes, E; Artiss, J D

1995-01-01

Blockade of adenosine receptors by 3-isobutyl-1-methylxanthine or degradation of endogenous adenosine with adenosine deaminase increased the phosphatidylcholine concentration in isolated rat adipocyte plasma membranes, an effect which was suppressed by the phosphatidylethanolamine methyltransferase inhibitor, S-adenosyl-L-homocysteine, and reversed by the adenosine analogue, N6-(L-phenylisopropyl)-adenosine. For example, the addition of N6-(L-phenylisopropyl)-adenosine to adenosine deaminase pretreated plasma membranes rapidly lowered the concentration of phosphatidylcholine by 171 nmol/mg at 30 seconds compared to control. Insulin-induced stimulation of phospholipid methylation in membranes treated with 3-isobutyl-1-methylxanthine or adenosine deaminase was achieved only after the addition of N6-(L-phenylisopropyl)-adenosine. These results suggest that adenosine receptor occupancy inhibits phospholipid methylation, is required for insulin stimulation of phospholipid methylation, and may perhaps activate a phosphatidylcholine-specific phospholipase C or phospholipase D.

Cobalt oxide nanoparticles can enter inside the cells by crossing plasma membranes

PubMed Central

Bossi, Elena; Zanella, Daniele; Gornati, Rosalba; Bernardini, Giovanni

2016-01-01

The ability of nanoparticles (NPs) to be promptly uptaken by the cells makes them both dangerous and useful to human health. It was recently postulated that some NPs might cross the plasma membrane also by a non-endocytotic pathway gaining access to the cytoplasm. To this aim, after having filled mature Xenopus oocytes with Calcein, whose fluorescence is strongly quenched by divalent metal ions, we have exposed them to different cobalt NPs quantifying quenching as evidence of the increase of the concentration of Co2+ released by the NPs that entered into the cytoplasm. We demonstrated that cobalt oxide NPs, but not cobalt nor cobalt oxide NPs that were surrounded by a protein corona, can indeed cross plasma membranes. PMID:26924527

Plasma Membrane is Compartmentalized by a Self-Similar Cortical Actin Meshwork

NASA Astrophysics Data System (ADS)

Sadegh, Sanaz; Higgins, Jenny L.; Mannion, Patrick C.; Tamkun, Michael M.; Krapf, Diego

2017-01-01

A broad range of membrane proteins display anomalous diffusion on the cell surface. Different methods provide evidence for obstructed subdiffusion and diffusion on a fractal space, but the underlying structure inducing anomalous diffusion has never been visualized because of experimental challenges. We addressed this problem by imaging the cortical actin at high resolution while simultaneously tracking individual membrane proteins in live mammalian cells. Our data confirm that actin introduces barriers leading to compartmentalization of the plasma membrane and that membrane proteins are transiently confined within actin fences. Furthermore, superresolution imaging shows that the cortical actin is organized into a self-similar meshwork. These results present a hierarchical nanoscale picture of the plasma membrane.

Magnesium Oxide (MgO) pH-sensitive Sensing Membrane in Electrolyte-Insulator-Semiconductor Structures with CF4 Plasma Treatment.

PubMed

Kao, Chyuan-Haur; Chang, Chia Lung; Su, Wei Ming; Chen, Yu Tzu; Lu, Chien Cheng; Lee, Yu Shan; Hong, Chen Hao; Lin, Chan-Yu; Chen, Hsiang

2017-08-03

Magnesium oxide (MgO) sensing membranes in pH-sensitive electrolyte-insulator-semiconductor structures were fabricated on silicon substrate. To optimize the sensing capability of the membrane, CF 4 plasma was incorporated to improve the material quality of MgO films. Multiple material analyses including FESEM, XRD, AFM, and SIMS indicate that plasma treatment might enhance the crystallization and increase the grain size. Therefore, the sensing behaviors in terms of sensitivity, linearity, hysteresis effects, and drift rates might be improved. MgO-based EIS membranes with CF 4 plasma treatment show promise for future industrial biosensing applications.

Adaptation of H+-pumping and plasma membrane H+ ATPase activity in proteoid roots of white lupin under phosphate deficiency.

PubMed

Yan, Feng; Zhu, Yiyong; Müller, Caroline; Zörb, Christian; Schubert, Sven

2002-05-01

White lupin (Lupinus albus) is able to adapt to phosphorus deficiency by producing proteoid roots that release a huge amount of organic acids, resulting in mobilization of sparingly soluble soil phosphate in rhizosphere. The mechanisms responsible for the release of organic acids by proteoid root cells, especially the trans-membrane transport processes, have not been elucidated. Because of high cytosolic pH, the release of undissociated organic acids is not probable. In the present study, we focused on H+ export by plasma membrane H+ ATPase in active proteoid roots. In vivo, rhizosphere acidification of active proteoid roots was vanadate sensitive. Plasma membranes were isolated from proteoid roots and lateral roots from P-deficient and -sufficient plants. In vitro, in comparison with two types of lateral roots and proteoid roots of P-sufficient plants, the following increase of the various parameters was induced in active proteoid roots of P-deficient plants: (a) hydrolytic ATPase activity, (b) Vmax and Km, (c) H+ ATPase enzyme concentration of plasma membrane, (d) H+-pumping activity, (e) pH gradient across the membrane of plasmalemma vesicles, and (f) passive H+ permeability of plasma membrane. In addition, lower vanadate sensitivity and more acidic pH optimum were determined for plasma membrane ATPase of active proteoid roots. Our data support the hypothesis that in active proteoid root cells, H+ and organic anions are exported separately, and that modification of plasma membrane H+ ATPase is essential for enhanced rhizosphere acidification by active proteoid roots.

Estradiol's interesting life at the cell's plasma membrane.

PubMed

Caldwell, J D; Gebhart, V M; Jirikowski, G F

2016-07-01

Clearly, we have presented here evidence of a very complex set of mechanisms and proteins involved with various and intricate actions of steroids at the plasma membrane. Steroids do MUCH more at the plasma membrane than simply passing passively through it. They may sit in the membrane; they are bound by numerous proteins in the membrane, including ERs, SHBG, steroid-binding globulin receptors, and perhaps elements of cellular architecture such as tubulin. It also seems likely that the membrane itself responds graphically to the presence of steroids by actually changing its shape as well, perhaps, as accumulating steroids. Clara Szego suggested in the 1980s that actions of E2 at one level would act synergistically with its actions at another level (e.g. membrane actions would complement nuclear actions). Given the sheer number of proteins involved in steroid actions, just at the membrane level, it seems unlikely that every action of a steroid on every potential protein effector will act to the same end. It seems more likely that these multiple effects and sites of effect of steroids contribute to the confusion that exists as to what actions steroids always have. For example, there is confusion with regard to synthetic agents (SERMs etc.) that have different and often opposite actions depending on which organ they act upon. A better understanding of the basic actions of steroids should aid in understanding the variability of their clinical effects. Copyright © 2016 Elsevier Inc. All rights reserved.

Plasma deposition of silver nanoparticles on ultrafiltration membranes: antibacterial and anti-biofouling properties.

PubMed

Cruz, Mercedes Cecilia; Ruano, Gustavo; Wolf, Marcus; Hecker, Dominic; Vidaurre, Elza Castro; Schmittgens, Ralph; Rajal, Verónica Beatriz

2015-02-01

A novel and versatile plasma reactor was used to modify Polyethersulphone commercial membranes. The equipment was applied to: i) functionalize the membranes with low-temperature plasmas, ii) deposit a film of poly(methyl methacrylate) (PMMA) by Plasma Enhanced Chemical Vapor Deposition (PECVD) and, iii) deposit silver nanoparticles (SNP) by Gas Flow Sputtering. Each modification process was performed in the same reactor consecutively, without exposure of the membranes to atmospheric air. Scanning electron microscopy and transmission electron microscopy were used to characterize the particles and modified membranes. SNP are evenly distributed on the membrane surface. Particle fixation and transport inside membranes were assessed before- and after-washing assays by X-ray photoelectron spectroscopy depth profiling analysis. PMMA addition improved SNP fixation. Plasma-treated membranes showed higher hydrophilicity. Anti-biofouling activity was successfully achieved against Gram-positive ( Enterococcus faecalis ) and -negative ( Salmonella Typhimurium) bacteria. Therefore, disinfection by ultrafiltration showed substantial resistance to biofouling. The post-synthesis functionalization process developed provides a more efficient fabrication route for anti-biofouling and anti-bacterial membranes used in the water treatment field. To the best of our knowledge, this is the first report of a gas phase condensation process combined with a PECVD procedure in order to deposit SNP on commercial membranes to inhibit biofouling formation.

Plasma deposition of silver nanoparticles on ultrafiltration membranes: antibacterial and anti-biofouling properties

PubMed Central

Cruz, Mercedes Cecilia; Ruano, Gustavo; Wolf, Marcus; Hecker, Dominic; Vidaurre, Elza Castro; Schmittgens, Ralph; Rajal, Verónica Beatriz

2015-01-01

A novel and versatile plasma reactor was used to modify Polyethersulphone commercial membranes. The equipment was applied to: i) functionalize the membranes with low-temperature plasmas, ii) deposit a film of poly(methyl methacrylate) (PMMA) by Plasma Enhanced Chemical Vapor Deposition (PECVD) and, iii) deposit silver nanoparticles (SNP) by Gas Flow Sputtering. Each modification process was performed in the same reactor consecutively, without exposure of the membranes to atmospheric air. Scanning electron microscopy and transmission electron microscopy were used to characterize the particles and modified membranes. SNP are evenly distributed on the membrane surface. Particle fixation and transport inside membranes were assessed before- and after-washing assays by X-ray photoelectron spectroscopy depth profiling analysis. PMMA addition improved SNP fixation. Plasma-treated membranes showed higher hydrophilicity. Anti-biofouling activity was successfully achieved against Gram-positive (Enterococcus faecalis) and -negative (Salmonella Typhimurium) bacteria. Therefore, disinfection by ultrafiltration showed substantial resistance to biofouling. The post-synthesis functionalization process developed provides a more efficient fabrication route for anti-biofouling and anti-bacterial membranes used in the water treatment field. To the best of our knowledge, this is the first report of a gas phase condensation process combined with a PECVD procedure in order to deposit SNP on commercial membranes to inhibit biofouling formation. PMID:26166926

Nitrogen dioxide-induced alterations in ganglioside content and structure of pulmonary artery endothelial cell plasma membranes

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

Sekharam, M.; Patel, J.M.; Block, E.R.

1990-02-26

Nitrogen dioxide (NO{sub 2}), an environmental oxidant, is known to cause injury to the surface of pulmonary artery endothelial cells (PAEC). Because gangliosides are present in the outer leaflet of plasma membranes, the authors hypothesize that NO{sub 2} exposure may alter the ganglioside content and structure of PAEC plasma membranes. To test this, confluent porcine PAEC were exposed to 5 ppm NO{sub 2} containing 5% CO{sub 2} for 48 hours at 37 C in a CO{sub 2} incubator. Controls were exposed to air containing 5% Co{sub 2} under identical conditions. After exposure: (1) total lipids were extracted and ganglioside basesmore » were separated and estimated by fluorescamine, (2) the sialic acid content of intact cells was measured by the resorcinol method, and (3) freeze-fracture analysis of the intact cell plasma membrane was done by propane jet freezing and shadowing with platinum and carbon to form a replica. The ganglioside and sialic acid/{mu}g protein, respectively. In No{sub 2}-exposed cells, ganglioside content was reduced by 45% and sialic acid content was increased by 30%. Freeze-fracture analysis of the plasma membrane of control cells showed the presence of 160{+-}12 particles/cm area at 45000x. In contrast, the number of particles on the No{sub 2}-exposed plasma membrane was reduced to 68{+-}5 particles/cm at 45000x (p < 0.05). These results demonstrate that NO{sub 2} causes structural changes in the surface of PAEC plasma membranes, and these are temporally associated with a reduction in the number of gagliosides in these cells.« less

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

DOE PAGES

Frisz, Jessica F.; Klitzing, Haley A.; Lou, Kaiyan; ...

2013-04-22

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

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

PubMed Central

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

2012-01-01

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

Exploring the biophysical properties of phytosterols in the plasma membrane for novel cancer prevention strategies.

PubMed

Fakih, Omar; Sanver, Didem; Kane, David; Thorne, James L

2018-05-03

Cancer is a global problem with no sign that incidences are reducing. The great costs associated with curing cancer, through developing novel treatments and applying patented therapies, is an increasing burden to developed and developing nations alike. These financial and societal problems will be alleviated by research efforts into prevention, or treatments that utilise off-patent or repurposed agents. Phytosterols are natural components of the diet found in an array of seeds, nuts and vegetables and have been added to several consumer food products for the management of cardio-vascular disease through their ability to lower LDL-cholesterol levels. In this review, we provide a connected view between the fields of structural biophysics and cellular and molecular biology to evaluate the growing evidence that phytosterols impair oncogenic pathways in a range of cancer types. The current state of understanding of how phytosterols alter the biophysical properties of plasma membrane is described, and the potential for phytosterols to be repurposed from cardio-vascular to oncology therapeutics. Through an overview of the types of biophysical and molecular biology experiments that have been performed to date, this review informs the reader of the molecular and biophysical mechanisms through which phytosterols could have anti-cancer properties via their interactions with the plasma cell membrane. We also outline emerging and under-explored areas such as computational modelling, improved biomimetic membranes and ex vivo tissue evaluation. Focus of future research in these areas should improve understanding, not just of phytosterols in cancer cell biology but also to give insights into the interaction between the plasma membrane and the genome. These fields are increasingly providing meaningful biological and clinical data but iterative experiments between molecular biology assays, biosynthetic membrane studies and computational membrane modelling improve and refine our

Profiling of kidney vascular endothelial cell plasma membrane proteins by liquid chromatography-tandem mass spectrometry.

PubMed

Liu, Zan; Xu, Bo; Nameta, Masaaki; Zhang, Ying; Magdeldin, Sameh; Yoshida, Yutaka; Yamamoto, Keiko; Fujinaka, Hidehiko; Yaoita, Eishin; Tasaki, Masayuki; Nakagawa, Yuki; Saito, Kazuhide; Takahashi, Kota; Yamamoto, Tadashi

2013-06-01

Vascular endothelial cells (VECs) play crucial roles in physiological and pathologic conditions in tissues and organs. Most of these roles are related to VEC plasma membrane proteins. In the kidney, VECs are closely associated with structures and functions; however, plasma membrane proteins in kidney VECs remain to be fully elucidated. Rat kidneys were perfused with cationic colloidal silica nanoparticles (CCSN) to label the VEC plasma membrane. The CCSN-labeled plasma membrane fraction was collected by gradient ultracentrifugation. The VEC plasma membrane or whole-kidney lysate proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and digested with trypsin in gels for liquid chromatography-tandem mass spectrometry. Enrichment analysis was then performed. The VEC plasma membrane proteins were purified by the CCSN method with high yield (approximately 20 μg from 1 g of rat kidney). By Mascot search, 582 proteins were identified in the VEC plasma membrane fraction, and 1,205 proteins were identified in the kidney lysate. In addition to 16 VEC marker proteins such as integrin beta-1 and intercellular adhesion molecule-2 (ICAM-2), 8 novel proteins such as Deltex 3-like protein and phosphatidylinositol binding clathrin assembly protein (PICALM) were identified. As expected, many key functions of plasma membranes in general and of endothelial cells in particular (i.e., leukocyte adhesion) were significantly overrepresented in the proteome of CCSN-labeled kidney VEC fraction. The CCSN method is a reliable technique for isolation of VEC plasma membrane from the kidney, and proteomic analysis followed by bioinformatics revealed the characteristics of in vivo VECs in the kidney.

Simulations of simple linoleic acid-containing lipid membranes and models for the soybean plasma membranes.

PubMed

Zhuang, Xiaohong; Ou, Anna; Klauda, Jeffery B

2017-06-07

The all-atom CHARMM36 lipid force field (C36FF) has been tested with saturated, monounsaturated, and polyunsaturated lipids; however, it has not been validated against the 18:2 linoleoyl lipids with an unsaturated sn-1 chain. The linoleoyl lipids are common in plants and the main component of the soybean membrane. The lipid composition of soybean plasma membranes has been thoroughly characterized with experimental studies. However, there is comparatively less work done with computational modeling. Our molecular dynamics (MD) simulation results show that the pure linoleoyl lipids, 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (18:0/18:2) and 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (di-18:2), agree very well with the experiments, which demonstrates the accuracy of the C36FF for the computational study of soybean membranes. Based on the experimental composition, the soybean hypocotyl and root plasma membrane models are developed with each containing seven or eight types of linoleoyl phospholipids and two types of sterols (sitosterol and stigmasterol). MD simulations are performed to characterize soybean membranes, and the hydrogen bonds and clustering results demonstrate that the lipids prefer to interact with the lipids of the same/similar tail unsaturation. All the results suggest that these two soybean membrane models can be used as a basis for further research in soybean and higher plant membranes involving membrane-associated proteins.

Simulations of simple linoleic acid-containing lipid membranes and models for the soybean plasma membranes

NASA Astrophysics Data System (ADS)

Zhuang, Xiaohong; Ou, Anna; Klauda, Jeffery B.

2017-06-01

The all-atom CHARMM36 lipid force field (C36FF) has been tested with saturated, monounsaturated, and polyunsaturated lipids; however, it has not been validated against the 18:2 linoleoyl lipids with an unsaturated sn-1 chain. The linoleoyl lipids are common in plants and the main component of the soybean membrane. The lipid composition of soybean plasma membranes has been thoroughly characterized with experimental studies. However, there is comparatively less work done with computational modeling. Our molecular dynamics (MD) simulation results show that the pure linoleoyl lipids, 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (18:0/18:2) and 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (di-18:2), agree very well with the experiments, which demonstrates the accuracy of the C36FF for the computational study of soybean membranes. Based on the experimental composition, the soybean hypocotyl and root plasma membrane models are developed with each containing seven or eight types of linoleoyl phospholipids and two types of sterols (sitosterol and stigmasterol). MD simulations are performed to characterize soybean membranes, and the hydrogen bonds and clustering results demonstrate that the lipids prefer to interact with the lipids of the same/similar tail unsaturation. All the results suggest that these two soybean membrane models can be used as a basis for further research in soybean and higher plant membranes involving membrane-associated proteins.

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

PubMed

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

2015-10-20

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

The plasma membrane calcium pumps: focus on the role in (neuro)pathology.

PubMed

Brini, Marisa; Carafoli, Ernesto; Calì, Tito

2017-02-19

The plasma membrane Ca 2+ ATPase (PMCA pump) is a member of the superfamily of P-type pumps. It is organized in the plasma membrane with ten transmembrane helices and two main cytosolic loops, one of which contains the catalytic center. It also contains a long C-terminal tail that houses the binding site for calmodulin, the main regulator of the activity of the pump. The pump also contains a number of other regulators, among them acidic phospholipids, kinases, and numerous protein interactors. Separate genes code for 4 basic pump isoforms in mammals, additional isoform complexity being generated by the alternative splicing of primary transcripts. Pumps 1 and 4 are expressed ubiquitously, pumps 2 and 3 are tissue restricted, with preference for the nervous system. In essentially all cells, the pump coexists with much more powerful systems that clear Ca 2+ from the cytosol, e.g. the SERCA pump and the Na + /Ca 2+ exchanger. Its role in the global regulation of cellular Ca 2+ homeostasis is thus quantitatively marginal: its main function is the regulation of Ca 2+ signaling in selected sub-plasma membrane microdomains where Ca 2+ modulated interactors also reside. Malfunctions of the pump linked to genetic mutations are now described with increasing frequency, the disease phenotypes being especially severe in the nervous system where isoforms 2 and 3 predominate. The analysis of the pump defects suggests that the disease phenotypes are likely to be related to the imperfect modulation of Ca 2+ signaling in selected sub-plasma membrane microdomains, leading to the defective control of the activity of important Ca 2+ dependent interactors. Copyright © 2016 Elsevier Inc. All rights reserved.

Grafting of molecularly imprinted polymer to porous polyethylene filtration membranes by plasma polymerization.

PubMed

Cowieson, D; Piletska, E; Moczko, E; Piletsky, S

2013-08-01

An application of plasma-induced grafting of polyethylene membranes with a thin layer of molecularly imprinted polymer (MIP) was presented. High-density polyethylene (HDPE) membranes, "Vyon," were used as a substrate for plasma grafting modification. The herbicide atrazine, one of the most popular targets of the molecular imprinting, was chosen as a template. The parameters of the plasma treatment were optimized in order to achieve a good balance between polymerization and ablation processes. Modified HDPE membranes were characterized, and the presence of the grafted polymeric layer was confirmed based on the observed weight gain, pore size measurements, and infrared spectrometry. Since there was no significant change in the porosity of the modified membranes, it was assumed that only a thin layer of the polymer was introduced on the surface. The experiments on the re-binding of the template atrazine to the membranes modified with MIP and blank polymers were performed. HDPE membranes which were grafted with polymer using continuous plasma polymerization demonstrated the best result which was expressed in an imprinted factor equal to 3, suggesting that molecular imprinting was successfully achieved.

Uniform structure of eukaryotic plasma membrane: lateral domains in plants.

PubMed

Malínská, Kateŕina; Zažímalová, Eva

2011-03-01

Current models of the plasma membrane (PM) organization focus on the lateral heterogeneity of the membrane and its relation to the cell function. Increasing evidence in mammals and yeast supports the direct relationship between PM lateral microdomains and specific cell processes and functions (nutrient transport, signaling, protein and lipid sorting, endocytosis, pathogen entry etc.). However, for the present the functional significance of an enrichment of specific proteins and possibly lipids in plant PM domains as well as the underlying molecular mechanism driving the lateral PM segregation remain unaddressed. Here we summarize recent findings on the plant PM organization and its role in signaling pathways, with the special emphasis on auxin transport.

Interactions of Ras proteins with the plasma membrane and their roles in signaling.

PubMed

Eisenberg, Sharon; Henis, Yoav I

2008-01-01

The complex dynamic structure of the plasma membrane plays critical roles in cellular signaling; interactions with the membrane lipid milieu, spatial segregation within and between cellular membranes and/or targeting to specific membrane-associated scaffolds are intimately involved in many signal transduction pathways. In this review, we focus on the membrane interactions of Ras proteins. These small GTPases play central roles in the regulation of cell growth and proliferation, and their excessive activation is commonly encountered in human tumors. Ras proteins associate with the membrane continuously via C-terminal lipidation and additional interactions in both their inactive and active forms; this association, as well as the targeting of specific Ras isoforms to plasma membrane microdomains and to intracellular organelles, have recently been implicated in Ras signaling and oncogenic potential. We discuss biochemical and biophysical evidence for the roles of specific domains of Ras proteins in mediating their association with the plasma membrane, and consider the potential effects of lateral segregation and interactions with membrane-associated protein assemblies on the signaling outcomes.

Hemagglutinin Clusters in the Plasma Membrane Are Not Enriched with Cholesterol and Sphingolipids

DOE PAGES

Wilson, Robert L.; Frisz, Jessica F.; Klitzing, Haley A.; ...

2015-04-07

The clusters of the influenza envelope protein, hemagglutinin, within the plasma membrane are hypothesized to be enriched with cholesterol and sphingolipids. Here in this paper, we directly tested this hypothesis by using high-resolution secondary ion mass spectrometry to image the distributions of antibody-labeled hemagglutinin and isotope-labeled cholesterol and sphingolipids in the plasma membranes of fibroblast cells that stably express hemagglutinin. We found that the hemagglutinin clusters were neither enriched with cholesterol nor colocalized with sphingolipid domains. Thus, hemagglutinin clustering and localization in the plasma membrane is not controlled by cohesive interactions between hemagglutinin and liquid-ordered domains enriched with cholesterol andmore » sphingolipids, or from specific binding interactions between hemagglutinin, cholesterol, and/or the majority of sphingolipid species in the plasma membrane.« less

A practical guide for the identification of membrane and plasma membrane proteins in human embryonic stem cells and human embryonal carcinoma cells.

PubMed

Dormeyer, Wilma; van Hoof, Dennis; Mummery, Christine L; Krijgsveld, Jeroen; Heck, Albert J R

2008-10-01

The identification of (plasma) membrane proteins in cells can provide valuable insights into the regulation of their biological processes. Pluripotent cells such as human embryonic stem cells and embryonal carcinoma cells are capable of unlimited self-renewal and share many of the biological mechanisms that regulate proliferation and differentiation. The comparison of their membrane proteomes will help unravel the biological principles of pluripotency, and the identification of biomarker proteins in their plasma membranes is considered a crucial step to fully exploit pluripotent cells for therapeutic purposes. For these tasks, membrane proteomics is the method of choice, but as indicated by the scarce identification of membrane and plasma membrane proteins in global proteomic surveys it is not an easy task. In this minireview, we first describe the general challenges of membrane proteomics. We then review current sample preparation steps and discuss protocols that we found particularly beneficial for the identification of large numbers of (plasma) membrane proteins in human tumour- and embryo-derived stem cells. Our optimized assembled protocol led to the identification of a large number of membrane proteins. However, as the composition of cells and membranes is highly variable we still recommend adapting the sample preparation protocol for each individual system.

Plasma membrane organization promotes virulence of the human fungal pathogen Candida albicans

PubMed Central

Douglas, Lois M.; Konopka, James. B.

2017-01-01

Candida albicans is a human fungal pathogen capable of causing lethal systemic infections. The plasma membrane plays key roles in virulence because it not only functions as a protective barrier, it also mediates dynamic functions including secretion of virulence factors, cell wall synthesis, invasive hyphal morphogenesis, endocytosis, and nutrient uptake. Consistent with this functional complexity, the plasma membrane is composed of a wide array of lipids and proteins. These components are organized into distinct domains that will be the topic of this review. Some of the plasma membrane domains that will be described are known to act as scaffolds or barriers to diffusion, such as MCC/eisosomes, septins, and sites of contact with the endoplasmic reticulum. Other zones mediate dynamic processes, including secretion, endocytosis, and a special region at hyphal tips that facilitates rapid growth. The highly organized architecture of the plasma membrane facilitates the coordination of diverse functions and promotes the pathogenesis of C. albicans. PMID:26920878

Characterization of Bufo arenarum oocyte plasma membrane proteins that interact with sperm.

PubMed

Coux, Gabriela; Cabada, Marcelo O

2006-04-28

Sperm-oocyte plasma membrane interaction is an essential step in fertilization. In amphibians, the molecules involved have not been identified. Our aim was to detect and characterize oocyte molecules with binding affinity for sperm. We isolated plasma membranes free from vitelline envelope and yolk proteins from surface-biotinylated Bufo arenarum oocytes. Using binding assays we detected a biotinylated 100 kDa plasma membrane protein that consistently bound to sperm. Chromatographic studies confirmed the 100 kDa protein and detected two additional oocyte molecules of 30 and 70 kDa with affinity for sperm. Competition studies with an integrin-interacting peptide and cross-reaction with an anti-HSP70 antibody suggested that the 100 and 70 kDa proteins are members of the integrin family and HSP70, respectively. MS/MS analysis suggested extra candidates for a role in this step of fertilization. In conclusion, we provide evidence for the involvement of several proteins, including integrins and HSP70, in B. arenarum sperm-oocyte plasma membrane interactions.

Plasma membrane organization promotes virulence of the human fungal pathogen Candida albicans.

PubMed

Douglas, Lois M; Konopka, James B

2016-03-01

Candida albicans is a human fungal pathogen capable of causing lethal systemic infections. The plasma membrane plays key roles in virulence because it not only functions as a protective barrier, it also mediates dynamic functions including secretion of virulence factors, cell wall synthesis, invasive hyphal morphogenesis, endocytosis, and nutrient uptake. Consistent with this functional complexity, the plasma membrane is composed of a wide array of lipids and proteins. These components are organized into distinct domains that will be the topic of this review. Some of the plasma membrane domains that will be described are known to act as scaffolds or barriers to diffusion, such as MCC/eisosomes, septins, and sites of contact with the endoplasmic reticulum. Other zones mediate dynamic processes, including secretion, endocytosis, and a special region at hyphal tips that facilitates rapid growth. The highly organized architecture of the plasma membrane facilitates the coordination of diverse functions and promotes the pathogenesis of C. albicans.

Palmitoylated SCP1 is targeted to the plasma membrane and negatively regulates angiogenesis

PubMed Central

Liao, Peng; Wang, Weichao; Li, Yu; Wang, Rui; Jin, Jiali; Pang, Weijuan; Chen, Yunfei; Shen, Mingyue; Wang, Xinbo; Jiang, Dongyang; Pang, Jinjiang; Liu, Mingyao; Lin, Xia; Feng, Xin-Hua; Wang, Ping; Ge, Xin

2017-01-01

SCP1 as a nuclear transcriptional regulator acts globally to silence neuronal genes and to affect the dephosphorylation of RNA Pol ll. However, we report the first finding and description of SCP1 as a plasma membrane-localized protein in various cancer cells using EGFP- or other epitope-fused SCP1. Membrane-located SCP1 dephosphorylates AKT at serine 473, leading to the abolishment of serine 473 phosphorylation that results in suppressed angiogenesis and a decreased risk of tumorigenesis. Consistently, we observed increased AKT phosphorylation and angiogenesis followed by enhanced tumorigenesis in Ctdsp1 (which encodes SCP1) gene - knockout mice. Importantly, we discovered that the membrane localization of SCP1 is crucial for impeding angiogenesis and tumor growth, and this localization depends on palmitoylation of a conserved cysteine motif within its NH2 terminus. Thus, our study discovers a novel mechanism underlying SCP1 shuttling between the plasma membrane and nucleus, which constitutes a unique pathway in transducing AKT signaling that is closely linked to angiogenesis and tumorigenesis. DOI: http://dx.doi.org/10.7554/eLife.22058.001 PMID:28440748

Detection of cholesterol-rich microdomains in the inner leaflet of the plasma membrane

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

Hayashi, Masami; Shimada, Yukiko; Inomata, Mitsushi

2006-12-22

The C-terminal domain (D4) of perfringolysin O binds selectively to cholesterol in cholesterol-rich microdomains. To address the issue of whether cholesterol-rich microdomains exist in the inner leaflet of the plasma membrane, we expressed D4 as a fusion protein with EGFP in MEF cells. More than half of the EGFP-D4 expressed in stable cell clones was bound to membranes in raft fractions. Depletion of membrane cholesterol with {beta}-cyclodextrin reduced the amount of EGFP-D4 localized in raft fractions, confirming EGFP-D4 binding to cholesterol-rich microdomains. Subfractionation of the raft fractions showed most of the EGFP-D4 bound to the plasma membrane rather than tomore » intracellular membranes. Taken together, these results strongly suggest the existence of cholesterol-rich microdomains in the inner leaflet of the plasma membrane.« less

Topographical analysis of the plasma membrane-associated sucrose binding protein from soybean.

PubMed

Overvoorde, P J; Grimes, H D

1994-05-27

Plasma membranes of soybean cells actively engaged in sucrose transport have a sucrose binding protein (SBP) that does not appear to be an integral membrane protein. Experiments were undertaken to analyze the topographical association of this protein with the membrane. Treatment of purified plasma membrane vesicles with either 1 M KCl or KI released less than 35% of the sucrose binding protein from the membrane whereas treatment with either 4 M urea or 0.1 M Na2CO3, pH 11.5, disassociated between 50 and 70%, respectively, of this protein from the membrane. SDS, at either 0.5x, 1x, or 10x of its critical micelle concentration, effectively solubilized the sucrose binding protein. The nonionic detergents Triton X-100 and CHAPS, at either 0.5x, 1x, or 10x of their critical micelle concentration, solubilized between 65 and 75% of this protein. When either native plasma membrane-associated or in vitro-transcribed and -translated SBP were subjected to Triton X-114 phase separation, 80% partitioned into the detergent-poor aqueous phase. These results indicate that the SBP is a peripheral membrane protein but also suggest that there is a population of this protein that is tethered to the membrane.

Selective production of sealed plasma membrane vesicles from red beet (Beta vulgaris L.) storage tissue.

PubMed

Giannini, J L; Gildensoph, L H; Briskin, D P

1987-05-01

Modification of our previous procedure for the isolation of microsomal membrane vesicles from red beet (Beta vulgaris L.) storage tissue allowed the recovery of sealed membrane vesicles displaying proton transport activity sensitive to both nitrate and orthovanadate. In the absence of a high salt concentration in the homogenization medium, contributions of nitrate-sensitive (tonoplast) and vanadate-sensitive (plasma membrane) proton transport were roughly equal. The addition of 0.25 M KCl to the homogenization medium increased the relative amount of nitrate-inhibited proton transport activity while the addition of 0.25 M KI resulted in proton pumping vesicles displaying inhibition by vanadate but stimulation by nitrate. These effects appeared to result from selective sealing of either plasma membrane or tonoplast membrane vesicles during homogenization in the presence of the two salts. Following centrifugation on linear sucrose gradients it was shown that the nitrate-sensitive, proton-transporting vesicles banded at low density and comigrated with nitrate-sensitive ATPase activity while the vanadate-sensitive, proton-transporting vesicles banded at a much higher density and comigrated with vanadate-sensitive ATPase. The properties of the vanadate-sensitive proton pumping vesicles were further characterized in microsomal membrane fractions produced by homogenization in the presence of 0.25 M KI and centrifugation on discontinuous sucrose density gradients. Proton transport was substrate specific for ATP, displayed a sharp pH optimum at 6.5, and was insensitive to azide but inhibited by N'-N-dicyclohexylcarbodiimide, diethylstilbestrol, and fluoride. The Km of proton transport for Mg:ATP was 0.67 mM and the K0.5 for vanadate inhibition was at about 50 microM. These properties are identical to those displayed by the plasma membrane ATPase and confirm a plasma membrane origin for the vesicles.

Polyphosphoinositides are present in plasma membranes isolated from fusogenic carrot cells

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

Wheeler, J.J.; Boss, W.F.

1987-10-01

Fusogenic carrot cells grown in suspension culture were labeled 12 hours with myo-(2-/sup 3/H)inositol. Plasma membranes were isolated from the prelabeled fusogenic carrot cells by both aqueous polymer two-phase partitioning and Renografin density gradients. With both methods, the plasma membrane-enriched fractions, as identified by marker enzymes, were enriched in (/sup 3/H)inositol-labeled phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP/sub 2/). An additional (/sup 3/H)inositol-labeled lipid, lysophosphatidylinositol monophosphate, which migrated between PIP and PIP/sub 2/ on thin layer plates, was found primarily in the plasma membrane-rich fraction of the fusogenic cells. This was in contrast to lysophosphatidylinositol which is found primarily inmore » the lower phase, microsomal/mitchrondrial-rich fraction.« less

Plasma membrane lipids and their role in fungal virulence.

PubMed

Rella, Antonella; Farnoud, Amir M; Del Poeta, Maurizio

2016-01-01

There has been considerable evidence in recent years suggesting that plasma membrane lipids are important regulators of fungal pathogenicity. Various glycolipids have been shown to impart virulent properties in several fungal species, while others have been shown to play a role in host defense. In addition to their role as virulence factors, lipids also contribute to other virulence mechanisms such as drug resistance, biofilm formation, and release of extracellular vesicles. In addition, lipids also affect the mechanical properties of the plasma membrane through the formation of packed microdomains composed mainly of sphingolipids and sterols. Changes in the composition of lipid microdomains have been shown to disrupt the localization of virulence factors and affect fungal pathogenicity. This review gathers evidence on the various roles of plasma membrane lipids in fungal virulence and how lipids might contribute to the different processes that occur during infection and treatment. Insight into the role of lipids in fungal virulence can lead to an improved understanding of the process of fungal pathogenesis and the development of new lipid-mediated therapeutic strategies. Published by Elsevier Ltd.

Signal transduction, plasma membrane calcium movements, and pigment translocation in freshwater shrimp chromatophores.

PubMed

Milograna, Sarah Ribeiro; Bell, Fernanda Tinti; McNamara, John Campbell

2010-11-01

Crustacean color change results from the differential translocation of chromatophore pigments, regulated by neurosecretory peptides like red pigment concentrating hormone (RPCH) that, in the red ovarian chromatophores of the freshwater shrimp Macrobrachium olfersi, triggers pigment aggregation via increased cytosolic cGMP and Ca(2+) of both smooth endoplasmatic reticulum (SER) and extracellular origin. However, Ca(2+) movements during RPCH signaling and the mechanisms that regulate intracellular [Ca(2+)] are enigmatic. We investigate Ca(2+) transporters in the chromatophore plasma membrane and Ca(2+) movements that occur during RPCH signal transduction. Inhibition of the plasma membrane Ca(2+)-ATPase by La(3+) and indirect inhibition of the Na(+)/Ca(2+) exchanger by ouabain induce pigment aggregation, revealing a role for both in Ca(2+) extrusion. Ca(2+) channel blockade by La(3+) or Cd(2+) strongly inhibits slow-phase RPCH-triggered aggregation during which pigments disperse spontaneously. L-type Ca(2+) channel blockade by gabapentin markedly reduces rapid-phase translocation velocity; N- or P/Q-type blockade by ω-conotoxin MVIIC strongly inhibits RPCH-triggered aggregation and reduces velocity, effects revealing RPCH-signaled influx of extracellular Ca(2+). Plasma membrane depolarization, induced by increasing external K(+) from 5 to 50 mM, produces Ca(2+)-dependent pigment aggregation, whereas removal of K(+) from the perfusate causes pigment hyperdispersion, disclosing a clear correlation between membrane depolarization and pigment aggregation; K(+) channel blockade by Ba(2+) also partially inhibits RPCH action. We suggest that, during RPCH signal transduction, Ca(2+) released from the SER, together with K(+) channel closure, causes chromatophore membrane depolarization, leading to the opening of predominantly N- and/or P/Q-type voltage-gated Ca(2+) channels, and a Ca(2+)/cGMP cascade, resulting in pigment aggregation.

Antifouling coatings via plasma polymerization and atom transfer radical polymerization on thin film composite membranes for reverse osmosis

NASA Astrophysics Data System (ADS)

Hirsch, Ulrike; Ruehl, Marco; Teuscher, Nico; Heilmann, Andreas

2018-04-01

A major drawback to otherwise highly efficient membrane-based desalination techniques like reverse osmosis (RO) is the susceptibility of the membranes to biofouling. In this work, a combination of plasma activation, plasma bromination and surface-initiated atom transfer radical polymerization (si-ATRP) of hydrophilic and zwitterionic monomers, namely hydroxyethyl methacrylate (HEMA), 2-methacryloyloxyethyl phosphorylcholine (MPC) and [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)ammonium hydroxide (SBMA), was applied to generate non-specific, anti-adhesive coatings on thin film composite (TFC) membranes. The antifouling effect of the coatings was shown by short-time batch as well as long-time steady state cultivation experiments with the microorganism Pseudomonas fluorescens. It could be shown that plasma functionalization and polymerization is possible on delicate thin film composite membranes without restricting their filtration performance. All modified membranes showed an increased resistance towards the adhesion of Pseudomonas fluorescens. On average, the biofilm coverage was reduced by 51.4-12.6% (for HEMA, SBMA, and MPC), the highest reduction was monitored for MPC with a biofilm reduction by 85.4%. The hydrophilic coatings applied did not only suppress the adhesion of Pseudomonas fluorescens, but also significantly increase the permeate flux of the membranes relative to uncoated membranes. The stability of the coatings was however not ideal and will have to be improved for future commercial use.

Vesicle-associated membrane protein 2 mediates trafficking of {alpha}5{beta}1 integrin to the plasma membrane

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

Hasan, Nazarul; Hu, Chuan, E-mail: chuan.hu@louisville.edu

2010-01-01

Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of {alpha}5{beta}1 integrin. VAMP2 was present on vesicles containing endocytosed {beta}1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cellmore » surface {alpha}5{beta}1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of {alpha}5{beta}1, without altering cell surface expression of {alpha}2{beta}1 integrin or {alpha}3{beta}1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of {alpha}5{beta}1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.« less

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

PubMed

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

2015-08-01

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

Neurotensin-metabolizing peptidases in rat fundus plasma membranes.

PubMed

Checler, F; Barelli, H; Kwan, C Y; Kitabgi, P; Vincent, J P

1987-08-01

The mechanisms by which neurotensin (NT) was inactivated by rat fundus plasma membranes were characterized. Primary inactivating cleavages occurred at the Arg8-Arg9, Pro10-Tyr11, and Ile12-Leu13 peptidyl bonds. Hydrolysis at the Arg8-Arg9 bond was fully abolished by the use of N-[1(R,S)-carboxy-2-phenylethyl]-alanyl-alanyl-phenylalanine-p- aminobenzoate, a result indicating the involvement at this site of a recently purified soluble metallopeptidase. Hydrolysis of the Pro10-Tyr11 bond was totally resistant to N-benzyloxycarbonyl-prolyl-prolinal and thiorphan, an observation suggesting that the peptidase responsible for this cleavage was different from proline endopeptidase and endopeptidase 24.11 and might correspond to a NT-degrading neutral metallopeptidase recently isolated from rat brain synaptic membranes. The enzyme acting at the Ile12-Leu13 bond has not yet been identified. Secondary cleavages occurring on NT degradation products were mainly generated by bestatin-sensitive aminopeptidases and post-proline dipeptidyl aminopeptidase. The content in NT-metabolizing peptidases present in rat fundus plasma membranes is compared with that previously established for purified rat brain synaptic membranes.

NEU3 Sialidase Protein Interactors in the Plasma Membrane and in the Endosomes.

PubMed

Cirillo, Federica; Ghiroldi, Andrea; Fania, Chiara; Piccoli, Marco; Torretta, Enrica; Tettamanti, Guido; Gelfi, Cecilia; Anastasia, Luigi

2016-05-13

NEU3 sialidase has been shown to be a key player in many physio- and pathological processes, including cell differentiation, cellular response to hypoxic stress, and carcinogenesis. The enzyme, peculiarly localized on the outer leaflet of the plasma membrane, has been shown to be able to remove sialic acid residues from the gangliosides present on adjacent cells, thus creating cell to cell interactions. Nonetheless, herein we report that the enzyme localization is dynamically regulated between the plasma membrane and the endosomes, where a substantial amount of NEU3 is stored with low enzymatic activity. However, under opportune stimuli, NEU3 is shifted from the endosomes to the plasma membrane, where it greatly increases the sialidase activity. Finally, we found that NEU3 possesses also the ability to interact with specific proteins, many of which are different in each cell compartment. They were identified by mass spectrometry, and some selected ones were also confirmed by cross-immunoprecipitation with the enzyme, supporting NEU3 involvement in the cell stress response, protein folding, and intracellular trafficking. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Multi-protein assemblies underlie the mesoscale organization of the plasma membrane

PubMed Central

Saka, Sinem K.; Honigmann, Alf; Eggeling, Christian; Hell, Stefan W.; Lang, Thorsten; Rizzoli, Silvio O.

2014-01-01

Most proteins have uneven distributions in the plasma membrane. Broadly speaking, this may be caused by mechanisms specific to each protein, or may be a consequence of a general pattern that affects the distribution of all membrane proteins. The latter hypothesis has been difficult to test in the past. Here, we introduce several approaches based on click chemistry, through which we study the distribution of membrane proteins in living cells, as well as in membrane sheets. We found that the plasma membrane proteins form multi-protein assemblies that are long lived (minutes), and in which protein diffusion is restricted. The formation of the assemblies is dependent on cholesterol. They are separated and anchored by the actin cytoskeleton. Specific proteins are preferentially located in different regions of the assemblies, from their cores to their edges. We conclude that the assemblies constitute a basic mesoscale feature of the membrane, which affects the patterning of most membrane proteins, and possibly also their activity. PMID:25060237

Dietary fat and the diabetic state alter insulin binding and the fatty acyl composition of the adipocyte plasma membrane.

PubMed Central

Field, C J; Ryan, E A; Thomson, A B; Clandinin, M T

1988-01-01

Control and diabetic rats were fed on semi-purified high-fat diets providing a polyunsaturated/saturated fatty acid ratio (P/S) of 1.0 or 0.25, to examine the effect of diet on the fatty acid composition of major phospholipids of the adipocyte plasma membrane. Feeding the high-P/S diet (P/S = 1.0) compared with the low-P/S diet (P/S = 0.25) increased the content of polyunsaturated fatty acids in membrane phospholipids in both control and diabetic animals. The diabetic state decreased the content of polyunsaturated fatty acids, particularly arachidonic acid, in adipocyte membrane phospholipids. The decrease in arachidonic acid in membrane phospholipids of diabetic animals tended to be normalized to within the control values when high-P/S diets were given. For control animals, altered plasma-membrane composition was associated with change in insulin binding, suggesting that change in plasma-membrane composition may have physiological consequences for insulin-stimulated functions in the adipocyte. PMID:3052424

Pdr18 is involved in yeast response to acetic acid stress counteracting the decrease of plasma membrane ergosterol content and order.

PubMed

Godinho, Cláudia P; Prata, Catarina S; Pinto, Sandra N; Cardoso, Carlos; Bandarra, Narcisa M; Fernandes, Fábio; Sá-Correia, Isabel

2018-05-18

Saccharomyces cerevisiae has the ability to become less sensitive to a broad range of chemically and functionally unrelated cytotoxic compounds. Among multistress resistance mechanisms is the one mediated by plasma membrane efflux pump proteins belonging to the ABC superfamily, questionably proposed to enhance the kinetics of extrusion of all these compounds. This study provides new insights into the biological role and impact in yeast response to acetic acid stress of the multistress resistance determinant Pdr18 proposed to mediate ergosterol incorporation in plasma membrane. The described coordinated activation of the transcription of PDR18 and of several ergosterol biosynthetic genes (ERG2-4, ERG6, ERG24) during the period of adaptation to acetic acid inhibited growth provides further support to the involvement of Pdr18 in yeast response to maintain plasma membrane ergosterol content in stressed cells. Pdr18 role in ergosterol homeostasis helps the cell to counteract acetic acid-induced decrease of plasma membrane lipid order, increase of the non-specific membrane permeability and decrease of transmembrane electrochemical potential. Collectively, our results support the notion that Pdr18-mediated multistress resistance is closely linked to the status of plasma membrane lipid environment related with ergosterol content and the associated plasma membrane properties.

Amine Enrichment of Thin-Film Composite Membranes via Low Pressure Plasma Polymerization for Antimicrobial Adhesion.

PubMed

Reis, Rackel; Dumée, Ludovic F; He, Li; She, Fenghua; Orbell, John D; Winther-Jensen, Bjorn; Duke, Mikel C

2015-07-15

Thin-film composite membranes, primarily based on poly(amide) (PA) semipermeable materials, are nowadays the dominant technology used in pressure driven water desalination systems. Despite offering superior water permeation and salt selectivity, their surface properties, such as their charge and roughness, cannot be extensively tuned due to the intrinsic fabrication process of the membranes by interfacial polymerization. The alteration of these properties would lead to a better control of the materials surface zeta potential, which is critical to finely tune selectivity and enhance the membrane materials stability when exposed to complex industrial waste streams. Low pressure plasma was employed to introduce amine functionalities onto the PA surface of commercially available thin-film composite (TFC) membranes. Morphological changes after plasma polymerization were analyzed by SEM and AFM, and average surface roughness decreased by 29%. Amine enrichment provided isoelectric point changes from pH 3.7 to 5.2 for 5 to 15 min of plasma polymerization time. Synchrotron FTIR mappings of the amine-modified surface indicated the addition of a discrete 60 nm film to the PA layer. Furthermore, metal affinity was confirmed by the enhanced binding of silver to the modified surface, supported by an increased antimicrobial functionality with demonstrable elimination of E. coli growth. Essential salt rejection was shown minimally compromised for faster polymerization processes. Plasma polymerization is therefore a viable route to producing functional amine enriched thin-film composite PA membrane surfaces.

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.

Emerging role of chemoprotective agents in the dynamic shaping of plasma membrane organization.

PubMed

Fuentes, Natividad R; Salinas, Michael L; Kim, Eunjoo; Chapkin, Robert S

2017-09-01

In the context of an organism, epithelial cells by nature are designed to be the defining barrier between self and the outside world. This is especially true for the epithelial cells that form the lining of the digestive tract, which absorb nutrients and serve as a barrier against harmful substances. These cells are constantly bathed by a complex mixture of endogenous (bile acids, mucus, microbial metabolites) and exogenous (food, nutrients, drugs) bioactive compounds. From a cell biology perspective, this type of exposure would directly impact the plasma membrane, which consists of a myriad of complex lipids and proteins. The plasma membrane not only functions as a barrier but also as the medium in which cellular signaling complexes form and function. This property is mediated by the organization of the plasma membrane, which is exquisitely temporally (nanoseconds to minutes) and spatially (nanometers to micrometers) regulated. Since numerous bioactive compounds found in the intestinal lumen can directly interact with lipid membranes, we hypothesize that the dynamic reshaping of plasma membrane organization underlies the chemoprotective effect of select membrane targeted dietary bioactives (MTDBs). This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá. Copyright © 2017 Elsevier B.V. All rights reserved.

Functional domains of the T lymphocyte plasma membrane: characterization of the polypeptide composition.

PubMed

Szamel, M; Kaever, V; Resch, K

1987-01-01

Highly purified plasma membranes from calf thymocytes were fractionated by affinity chromatography on Concanavalin A-Sepharose into two subfractions, one eluting freely from the affinity column (MF1) and a second being specifically retained (MF2). SDS-polyacrylamide-gel-electrophoresis revealed different polypeptide patterns of the two plasma membrane subfractions. Polypeptides of apparent molecular weights of 170, 150, 110, 94, 39, and 30 kDa were several-fold enriched in the adherent fraction, MF2. In contrast, several proteins in the 55-65 kDa range were preferentially recovered in the non-adherent fraction. Five Five of the six polypeptides, preferentially recovered in MF2 proved to be glycoproteins, the 39 kDa peptide was non-glycosilated. The differences in the amounts of the polypeptides specifically enriched in the adherent fraction MF2 became even more clear-cut when plasma membranes solubilized with non-ionic detergents (lysolecithin, ET-18-2H, Triton-X-100) were separated by affinity chromatography on Concanavalin A-Sepharose. The non-glycosilated peptide of apparent molecular weight of 39 kDa was recovered together with several glycoproteins in the adherent fraction, MF2, suggesting that not single glycoproteins, but plasma membrane domains were separated by Concanavalin A-Sepharose. Although the glycoproteins of the non-adherent fraction MF1 bound significant amounts of Concanavalin A, the major Concanavalin A binding glycoproteins were recovered in the adherent fraction, MF2. The plasma membrane subfractions showed also different functional properties, the specific activities [Na+ + K+]AT-Pase, Ca2+ ATPase and lysolecithin acyltransferase were several-fold enriched in the adherent fraction, MF2, as compared to MF1. The data suggest the existence of plasma membrane domains in the plasma membranes of thymocytes consisting of a different set of proteins, among others the major Concanavalin A binding glycoproteins with some membrane bound enzymes

Study of hepatocyte plasma membrane mechanical properties using optical trapping

NASA Astrophysics Data System (ADS)

Vedyaykin, A. D.; Morozova, N. E.; Pobegalov, G. E.; Arseniev, A. N.; Khodorkoskii, M. A.; Sabantsev, A. V.

2014-12-01

In this paper we describe the use of membrane tether formation technique which is widely used to study mechanical properties of plasma membranes. This method was successfully used for the direct measurement of parameters characterizing membranes mechanical properties (static tether tension force and effective membrane viscosity) of human hepatocytes (HepG2 hepatocellular carcinoma line). These results allow using this method in future for diagnostics of the cell membrane, evaluating the influence on the mechanical parameters of various factors, including toxins and drugs.

Detecting Subtle Plasma Membrane Perturbation in Living Cells Using Second Harmonic Generation Imaging

PubMed Central

Moen, Erick K.; Ibey, Bennett L.; Beier, Hope T.

2014-01-01

The requirement of center asymmetry for the creation of second harmonic generation (SHG) signals makes it an attractive technique for visualizing changes in interfacial layers such as the plasma membrane of biological cells. In this article, we explore the use of lipophilic SHG probes to detect minute perturbations in the plasma membrane. Three candidate probes, Di-4-ANEPPDHQ (Di-4), FM4-64, and all-trans-retinol, were evaluated for SHG effectiveness in Jurkat cells. Di-4 proved superior with both strong SHG signal and limited bleaching artifacts. To test whether rapid changes in membrane symmetry could be detected using SHG, we exposed cells to nanosecond-pulsed electric fields, which are believed to cause formation of nanopores in the plasma membrane. Upon nanosecond-pulsed electric fields exposure, we observed an instantaneous drop of ∼50% in SHG signal from the anodic pole of the cell. When compared to the simultaneously acquired fluorescence signals, it appears that the signal change was not due to the probe diffusing out of the membrane or changes in membrane potential or fluidity. We hypothesize that this loss in SHG signal is due to disruption in the interfacial nature of the membrane. The results show that SHG imaging has great potential as a tool for measuring rapid and subtle plasma membrane disturbance in living cells. PMID:24853757

Detecting subtle plasma membrane perturbation in living cells using second harmonic generation imaging.

PubMed

Moen, Erick K; Ibey, Bennett L; Beier, Hope T

2014-05-20

The requirement of center asymmetry for the creation of second harmonic generation (SHG) signals makes it an attractive technique for visualizing changes in interfacial layers such as the plasma membrane of biological cells. In this article, we explore the use of lipophilic SHG probes to detect minute perturbations in the plasma membrane. Three candidate probes, Di-4-ANEPPDHQ (Di-4), FM4-64, and all-trans-retinol, were evaluated for SHG effectiveness in Jurkat cells. Di-4 proved superior with both strong SHG signal and limited bleaching artifacts. To test whether rapid changes in membrane symmetry could be detected using SHG, we exposed cells to nanosecond-pulsed electric fields, which are believed to cause formation of nanopores in the plasma membrane. Upon nanosecond-pulsed electric fields exposure, we observed an instantaneous drop of ~50% in SHG signal from the anodic pole of the cell. When compared to the simultaneously acquired fluorescence signals, it appears that the signal change was not due to the probe diffusing out of the membrane or changes in membrane potential or fluidity. We hypothesize that this loss in SHG signal is due to disruption in the interfacial nature of the membrane. The results show that SHG imaging has great potential as a tool for measuring rapid and subtle plasma membrane disturbance in living cells. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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.

Expression of plasma membrane receptor genes during megakaryocyte development

PubMed Central

Sun, Sijie; Wang, Wenjing; Latchman, Yvette; Gao, Dayong; Aronow, Bruce

2013-01-01

Megakaryocyte (MK) development is critically informed by plasma membrane-localized receptors that integrate a multiplicity of environmental cues. Given that the current understanding about receptors and ligands involved in megakaryocytopoiesis is based on single targets, we performed a genome-wide search to identify a plasma membrane receptome for developing MKs. We identified 40 transmembrane receptor genes as being upregulated during MK development. Seven of the 40 receptor-associated genes were selected to validate the dataset. These genes included: interleukin-9 receptor (IL9R), transforming growth factor, β receptor II (TGFBR2), interleukin-4 receptor (IL4R), colony stimulating factor-2 receptor-beta (CSFR2B), adiponectin receptor (ADIPOR2), thrombin receptor (F2R), and interleukin-21 receptor (IL21R). RNA and protein analyses confirmed their expression in primary human MKs. Matched ligands to IL9R, TGFBR2, IL4R, CSFR2B, and ADIPOR2 affected megakaryocytopoiesis. IL9 was unique in its ability to increase the number of MKs formed. In contrast, MK colony formation was inhibited by adiponectin, TGF-β, IL4, and GM-CSF. The thrombin-F2R axis affected platelet function, but not MK development, while IL21 had no apparent detectable effects. ADP-induced platelet aggregation was suppressed by IL9, TGF-β, IL4, and adiponectin. Overall, six of seven of the plasma membrane receptors were confirmed to have functional roles in MK and platelet biology. Also, results show for the first time that adiponectin plays a regulatory role in MK development. Together these data support a strong likelihood that the 40 transmembrane genes identified as being upregulated during MK development will be an important resource to the research community for deciphering the complex repertoire of environmental cues regulating megakaryocytopoiesis and/or platelet function. PMID:23321270

Endoplasmic Reticulum-Plasma Membrane Contact Sites.

PubMed

Saheki, Yasunori; De Camilli, Pietro

2017-06-20

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

Alteration in lipid composition of plasma membranes of sensitive and resistant Guerin carcinoma cells due to the action of free and liposomal form of cisplatin.

PubMed

Naleskina, L A; Todor, I N; Nosko, M M; Lukianova, N Y; Pivnyuk, V M; Chekhun, V F

2013-09-01

To study in vivo changes of lipid composition of plasma membranes of sensitive and resistant to cisplatin Guerin carcinoma cells under influence of free and liposomal cisplatin forms. The isolation of plasma membranes from parental (sensitive) and resistant to cisplatin Guerin carcinoma cells was by differential ultracentrifugation in sucrose density gradient. Lipids were detected by method of thin-layer chromatography. It was determined that more effective action of cisplatin liposomal form on resistant cells is associated with essential abnormalities of conformation of plasma membrane due to change of lipid components and architectonics of rafts. It results in the increase of membrane fluidity. Reconstructions in lipid composition of plasma membranes of cisplatin-resistant Guerin carcinoma cells provide more intensive delivery of drug into the cells, increase of its concentration and more effective interaction with cellular structural elements.

Supramolecular organization of the sperm plasma membrane during maturation and capacitation.

PubMed

Jones, Roy; James, Peter S; Howes, Liz; Bruckbauer, Andreas; Klenerman, David

2007-07-01

In the present study, a variety of high resolution microscopy techniques were used to visualize the organization and motion of lipids and proteins in the sperm's plasma membrane. We have addressed questions such as the presence of diffusion barriers, confinement of molecules to specific surface domains, polarized diffusion and the role of cholesterol in regulating lipid rafts and signal transduction during capacitation. Atomic force microscopy identified a novel region (EqSS) within the equatorial segment of bovine, porcine and ovine spermatozoa that was enriched in constitutively phosphorylated proteins. The EqSS was assembled during epididymal maturation. Fluorescence imaging techniques were then used to follow molecular diffusion on the sperm head. Single lipid molecules were freely exchangeable throughout the plasma membrane and showed no evidence for confinement within domains. Large lipid aggregates, however, did not cross over the boundary between the post-acrosome and equatorial segment suggesting the presence of a molecular filter between these two domains. A small reduction in membrane cholesterol enlarges or increases lipid rafts concomitant with phosphorylation of intracellular proteins. Excessive removal of cholesterol, however, disorganizes rafts with a cessation of phosphorylation. These techniques are forcing a revision of long-held views on how lipids and proteins in sperm membranes are assembled into larger complexes that mediate recognition and fusion with the egg.

[Changes in polarization of myometrial cells plasma and internal mitochondrial membranes under calixarenes action as inhibitors of plasma membrane Na+, K+-ATPase].

PubMed

Danylovych, H V; Danylovych, Iu V; Kolomiiets', O V; Kosterin, S O; Rodik, R V; Cherenok, S O; Kal'chenko, V I; Chunikhin, O Iu; Horchev, V F; Karakhim, S O

2012-01-01

The influence of supramolecular macrocyclic compounds--calix[4]arenes C-97, C-99, C-107, which are ouabainomymetic high affinity inhibitors of Na+, K(+)-ATPase, on the polarization level of plasmic and mitochondrial membranes of rat uterine smooth muscle cells was investigated. The influence of these compounds on the myocytes characteristic size was studied. By using a confocal microscopy and specific for mitochondrial MitoTracker Orange CM-H2TMRos dye it was proved that the potential-sensitive fluorescent probe DiOC6(3) interacts with mitochondria. Artificial potential collapse of plasmic membrane in this case was modeled by myocytes preincubation with ouabain (1 mM). Further experiments performed using the method of flow cytometry with DiOC6(3) have shown that the compounds C-97, C-99 and C-107 at concentration 50-100 nM caused depolarization of the plasma membrane (at the level of 30% relative to control values) in conditions of artificial collapse of mitochondrial potential by myocytes preincubation in the presence of 5 mM of sodium azide. Under artificial sarcolemma depolarization by ouabain, calixarenes C-97, C-99 and C-107 at 100 nM concentrations caused a transient increase of mitochondrial membrane potential, that is 40% of the control level and lasted about 5 minutes. Calixarenes C-99 and C-107 caused a significant increase in fluorescence of myocytes in these conditions, which was confirmed by confocal microscopy too. It was proved by photon correlation spectroscopy method that the C-99 and C-107 caused an increase of characteristic size of myocytes.

Fatty acid composition of plasma lipids and erythrocyte membranes during simulated extravehicular activity

NASA Astrophysics Data System (ADS)

Skedina, M. A.; Katuntsev, V. P.; Buravkova, L. B.; Naidina, V. P.

Ten subjects (from 27 to 41 years) have been participated in 32 experiments. They were decompressed from ground level to 40-35 kPa in altitude chamber when breathed 100% oxygen by mask and performed repeated cycles of exercises (3.0 Kcal/min). The intervals between decompressions were 3-5 days. Plasma lipid and erythrocyte membrane fatty acid composition was evaluated in the fasting venous blood before and immediately after hypobaric exposure. There were 7 cases decompression sickness (DCS). Venous gas bubbles (GB) were detected in 27 cases (84.4%). Any significant changes in the fatty acid composition of erythrocyte membranes and plasma didn't practically induce after the first decompression. However, by the beginning of the second decompression the total lipid level in erythrocyte membranes decreased from 54.6 mg% to 40.4 mg% in group with DCS symptoms and from 51.2 mg% to 35.2 mg% (p < 0.05) without DCS symptoms. In group with DCS symptoms a tendency to increased level of saturated fatty acids in erythrocyte membranes (16:0, 18:0), the level of the polyunsaturated linoleic fatty acid (18:2) and arachidonic acid (20:4) tended to be decreased by the beginning of the second decompression. Insignificant changes in blood plasma fatty acid composition was observed in both groups. The obtained biochemical data that indicated the simulated extravehicular activity (EVA) condition is accompanied by the certain changes in the blood lipid metabolism, structural and functional state of erythrocyte membranes, which are reversible. The most pronounced changes are found in subjects with DCS symptoms.

Fatty acid composition of plasma lipids and erythrocyte membranes during simulated extravehicular activity.

PubMed

Skedina, M A; Katuntsev, V P; Buravkova, L B; Naidina, V P

1998-01-01

Ten subjects (from 27 to 41 years) have been participated in 32 experiments. They were decompressed from ground level to 40-35 kPa in altitude chamber when breathed 100% oxygen by mask and performed repeated cycles of exercises (3.0 Kcal/min). The intervals between decompressions were 3-5 days. Plasma lipid and erythrocyte membrane fatty acid composition was evaluated in the fasting venous blood before and immediately after hypobaric exposure. There were 7 cases decompression sickness (DCS). Venous gas bubbles (GB) were detected in 27 cases (84.4%). Any significant changes in the fatty acid composition of erythrocyte membranes and plasma didn't practically induce after the first decompression. However, by the beginning of the second decompression the total lipid level in erythrocyte membranes decreased from 54.6 mg% to 40.4 mg% in group with DCS symptoms and from 51.2 mg% to 35.2 mg% (p<0.05) without DCS symptoms. In group with DCS symptoms a tendency to increased level of saturated fatty acids in erythrocyte membranes (16:0, 18:0), the level of the polyunsaturated linoleic fatty acid (18:2) and arachidonic acid (20:4) tended to be decreased by the beginning of the second decompression. Insignificant changes in blood plasma fatty acid composition was observed in both groups. The obtained biochemical data that indicated the simulated extravehicular activity (EVA) condition is accompanied by the certain changes in the blood lipid metabolism, structural and functional state of erythrocyte membranes, which are reversible. The most pronounced changes are found in subjects with DCS symptoms.

Super-resolution microscopy reveals the insulin-resistance-regulated reorganization of GLUT4 on plasma membranes.

PubMed

Gao, Lan; Chen, Junling; Gao, Jing; Wang, Hongda; Xiong, Wenyong

2017-01-15

GLUT4 (also known as SLC2A4) is essential for glucose uptake in skeletal muscles and adipocytes, which play central roles in whole-body glucose metabolism. Here, using direct stochastic optical reconstruction microscopy (dSTORM) to investigate the characteristics of plasma-membrane-fused GLUT4 at the single-molecule level, we have demonstrated that insulin and insulin resistance regulate the spatial organization of GLUT4 in adipocytes. Stimulation with insulin shifted the balance of GLUT4 on the plasma membrane toward a more dispersed configuration. In contrast, insulin resistance induced a more clustered distribution of GLUT4 and increased the mean number of molecules per cluster. Furthermore, our data demonstrate that the F 5 QQI motif and lipid rafts mediate the maintenance of GLUT4 clusters on the plasma membrane. Mutation of F 5 QQI (F 5 QQA-GLUT4) induced a more clustered distribution of GLUT4; moreover, destruction of lipid rafts in adipocytes expressing F 5 QQA-GLUT4 dramatically decreased the percentage of large clusters and the mean number of molecules per cluster. In conclusion, our data clarify the effects of insulin stimulation or insulin resistance on GLUT4 reorganization on the plasma membrane and reveal new pathogenic mechanisms of insulin resistance. © 2017. Published by The Company of Biologists Ltd.

A cell-free assay to determine the stoichiometry of plasma membrane proteins.

PubMed

Trigo, Cesar; Vivar, Juan P; Gonzalez, Carlos B; Brauchi, Sebastian

2013-04-01

Plasma membrane receptors, transporters, and ion channel molecules are often found as oligomeric structures that participate in signaling cascades essential for cell survival. Different states of protein oligomerization may play a role in functional control and allosteric regulation. Stochastic GFP-photobleaching (SGP) has emerged as an affordable and simple method to determine the stoichiometry of proteins at the plasma membrane. This non-invasive optical approach can be useful for total internal reflection of fluorescence microscopy (TIRFM), where signal-to-noise ratio is very high at the plasma membrane. Here, we report an alternative methodology implemented on a standard laser scanning confocal microscope (LSCM). The simplicity of our method will allow for its implementation in any epifluorescence microscope of choice.

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

Alteration of the lipid composition of rat testicular plasma membranes by dietary (n-3) fatty acids changes the responsiveness of Leydig cells and testosterone synthesis.

PubMed

Sebokova, E; Garg, M L; Wierzbicki, A; Thomson, A B; Clandinin, M T

1990-06-01

Experiments were conducted to assess whether changing dietary fat composition altered phospholipid composition of rat testicular plasma membranes in a manner that altered receptor-mediated action of luteinizing hormone (LH)/human chorionic gonadotropin (hCG). Weanling rats were fed diets that provided high or low cholesterol intakes and that were enriched with linseed oil, fish oil or beef tallow for 4 wk. Feeding diets high in (n-3) fatty acids decreased plasma and testicular plasma membrane 20:4(n-6) content. A marked reduction of the 22:5(n-6) content and an increase in the 22:6(n-3) content of testicular plasma membrane was found only in animals fed fish oil. A decrease in binding capacity of the gonadotropin (LH/hCG) receptor in the plasma membrane, with no change in receptor affinity, was observed for animals fed either linseed oil or fish oil diets. Dietary treatments that raised plasma membrane cholesterol content and the cholesterol to phospholipid ratio in the membrane were associated with increased binding capacity of the gonadotropin receptor. Feeding diets high in 18:3(n-3) vs. those high in fish oil altered receptor-mediated adenylate cyclase activity in a manner that depended on the level of dietary cholesterol. Feeding diets high in cholesterol or fish oil increased basal and LH-stimulated testosterone synthesis relative to that in animals fed the low cholesterol diet containing linseed oil. It is concluded that changing the fat composition of the diet alters the phospholipid composition of rat testicular plasma membranes and that this change in composition influences membrane-mediated unmasking of gonadotropin receptor-mediated action in testicular tissue.

Effects of plasma proteins on sieving of tracer macromolecules in glomerular basement membrane.

PubMed

Lazzara, M J; Deen, W M

2001-11-01

It was found previously that the sieving coefficients of Ficoll and Ficoll sulfate across isolated glomerular basement membrane (GBM) were greatly elevated when BSA was present at physiological levels, and it was suggested that most of this increase might have been the result of steric interactions between BSA and the tracers (5). To test this hypothesis, we extended the theory for the sieving of macromolecular tracers to account for the presence of a second, abundant solute. Increasing the concentration of an abundant solute is predicted to increase the equilibrium partition coefficient of a tracer in a porous or fibrous membrane, thereby increasing the sieving coefficient. The magnitude of this partitioning effect depends on solute size and membrane structure. The osmotic reduction in filtrate velocity caused by an abundant, mostly retained solute will also tend to elevate the tracer sieving coefficient. The osmotic effect alone explained only about one-third of the observed increase in the sieving coefficients of Ficoll and Ficoll sulfate, whereas the effect of BSA on tracer partitioning was sufficient to account for the remainder. At physiological concentrations, predictions for tracer sieving in the presence of BSA were found to be insensitive to the assumed shape of the protein (sphere or prolate spheroid). For protein mixtures, the theoretical effect of 6 g/dl BSA on the partitioning of spherical tracers was indistinguishable from that of 3 g/dl BSA and 3 g/dl IgG. This suggests that for partitioning and sieving studies in vitro, a good experimental model for plasma is a BSA solution with a mass concentration matching that of total plasma protein. The effect of plasma proteins on tracer partitioning is expected to influence sieving not only in isolated GBM but also in intact glomerular capillaries in vivo.

The cell-based L-glutathione protection assays to study endocytosis and recycling of plasma membrane proteins.

PubMed

Cihil, Kristine M; Swiatecka-Urban, Agnieszka

2013-12-13

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Surface modification of PTMSP membranes by plasma treatment: Asymmetry of transport in organic solvent nanofiltration.

PubMed

Volkov, A V; Tsarkov, S E; Gilman, A B; Khotimsky, V S; Roldughin, V I; Volkov, V V

2015-08-01

For the first time, the effect of asymmetry of the membrane transport was studied for organic solvents and solutes upon their nanofiltration through the plasma-modified membranes based on poly(1-trimethylsilyl-1-propyne) (PTMSP). Plasma treatment is shown to provide a marked hydrophilization of the hydrophobic PTMSP surface (the contact angle of water decreases from 88 down to 20°) and leads to the development of a negative charge of -5.2 nC/cm(2). The XPS measurements prove the formation of the oxygen-containing groups (Si-O and C-O) due to the surface modification. The AFM images show that the small-scale surface roughness of the plasma-treated PTMSP sample is reduced but the large-scale surface heterogeneities become more pronounced. The modified membranes retain their hydrophilic surface properties even after the nanofiltration tests and 30-day storage under ambient conditions. The results of the filtration tests show that when the membrane is oriented so that its modified layer contacts the feed solution, the membrane permeability for linear alcohols (methanol-propanol) and acetone decreases nearly two times. When the modified membrane surface faces the permeate, the membrane is seen to regain its transport characteristics: the flux becomes equal to that of the unmodified PTMSP. The well-pronounced effect of the transport asymmetry is observed for the solution of the neutral dye Solvent Blue 35 in methanol, ethanol, and acetone. For example, the initial membrane shows the negative retention for the Solvent Blue 35 dye (-16%) upon its filtration from the ethanol solution whereas, for the modified PTMSP membrane, the retention increases up to 17%. Various effects contributing to the asymmetry of the membrane transport characteristics are discussed. Copyright © 2014 Elsevier B.V. All rights reserved.

Comparative analysis of soybean plasma membrane proteins under osmotic stress using gel-based and LC MS/MS-based proteomics approaches.

PubMed

Nouri, Mohammad-Zaman; Komatsu, Setsuko

2010-05-01

To study the soybean plasma membrane proteome under osmotic stress, two methods were used: a gel-based and a LC MS/MS-based proteomics method. Two-day-old seedlings were subjected to 10% PEG for 2 days. Plasma membranes were purified from seedlings using a two-phase partitioning method and their purity was verified by measuring ATPase activity. Using the gel-based proteomics, four and eight protein spots were identified as up- and downregulated, respectively, whereas in the nanoLC MS/MS approach, 11 and 75 proteins were identified as up- and downregulated, respectively, under PEG treatment. Out of osmotic stress responsive proteins, most of the transporter proteins and all proteins with high number of transmembrane helices as well as low-abundance proteins could be identified by the LC MS/MS-based method. Three homologues of plasma membrane H(+)-ATPase, which are transporter proteins involved in ion efflux, were upregulated under osmotic stress. Gene expression of this protein was increased after 12 h of stress exposure. Among the identified proteins, seven proteins were mutual in two proteomics techniques, in which calnexin was the highly upregulated protein. Accumulation of calnexin in plasma membrane was confirmed by immunoblot analysis. These results suggest that under hyperosmotic conditions, calnexin accumulates in the plasma membrane and ion efflux accelerates by upregulation of plasma membrane H(+)-ATPase protein.

Plasma Membrane Protein Profiling in Beta-Amyloid-Treated Microglia Cell Line.

PubMed

Correani, Virginia; Di Francesco, Laura; Mignogna, Giuseppina; Fabrizi, Cinzia; Leone, Stefano; Giorgi, Alessandra; Passeri, Alessia; Casata, Roberto; Fumagalli, Lorenzo; Maras, Bruno; Schininà, M Eugenia

2017-09-01

In the responsiveness of microglia to toxic stimuli, plasma membrane proteins play a key role. In this study we treated with a synthetic beta amyloid peptide murine microglial cells metabolically differently labelled with stable isotope amino acids (SILAC). The plasma membrane was selectively enriched by a multi-stage aqueous two-phase partition system. We were able to identify by 1D-LC-MS/MS analyses 1577 proteins, most of them are plasma membrane proteins according to the Gene Ontology annotation. An unchanged level of amyloid receptors in this data set suggests that microglia preserve their responsiveness capability to the environment even after 24-h challenge with amyloid peptides. On the other hand, 14 proteins were observed to change their plasma membrane abundance to a statistically significant extent. Among these, we proposed as reliable biomarkers of the inflammatory microglia phenotype in AD damaged tissues MAP/microtubule affinity-regulating kinase 3 (MARK3), Interferon-induced transmembrane protein 3 (IFITM3), Annexins A5 and A7 (ANXA5, ANXA7) and Neuropilin-1 (NRP1), all proteins known to be involved in the inflammation processes and in microtubule network assembly rate. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A fluorogenic probe for SNAP-tagged plasma membrane proteins based on the solvatochromic molecule Nile Red.

PubMed

Prifti, Efthymia; Reymond, Luc; Umebayashi, Miwa; Hovius, Ruud; Riezman, Howard; Johnsson, Kai

2014-03-21

A fluorogenic probe for plasma membrane proteins based on the dye Nile Red and SNAP-tag is introduced. It takes advantage of Nile Red, a solvatochromic molecule highly fluorescent in an apolar environment, such as cellular membranes, but almost dark in a polar aqueous environment. The probe possesses a tuned affinity for membranes allowing its Nile Red moiety to insert into the lipid bilayer of the plasma membrane, becoming fluorescent, only after its conjugation to a SNAP-tagged plasma membrane protein. The fluorogenic character of the probe was demonstrated for different SNAP-tag fusion proteins, including the human insulin receptor. This work introduces a new approach for generating a powerful turn-on probe for "no-wash" labeling of plasma membrane proteins with numerous applications in bioimaging.

Compositional changes in lipid microdomains of air-blood barrier plasma membranes in pulmonary interstitial edema.

PubMed

Palestini, Paola; Calvi, Chiara; Conforti, Elena; Daffara, Rossella; Botto, Laura; Miserocchi, Giuseppe

2003-10-01

We evaluated in anesthetized rabbits the compositional changes of plasmalemmal lipid microdomains from lung tissue samples after inducing pulmonary interstitial edema (0.5 ml/kg for 3 h, leading to approximately 5% increase in extravascular water). Lipid microdomains (lipid rafts and caveolae) were present in the detergent-resistant fraction (DRF) obtained after discontinuous sucrose density gradient. DRF was enriched in caveolin-1, flotillin, aquaporin-1, GM1, cholesterol, sphingomyelin, and phosphatidylserine, and their contents significantly increased in interstitial edema. The higher DRF content in caveolin, flotillin, and aquaporin-1 and of the ganglioside GM1 suggests an increase both in caveolar domains and in lipid rafts, respectively. Compositional changes could be ascribed to endothelial and epithelial cells that provide most of plasma membrane surface area in the air-blood barrier. Alterations in lipid components in the plasma membrane may reflect rearrangement of floating lipid platforms within the membrane and/or lipid translocation from intracellular stores. Lipid traffic could be stimulated by the marked increase in hydraulic interstitial pressure after initial water accumulation, from approximately -10 to 5 cmH2O, due to the low compliance of the pulmonary tissue, in particular in the basement membranes and in the interfibrillar substance. Compositional changes in lipid microdomains represent a sign of cellular activation and suggest the potential role of mechanotransduction in response to developing interstitial edema.

The cytotoxic activity of miltefosine against Leishmania and macrophages is associated with dynamic changes in plasma membrane proteins.

PubMed

Fernandes, Kelly Souza; de Souza, Paulo Eduardo Narcizo; Dorta, Miriam Leandro; Alonso, Antonio

2017-01-01

In this study, we combined electron paramagnetic resonance (EPR) spectroscopy with an analysis of biophysical cellular parameters to study the mechanisms underlying the in vitro anti-leishmanial activity of miltefosine (MT). A thiol-specific spin label attached to membrane-bound proteins of Leishmania amazonensis and peritoneal macrophages indicated that MT may bind to plasma membrane proteins in large quantities via a detergent-like action and cause structural changes associated with a marked increase in dynamics and exposure to an aqueous environment. EPR spectra of a spin-labeled stearic acid indicated strong interactions between the probe and membrane proteins and a marked increase in the membrane fluidity of MT-treated cells. The cytotoxicity of MT was found to depend on the cell concentration used in the assay. This dependence was described by an equation involving the 50% inhibitory concentrations of MT in the aqueous medium (c w50 ) and the cell membrane (c m50 ) and the membrane-aqueous medium partition coefficient of MT (K). With a c w50 of 8.7μM, macrophages were less sensitive to MT than amastigotes and promastigotes of Leishmania, which had c w50 values of 2.4-3.1μM. The estimated c m50 of MT for Leishmania was 1.8M, which appears sufficient to cause ruptures or formation of pores in the plasma membrane. Additionally, we demonstrated that the changes in the plasma membrane detected by EPR spectroscopy occurred at cytotoxic concentrations of MT, as assessed through in vitro assays. Copyright © 2016 Elsevier B.V. All rights reserved.

Lateral Organization of Influenza Virus Proteins in the Budozone Region of the Plasma Membrane.

PubMed

Leser, George P; Lamb, Robert A

2017-05-01

Influenza virus assembles and buds at the plasma membrane of virus-infected cells. The viral proteins assemble at the same site on the plasma membrane for budding to occur. This involves a complex web of interactions among viral proteins. Some proteins, like hemagglutinin (HA), NA, and M2, are integral membrane proteins. M1 is peripherally membrane associated, whereas NP associates with viral RNA to form an RNP complex that associates with the cytoplasmic face of the plasma membrane. Furthermore, HA and NP have been shown to be concentrated in cholesterol-rich membrane raft domains, whereas M2, although containing a cholesterol binding motif, is not raft associated. Here we identify viral proteins in planar sheets of plasma membrane using immunogold staining. The distribution of these proteins was examined individually and pairwise by using the Ripley K function, a type of nearest-neighbor analysis. Individually, HA, NA, M1, M2, and NP were shown to self-associate in or on the plasma membrane. HA and M2 are strongly coclustered in the plasma membrane; however, in the case of NA and M2, clustering depends upon the expression system used. Despite both proteins being raft resident, HA and NA occupy distinct but adjacent membrane domains. M2 and M1 strongly cocluster, but the association of M1 with HA or NA is dependent upon the means of expression. The presence of HA and NP at the site of budding depends upon the coexpression of other viral proteins. Similarly, M2 and NP occupy separate compartments, but an association can be bridged by the coexpression of M1. IMPORTANCE The complement of influenza virus proteins necessary for the budding of progeny virions needs to accumulate at budozones. This is complicated by HA and NA residing in lipid raft-like domains, whereas M2, although an integral membrane protein, is not raft associated. Other necessary protein components such as M1 and NP are peripherally associated with the membrane. Our data define spatial relationships

[Does a lateral gradient of membrane potential on the plasma membrane of growing pollen tube of germinating pollen grain exist?].

PubMed

Andreev, I M

2011-01-01

The data presented in the article by Breigina et al. (2009) "Changes in the membrane potential during pollen grain germination and pollen tube growth" (Tsitologiya. 51 (10): 815-823) and concerning the measurement of electric membrane potential (Delta Psi) on the plasma membrane of growing pollen tube of germinating pollen grain with the use of fluorescent potential-sensitive dye, di-4-ANEPPS, were critically analyzed in order to clarify whether a lateral gradient of Delta Psi on this membrane indeed exists. This analysis showed that the main conclusion of the authors of the above article on the existence of polar distribution of Delta Psi along the pollen tube plasma membrane is not in accordance with a number of known peculiarities of di-4-ANEPPS behavior in biological membranes and requires a significant revision. The findings in question reported by the authors, in my opinion, might be interpreted as evidence for the presence on the plasma membrane of growing pollen tube not only the membrane potential Delta Psi but also lateral gradient of so called intra-membrane dipole potential. Based on the comments made, another interpretation of the experimental results described by Breigina et al. has been offered. In addition, some drawbacks in the methodology used by the authors for measurement of Delta Psi with other fluorescent potential-sensitive dye, DiBAC3(3), are also shortly considered.

Alteration of plasma membrane-bound redox systems of iron deficient pea roots by chitosan.

PubMed

Meisrimler, Claudia-Nicole; Planchon, Sebastien; Renaut, Jenny; Sergeant, Kjell; Lüthje, Sabine

2011-08-12

Iron is essential for all living organisms and plays a crucial role in pathogenicity. This study presents the first proteome analysis of plasma membranes isolated from pea roots. Protein profiles of four different samples (+Fe, +Fe/Chitosan, -Fe, and -Fe/Chitosan) were compared by native IEF-PAGE combined with in-gel activity stains and DIGE. Using DIGE, 89 proteins of interest were detected in plasma membrane fractions. Data revealed a differential abundance of several spots in all samples investigated. In comparison to the control and -FeCh the abundance of six protein spots increased whereas 56 spots decreased in +FeCh. Altered protein spots were analyzed by MALDI-TOF-TOF mass spectrometry. Besides stress-related proteins, transport proteins and redox enzymes were identified. Activity stains after native PAGE and spectrophotometric measurements demonstrated induction of a ferric-chelate reductase (-Fe) and a putative respiratory burst oxidase homolog (-FeCh). However, the activity of the ferric-chelate reductase decreased in -Fe plants after elicitor treatment. The activity of plasma membrane-bound class III peroxidases increased after elicitor treatment and decreased under iron-deficiency, whereas activity of quinone reductases decreased mostly after elicitor treatment. Possible functions of proteins identified and reasons for a weakened pathogen response of iron-deficient plants were discussed. Copyright © 2011 Elsevier B.V. All rights reserved.

The Plasma Membrane is Compartmentalized by a Self-Similar Cortical Actin Fractal

NASA Astrophysics Data System (ADS)

Sadegh, Sanaz; Higgin, Jenny; Mannion, Patrick; Tamkun, Michael; Krapf, Diego

A broad range of membrane proteins display anomalous diffusion on the cell surface. Different methods provide evidence for obstructed subdiffusion and diffusion on a fractal space, but the underlying structure inducing anomalous diffusion has never been visualized due to experimental challenges. We addressed this problem by imaging the cortical actin at high resolution while simultaneously tracking individual membrane proteins in live mammalian cells. Our data show that actin introduces barriers leading to compartmentalization of the plasma membrane and that membrane proteins are transiently confined within actin fences. Furthermore, superresolution imaging shows that the cortical actin is organized into a self-similar fractal. These results present a hierarchical nanoscale picture of the plasma membrane and demonstrate direct interactions between the actin cortex and the cell surface.

Isolation of plasma membrane fractions from the intestinal epithelial model T84.

PubMed

Kaoutzani, P; Parkos, C A; Delp-Archer, C; Madara, J L

1993-05-01

The human intestinal epithelial cell line T84 is widely used as a model for studies of Cl- secretion and crypt cell biology. We report a fractionation approach that permits separation of purified apical and basolateral T84 plasma membrane domains. T84 cellular membranes were isolated by nitrogen cavitation and differential centrifugation from monolayers grown on permeable supports. Membranes were then fractionated by isopycnic sucrose density gradient sedimentation, and fractions were assessed, using enzymatic and Western blot techniques, for apical (alkaline phosphatase) and basolateral (Na(+)-K(+)-ATPase) plasma membrane markers and for cytosolic, lysosomal, Golgi, and mitochondrial markers. Buffer conditions were defined that permitted separation of enriched apical and basolateral markers. The validity of the selected markers for the apical and basolateral domains was verified by selective apical and basolateral surface labeling studies using trace iodinated wheat germ agglutinin or biotinylation. This approach allows for separation of apical and basolateral plasma membranes of T84 cells for biochemical analyses and should thus be of broad utility in studies of this model polarized and transporting epithelium.

Toxin Pores Endocytosed During Plasma Membrane Repair Traffic into the Lumen of MVBs for Degradation

PubMed Central

Corrotte, Matthias; Fernandes, Maria Cecilia; Tam, Christina; Andrews, Norma W.

2012-01-01

Cells permeabilized by the bacterial pore-forming toxin streptolysin O (SLO) reseal their plasma membrane in a Ca2+-dependent manner. Resealing involves Ca2+-dependent exocytosis of lysosomes, release of acid sphingomyelinase and rapid formation of endosomes that carry the transmembrane pores into the cell. The intracellular fate of the toxin-carrying endocytic vesicles, however, is still unknown. Here, we show that SLO pores removed from the plasma membrane by endocytosis are sorted into the lumen of lysosomes, where they are degraded. SLO-permeabilized cells contain elevated numbers of total endosomes, which increase gradually in size while transitioning from endosomes with flat clathrin coats to large multivesicular bodies (MVBs). Under conditions that allow endocytosis and plasma membrane repair, SLO is rapidly ubiquitinated and gradually degraded, in a process sensitive to inhibitors of lysosomal hydrolysis but not of proteasomes. The endosomes induced by SLO permeabilization become increasingly acidified and promote SLO degradation under normal conditions, but not in cells silenced for expression of Vps24, an ESCRT-III complex component required for the release of intraluminal vesicles into MVBs. Thus, cells dispose of SLO transmembrane pores by ubiquitination/ESCRT-dependent sorting into the lumen of late endosomes/lysosomes. PMID:22212686

Involvement of plasma membrane-located calmodulin in the response decay of cyclic nucleotide-gated cation channel of cultured carrot cells.

PubMed

Kurosaki, F; Kaburaki, H; Nishi, A

1994-03-07

Increase in cytoplasmic cyclic AMP concentration stimulates Ca2+ influx through the cyclic AMP-gated cation channel in the plasma membrane of cultured carrot cells. However, the Ca2+ current terminated after a few minutes even in the presence of high concentrations of cyclic AMP indicating that hydrolysis of the nucleotide is not responsible for stop of the Ca2+ influx. Cyclic AMP evoked discharge of Ca2+ from inside-out sealed vesicles of carrot plasma membrane, and it was strongly inhibited when the suspension of the vesicles was supplemented with 1 microM of free Ca2+, while Ca2+ lower than 0.1 microM did not affect the Ca(2+)-release. The Ca2+ flux across plasma membrane was restored from this Ca(2+)-induced inhibition by the addition of calmodulin inhibitors or anti-calmodulin. These results suggest that Ca2+ influx initiated by the increase in intracellular cAMP in cultured carrot cells is terminated when the cytosolic Ca2+ concentration reaches the excitatory level in the cells, and calmodulin located in the plasma membrane plays an important role in the response decay of the cyclic nucleotide-gated Ca2+ channel.

Organization of Lipids in Fiber-Cell Plasma Membranes of the Eye Lens

PubMed Central

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

2016-01-01

The plasma membrane together with the cytoskeleton forms the only supramolecular structure of the matured fiber cell which accounts for mostly all fiber cell lipids. The purpose of this review is to inform researchers about the importance of the lipid bilayer portion of the lens fiber cell plasma membranes in the maintaining lens homeostasis, and thus protecting against cataract development. PMID:26988627

Protein kinase Cδ differentially regulates cAMP-dependent translocation of NTCP and MRP2 to the plasma membrane

PubMed Central

Park, Se Won; Schonhoff, Christopher M.; Webster, Cynthia R. L.

2012-01-01

Cyclic AMP stimulates translocation of Na+/taurocholate cotransporting polypeptide (NTCP) from the cytosol to the sinusoidal membrane and multidrug resistance-associated protein 2 (MRP2) to the canalicular membrane. A recent study suggested that protein kinase Cδ (PKCδ) may mediate cAMP-induced translocation of Ntcp and Mrp2. In addition, cAMP has been shown to stimulate NTCP translocation in part via Rab4. The aim of this study was to determine whether cAMP-induced translocation of NTCP and MRP2 require kinase activity of PKCδ and to test the hypothesis that cAMP-induced activation of Rab4 is mediated via PKCδ. Studies were conducted in HuH-NTCP cells (HuH-7 cells stably transfected with NTCP). Transfection of cells with wild-type PKCδ increased plasma membrane PKCδ and NTCP and increased Rab4 activity. Paradoxically, overexpression of kinase-dead dominant-negative PKCδ also increased plasma membrane PKCδ and NTCP as well as Rab4 activity. Similar results were obtained in PKCδ knockdown experiments, despite a decrease in total PKCδ. These results raised the possibility that plasma membrane localization rather than kinase activity of PKCδ is necessary for NTCP translocation and Rab4 activity. This hypothesis was supported by results showing that rottlerin, which has previously been shown to inhibit cAMP-induced membrane translocation of PKCδ and NTCP, inhibited cAMP-induced Rab4 activity. In addition, LY294002 (a phosphoinositide-3-kinase inhibitor), which has been shown to inhibit cAMP-induced NTCP translocation, also inhibited cAMP-induced PKCδ translocation. In contrast to the results with NTCP, cAMP-induced MRP2 translocation was inhibited in cells transfected with DN-PKCδ and small interfering RNA PKCδ. Taken together, these results suggest that the plasma membrane localization rather than kinase activity of PKCδ plays an important role in cAMP-induced NTCP translocation and Rab4 activity, whereas the kinase activity of PKCδ is necessary for c

Protein kinase Cδ differentially regulates cAMP-dependent translocation of NTCP and MRP2 to the plasma membrane.

PubMed

Park, Se Won; Schonhoff, Christopher M; Webster, Cynthia R L; Anwer, M Sawkat

2012-09-01

Cyclic AMP stimulates translocation of Na(+)/taurocholate cotransporting polypeptide (NTCP) from the cytosol to the sinusoidal membrane and multidrug resistance-associated protein 2 (MRP2) to the canalicular membrane. A recent study suggested that protein kinase Cδ (PKCδ) may mediate cAMP-induced translocation of Ntcp and Mrp2. In addition, cAMP has been shown to stimulate NTCP translocation in part via Rab4. The aim of this study was to determine whether cAMP-induced translocation of NTCP and MRP2 require kinase activity of PKCδ and to test the hypothesis that cAMP-induced activation of Rab4 is mediated via PKCδ. Studies were conducted in HuH-NTCP cells (HuH-7 cells stably transfected with NTCP). Transfection of cells with wild-type PKCδ increased plasma membrane PKCδ and NTCP and increased Rab4 activity. Paradoxically, overexpression of kinase-dead dominant-negative PKCδ also increased plasma membrane PKCδ and NTCP as well as Rab4 activity. Similar results were obtained in PKCδ knockdown experiments, despite a decrease in total PKCδ. These results raised the possibility that plasma membrane localization rather than kinase activity of PKCδ is necessary for NTCP translocation and Rab4 activity. This hypothesis was supported by results showing that rottlerin, which has previously been shown to inhibit cAMP-induced membrane translocation of PKCδ and NTCP, inhibited cAMP-induced Rab4 activity. In addition, LY294002 (a phosphoinositide-3-kinase inhibitor), which has been shown to inhibit cAMP-induced NTCP translocation, also inhibited cAMP-induced PKCδ translocation. In contrast to the results with NTCP, cAMP-induced MRP2 translocation was inhibited in cells transfected with DN-PKCδ and small interfering RNA PKCδ. Taken together, these results suggest that the plasma membrane localization rather than kinase activity of PKCδ plays an important role in cAMP-induced NTCP translocation and Rab4 activity, whereas the kinase activity of PKCδ is necessary for

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

PubMed Central

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

2015-01-01

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

Poly(hydroxyethyl methacrylate) based affinity membranes for in vitro removal of anti-dsDNA antibodies from SLE plasma.

PubMed

Uzun, Lokman; Yavuz, Handan; Osman, Bilgen; Celik, Hamdi; Denizli, Adil

2010-07-01

The preparation of polymeric membrane using affinity technology for application in blood filtration devices is described here. DNA attached poly(hydroxyethyl methacrylate) (PHEMA) based microporous affinity membrane was prepared for selective removal of anti-dsDNA antibodies from systemic lupus erythematosus (SLE) patient plasma in in vitro. In order to further increase blood-compatibility of affinity membrane, aminoacid based comonomer N-methacryloyl-L-alanine (MAAL) was included in the polymerization recipe. PHEMAAL membrane was produced by a photopolymerization technique and then characterized by swelling tests and scanning electron microscope (SEM) studies. Blood-compatibility tests were also performed. The water swelling ratio of PHEMAAL membrane increased significantly (133.2%) compared with PHEMA (58%). PHEMAAL membrane has large pores around in the range of 5-10 microm. All the clotting times increased when compared with PHEMA membrane. Loss of platelets and leukocytes was very low. DNA loading was 7.8 mg/g. There was a very low anti-dsDNA-antibody adsorption onto the plain PHEMAAL membrane, about 78 IU/g. The PHEMAAL-DNA membrane adsorbed anti-dsDNA-antibody in the range of 10-68 x 10(3)IU/g from SLE plasma. Anti-dsDNA-antibody concentration decreased significantly from 875 to 144 IU/ml with the time. Anti-dsDNA-antibodies could be repeatedly adsorbed and eluted without noticeable loss in the anti-dsDNA-antibody adsorption amount. (c) 2010 Elsevier B.V. All rights reserved.

Mechanism and structure of the plant plasma membrane Ca{sup 2+}-ATPase

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

Briskin, D.P.

1993-12-31

Objectives of this project were the following: development of an enriched preparation of the red beet plasma membrane Ca{sup 2+} ATPase in order to develop a procedure for detergent solubilization of the enzyme from the membrane using detergents, resolution by a method which could be upscaled for batch isolation, and then reconstitution into liposomes to allow characterization of Ca{sup 2+} transport by the purified enzyme and; characterization of the reaction mechanism for the coupling of nucleoside triphosphate hydrolysis to Ca{sup 2+} transport as mediated by the plasma membrane Ca{sup 2+} ATPase.

Structural changes in plasma membranes prepared from irradiated Chinese hamster V79 cells as revealed by Raman spectroscopy

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

Verma, S.P.; Sonwalkar, N.

1991-04-01

The effect of gamma irradiation on the integrity of plasma membranes isolated from Chinese hamster V79 cells was investigated by Raman spectroscopy. Plasma membranes of control V79 cells show transitions between {minus}10 and 5{degree}C (low-temperature transition), 10 and 22{degree}C (middle-temperature transition), and 32 and 40{degree}C (high-temperature transition). Irradiation (5 Gy) alters these transitions markedly. First, the low-temperature transition shifts to higher temperature (onset and completion temperatures 4 and 14{degree}C). Second, the middle-temperature transition shifts up to the range of about 20-32{degree}C, but the width remains unchanged. Third, the higher temperature transition broadens markedly and shifts to the range of aboutmore » 15-40{degree}C. Protein secondary structure as determined by least-squares analysis of the amide I bands shows 36% total helix, 55% total beta-strand, and 9% turn plus undefined for control plasma membrane proteins. Plasma membrane proteins of irradiated V79 cells show an increase in total helix (40 and 45% at 5 and 10 Gy, respectively) and a decrease in the total beta-strand (48 and 44% at 5 and 10 Gy, respectively) structures. The qualitative analysis of the Raman features of plasma membranes and model compounds in the 1600 cm-1 region, assigned to tyrosine groups, revealed that irradiation alters the microenvironment of these groups. We conclude that the radiation dose used in the survival range of Chinese hamster V79 cells can cause damage to plasma membrane proteins without detectable lipid peroxidation, and that the altered proteins react differently with lipids, yielding a shift in the thermal transition properties.« less

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

PubMed Central

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

2016-01-01

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

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

PubMed

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

2016-12-06

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

Nonthermal dielectric-barrier discharge plasma-induced inactivation involves oxidative DNA damage and membrane lipid peroxidation in Escherichia coli.

PubMed

Joshi, Suresh G; Cooper, Moogega; Yost, Adam; Paff, Michelle; Ercan, Utku K; Fridman, Gregory; Friedman, Gary; Fridman, Alexander; Brooks, Ari D

2011-03-01

Oxidative stress leads to membrane lipid peroxidation, which yields products causing variable degrees of detrimental oxidative modifications in cells. Reactive oxygen species (ROS) are the key regulators in this process and induce lipid peroxidation in Escherichia coli. Application of nonthermal (cold) plasma is increasingly used for inactivation of surface contaminants. Recently, we reported a successful application of nonthermal plasma, using a floating-electrode dielectric-barrier discharge (FE-DBD) technique for rapid inactivation of bacterial contaminants in normal atmospheric air (S. G. Joshi et al., Am. J. Infect. Control 38:293-301, 2010). In the present report, we demonstrate that FE-DBD plasma-mediated inactivation involves membrane lipid peroxidation in E. coli. Dose-dependent ROS, such as singlet oxygen and hydrogen peroxide-like species generated during plasma-induced oxidative stress, were responsible for membrane lipid peroxidation, and ROS scavengers, such as α-tocopherol (vitamin E), were able to significantly inhibit the extent of lipid peroxidation and oxidative DNA damage. These findings indicate that this is a major mechanism involved in FE-DBD plasma-mediated inactivation of bacteria.

Nonthermal Dielectric-Barrier Discharge Plasma-Induced Inactivation Involves Oxidative DNA Damage and Membrane Lipid Peroxidation in Escherichia coli▿

PubMed Central

Joshi, Suresh G.; Cooper, Moogega; Yost, Adam; Paff, Michelle; Ercan, Utku K.; Fridman, Gregory; Friedman, Gary; Fridman, Alexander; Brooks, Ari D.

2011-01-01

Oxidative stress leads to membrane lipid peroxidation, which yields products causing variable degrees of detrimental oxidative modifications in cells. Reactive oxygen species (ROS) are the key regulators in this process and induce lipid peroxidation in Escherichia coli. Application of nonthermal (cold) plasma is increasingly used for inactivation of surface contaminants. Recently, we reported a successful application of nonthermal plasma, using a floating-electrode dielectric-barrier discharge (FE-DBD) technique for rapid inactivation of bacterial contaminants in normal atmospheric air (S. G. Joshi et al., Am. J. Infect. Control 38:293-301, 2010). In the present report, we demonstrate that FE-DBD plasma-mediated inactivation involves membrane lipid peroxidation in E. coli. Dose-dependent ROS, such as singlet oxygen and hydrogen peroxide-like species generated during plasma-induced oxidative stress, were responsible for membrane lipid peroxidation, and ROS scavengers, such as α-tocopherol (vitamin E), were able to significantly inhibit the extent of lipid peroxidation and oxidative DNA damage. These findings indicate that this is a major mechanism involved in FE-DBD plasma-mediated inactivation of bacteria. PMID:21199923

Asymmetry of plasma membrane lipid order in Madin-Darby Canine Kidney cells.

PubMed

Le Grimellec, C; Friedlander, G; Giocondi, M C

1988-07-01

Fluorescence anisotropy experiments have been done to estimate, in situ, the lipid order of the plasma membrane of polarized Madin-Darby Canine Kidney cells (MDCK) grown on glass cover slips and labeled by 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), a specific marker of the plasma membrane of living cells. Fluorescence microscopy, back-exchange, and quenching experiments indicated that TMA-DPH labeled the highly ordered (r greater than or equal to 0.32, 37 degrees C) apical domain of the plasma membrane of confluent monolayers. Opening of tight junctions or addition of the probe to cell suspensions resulted in a homogeneous distribution of TMA-DPH over the cell surface and in a marked decrease in anisotropy (0.27 less than or equal to r less than or equal to 0.29) that was due neither to a direct effect of Ca2+ on the probe nor to a change in fluorescence lifetime. Our data indicate that the apical domain, likely the external leaflet, of the plasma membrane of polarized MDCK cells is much more ordered than its basolateral counterpart.

Protein phosphorylation in human peripheral blood lymphocytes. Phosphorylation of endogenous plasma membrane and cytoplasmic proteins

PubMed Central

Chaplin, David D.; Wedner, H. James; Parker, Charles W.

1979-01-01

Phosphorylation of endogenous proteins in subcellular fractions of human peripheral-blood lymphocytes was studied by one- and two-dimensional polyacrylamide-gel electrophoresis. Studies using extensively purified subcellular fractions indicated that the endogenous phosphorylating activity in the particulate fractions was derived primarily from the plasma membrane. Electrophoresis of 32P-labelled subcellular fractions in two dimensions [O'Farrell (1975) J. Biol. Chem. 250, 4007–4021] provided much greater resolution of the endogenous phosphoproteins than electrophoresis in one dimension, facilitating their excision from gels for quantification of 32P content. More than 100 cytoplasmic and 20 plasma-membrane phosphorylated species were observed. Phosphorylation of more than 10 cytoplasmic proteins was absolutely dependent on cyclic AMP. In the plasma membrane, cyclic AMP-dependent phosphoproteins were observed with mol.wts. of 42000, 42000, 80000 and 90000 and pI values of 6.1, 6.3, 6.25 and 6.5 respectively. Phosphorylation of endogenous cytoplasmic and plasma-membrane proteins was rapid with t½=5–12s at 25°C. Between 40 and 70% of the 32P was recovered as phosphoserine and phosphothreonine when acid hydrolysates of isolated plasma-membrane phosphoproteins were analysed by high-voltage paper electrophoresis. The presence of cyclic AMP-dependent protein kinase and endogenous phosphate-acceptor proteins in the plasma membranes of lymphocytes provides a mechanism by which these cells might respond to plasma-membrane pools of cyclic AMP generated in response to stimulation by mitogens or physiological modulators of lymphocyte function. ImagesFig. 1.Fig. 2.Fig. 3.Fig. 4. PMID:228657

Plasma membrane lipid–protein interactions affect signaling processes in sterol-biosynthesis mutants in Arabidopsis thaliana

PubMed Central

Zauber, Henrik; Burgos, Asdrubal; Garapati, Prashanth; Schulze, Waltraud X.

2014-01-01

The plasma membrane is an important organelle providing structure, signaling and transport as major biological functions. Being composed of lipids and proteins with different physicochemical properties, the biological functions of membranes depend on specific protein–protein and protein–lipid interactions. Interactions of proteins with their specific sterol and lipid environment were shown to be important factors for protein recruitment into sub-compartmental structures of the plasma membrane. System-wide implications of altered endogenous sterol levels for membrane functions in living cells were not studied in higher plant cells. In particular, little is known how alterations in membrane sterol composition affect protein and lipid organization and interaction within membranes. Here, we conducted a comparative analysis of the plasma membrane protein and lipid composition in Arabidopsis sterol-biosynthesis mutants smt1 and ugt80A2;B1. smt1 shows general alterations in sterol composition while ugt80A2;B1 is significantly impaired in sterol glycosylation. By systematically analyzing different cellular fractions and combining proteomic with lipidomic data we were able to reveal contrasting alterations in lipid–protein interactions in both mutants, with resulting differential changes in plasma membrane signaling status. PMID:24672530

Proteomic analysis of plasma membranes isolated from undifferentiated and differentiated HepaRG cells

PubMed Central

2012-01-01

Liver infection with hepatitis B virus (HBV), a DNA virus of the Hepadnaviridae family, leads to severe disease, such as fibrosis, cirrhosis and hepatocellular carcinoma. The early steps of the viral life cycle are largely obscure and the host cell plasma membrane receptors are not known. HepaRG is the only proliferating cell line supporting HBV infection in vitro, following specific differentiation, allowing for investigation of new host host-cell factors involved in viral entry, within a more robust and reproducible environment. Viral infection generally begins with receptor recognition at the host cell surface, following highly specific cell-virus interactions. Most of these interactions are expected to take place at the plasma membrane of the HepaRG cells. In the present study, we used this cell line to explore changes between the plasma membrane of undifferentiated (−) and differentiated (+) cells and to identify differentially-regulated proteins or signaling networks that might potentially be involved in HBV entry. Our initial study identified a series of proteins that are differentially expressed in the plasma membrane of (−) and (+) cells and are good candidates for potential cell-virus interactions. To our knowledge, this is the first study using functional proteomics to study plasma membrane proteins from HepaRG cells, providing a platform for future experiments that will allow us to understand the cell-virus interaction and mechanism of HBV viral infection. PMID:22857383

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

Membrane proteins involved in transport, vesicle traffic and Ca(2+) signaling increase in beetroots grown in saline soils.

PubMed

Lino, Bárbara; Chagolla, Alicia; E González de la Vara, Luis

2016-07-01

By separating plasma membrane proteins according to their hydropathy from beetroots grown in saline soils, several proteins probably involved in salt tolerance were identified by mass spectrometry. Beetroots, as a salt-tolerant crop, have developed mechanisms to cope with stresses associated with saline soils. To observe which plasma membrane (PM) proteins were more abundant in beet roots grown in saline soils, beet root plants were irrigated with water or 0.2 M NaCl. PM-enriched membrane preparations were obtained from these plants, and their proteins were separated according to their hydropathy by serial phase partitioning with Triton X-114. Some proteins whose abundance increased visibly in membranes from salt-grown beetroots were identified by mass spectrometry. Among them, there was a V-type H(+)-ATPase (probably from contaminating vacuolar membranes), which increased with salt at all stages of beetroots' development. Proteins involved in solute transport (an H(+)-transporting PPase and annexins), vesicle traffic (clathrin and synaptotagmins), signal perception and transduction (protein kinases and phospholipases, mostly involved in calcium signaling) and metabolism, appeared to increase in salt-grown beetroot PM-enriched membranes. These results suggest that PM and vacuolar proteins involved in transport, metabolism and signal transduction increase in beet roots adapted to saline soils. In addition, these results show that serial phase partitioning with Triton X-114 is a useful method to separate membrane proteins for their identification by mass spectrometry.

Atomic force microscopy on plasma membranes from Xenopus laevis oocytes containing human aquaporin 4.

PubMed

Orsini, Francesco; Santacroce, Massimo; Cremona, Andrea; Gosvami, Nitya N; Lascialfari, Alessandro; Hoogenboom, Bart W

2014-11-01

Atomic force microscopy (AFM) is a unique tool for imaging membrane proteins in near-native environment (embedded in a membrane and in buffer solution) at ~1 nm spatial resolution. It has been most successful on membrane proteins reconstituted in 2D crystals and on some specialized and densely packed native membranes. Here, we report on AFM imaging of purified plasma membranes from Xenopus laevis oocytes, a commonly used system for the heterologous expression of membrane proteins. Isoform M23 of human aquaporin 4 (AQP4-M23) was expressed in the X. laevis oocytes following their injection with AQP4-M23 cRNA. AQP4-M23 expression and incorporation in the plasma membrane were confirmed by the changes in oocyte volume in response to applied osmotic gradients. Oocyte plasma membranes were then purified by ultracentrifugation on a discontinuous sucrose gradient, and the presence of AQP4-M23 proteins in the purified membranes was established by Western blotting analysis. Compared with membranes without over-expressed AQP4-M23, the membranes from AQP4-M23 cRNA injected oocytes showed clusters of structures with lateral size of about 10 nm in the AFM topography images, with a tendency to a fourfold symmetry as may be expected for higher-order arrays of AQP4-M23. In addition, but only infrequently, AQP4-M23 tetramers could be resolved in 2D arrays on top of the plasma membrane, in good quantitative agreement with transmission electron microscopy analysis and the current model of AQP4. Our results show the potential and the difficulties of AFM studies on cloned membrane proteins in native eukaryotic membranes. Copyright © 2014 John Wiley & Sons, Ltd.

The lipid composition modulates the influence of the bovine seminal plasma protein PDC-109 on membrane stability.

PubMed

Tannert, Astrid; Töpfer-Petersen, Edda; Herrmann, Andreas; Müller, Karin; Müller, Peter

2007-10-16

The bovine seminal plasma protein PDC-109 exerts an essential influence on the sperm cell plasma membrane during capacitation. However, by any mechanism, it has to be ensured that this function of the protein on sperm cells is not initiated too early, that is, upon ejaculation when PDC-109 and sperm cells come into first contact, but rather at later stages of sperm genesis in the female genital tract. To answer the question of whether changes of the bovine sperm lipid composition can modulate the effect of PDC-109 on sperm membranes, we have investigated the influence of PDC-109 on the integrity of (i) differently composed lipid vesicles and of (ii) membranes from human red blood cells and bovine spermatozoa. PDC-109 most effectively disturbed lipid membranes composed of choline-containing phospholipids and in the absence of cholesterol. The impact of the protein on lipid vesicles was attenuated in the presence of cholesterol or of noncholine-containing phospholipids, such as phosphatidylethanolamine or phosphatidylserine. An extraction of cholesterol from lipid or biological membranes using methyl-beta-cyclodextrin caused an increased membrane perturbation by PDC-109. Our results argue for a oppositional effect of PDC-109 during sperm cell genesis. We hypothesize that the lipid composition of ejaculated bull sperm cells allows a binding of PDC-109 without leading to an impairment of the plasma membrane. At later stages of sperm cell genesis upon release of cholesterol from sperm membranes, PDC-109 triggers a destabilization of the cells.

Neurospora crassa transcriptomics reveals oxidative stress and plasma membrane homeostasis biology genes as key targets in response to chitosan

PubMed Central

Lopez-Moya, Federico; Kowbel, David; Nueda, Ma José; Palma-Guerrero, Javier; Glass, N. Louise; Lopez-Llorca, Luis Vicente

2016-01-01

Chitosan is a natural polymer with antimicrobial activity. Chitosan causes plasma membrane permeabilization and induction of intracellular reactive oxygen species (ROS) in Neurospora crassa. We have determined the transcriptional profile of N. crassa to chitosan and identified the main gene targets involved in the cellular response to this compound. Global network analyses showed membrane, transport and oxidoreductase activity as key nodes affected by chitosan. Activation of oxidative metabolism indicates the importance of ROS and cell energy together with plasma membrane homeostasis in N. crassa response to chitosan. Deletion strain analysis of chitosan susceptibility pointed, NCU03639 encoding a class 3 lipase, involved in plasma membrane repair by lipid replacement and NCU04537 a MFS monosaccharide transporter related with assimilation of simple sugars, as main gene targets of chitosan. NCU10521, a glutathione S-transferase-4 involved in the generation of reducing power for scavenging intracellular ROS is also a determinant chitosan gene target. Ca2+ increased tolerance to chitosan in N. crassa. Growth of NCU10610 (fig 1 domain) and SYT1 (a synaptotagmin) deletion strains was significantly increased by Ca2+ in presence of chitosan. Both genes play a determinant role in N. crassa membrane homeostasis. Our results are of paramount importance for developing chitosan as antifungal. PMID:26694141

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

PubMed

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

2014-03-01

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

Lipid-Protein Interactions in Plasma Membranes of Fiber Cells Isolated from the Human Eye Lens

PubMed Central

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

2014-01-01

The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali,L., Raguz, M., O’Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed. PMID:24486794

Interactions of sugar-based bolaamphiphiles with biomimetic systems of plasma membranes.

PubMed

Nasir, Mehmet Nail; Crowet, Jean-Marc; Lins, Laurence; Obounou Akong, Firmin; Haudrechy, Arnaud; Bouquillon, Sandrine; Deleu, Magali

2016-11-01

Glycolipids constitute a class of molecules with various biological activities. Among them, sugar-based bolaamphiphiles characterized by their biocompatibility, biodegradability and lower toxicity, became interesting for the development of efficient and low cost lipid-based drug delivery systems. Their activity seems to be closely related to their interactions with the lipid components of the plasma membrane of target cells. Despite many works devoted to the chemical synthesis and characterization of sugar-based bolaamphiphiles, their interactions with plasma membrane have not been completely elucidated. In this work, two sugar-based bolaamphiphiles differing only at the level of their sugar residues were chemically synthetized. Their interactions with membranes have been investigated using model membranes containing or not sterol and with in silico approaches. Our findings indicate that the nature of sugar residues has no significant influence for their membrane interacting properties, while the presence of sterol attenuates the interactions of both bolaamphiphiles with the membrane systems. The understanding of this distinct behavior of bolaamphiphiles towards sterol-containing membrane systems could be useful for their applications as drug delivery systems. Copyright © 2016. Published by Elsevier B.V.

RAB-5- and RAB-11-dependent vesicle-trafficking pathways are required for plasma membrane repair after attack by bacterial pore-forming toxin.

PubMed

Los, Ferdinand C O; Kao, Cheng-Yuan; Smitham, Jane; McDonald, Kent L; Ha, Christine; Peixoto, Christina A; Aroian, Raffi V

2011-02-17

Pore-forming toxins (PFTs) secreted by pathogenic bacteria are the most common bacterial protein toxins and are important virulence factors for infection. PFTs punch holes in host cell plasma membranes, and although cells can counteract the resulting membrane damage, the underlying mechanisms at play remain unclear. Using Caenorhabditis elegans as a model, we demonstrate in vivo and in an intact epithelium that intestinal cells respond to PFTs by increasing levels of endocytosis, dependent upon RAB-5 and RAB-11, which are master regulators of endocytic and exocytic events. Furthermore, we find that RAB-5 and RAB-11 are required for protection against PFT and to restore integrity to the plasma membrane. One physical mechanism involved is the RAB-11-dependent expulsion of microvilli from the apical side of the intestinal epithelial cells. Specific vesicle-trafficking pathways thus protect cells against an attack by PFTs on plasma membrane integrity, via altered plasma membrane dynamics. Copyright © 2011 Elsevier Inc. All rights reserved.

Magnetic apatite for structural insights on the plasma membrane

NASA Astrophysics Data System (ADS)

Stanca, Sarmiza E.; Müller, Robert; Dellith, Jan; Nietzsche, Sandor; Stöckel, Stephan; Biskup, Christoph; Deckert, Volker; Krafft, Christoph; Popp, Jürgen; Fritzsche, Wolfgang

2015-01-01

The iron oxide-hydroxyapatite (FeOxHA) nanoparticles reported here differ from those reported before by their advantage of homogeneity and simple preparation; moreover, the presence of carboxymethyldextran (CMD), together with hydroxyapatite (HA), allows access to the cellular membrane, which makes our magnetic apatite unique. These nanoparticles combine magnetic behavior, Raman label ability and the property of interaction with the cellular membrane; they therefore represent an interesting material for structural differentiation of the cell membrane. It was observed by Raman spectroscopy, scanning electron microscopy (SEM) and fluorescence microscopy that FeOxHA adheres to the plasma membrane and does not penetrate the membrane. These insights make the nanoparticles a promising material for magnetic cell sorting, e.g. in microfluidic device applications.

Magnetic apatite for structural insights on the plasma membrane.

PubMed

Stanca, Sarmiza E; Müller, Robert; Dellith, Jan; Nietzsche, Sandor; Stöckel, Stephan; Biskup, Christoph; Deckert, Volker; Krafft, Christoph; Popp, Jürgen; Fritzsche, Wolfgang

2015-01-21

The iron oxide-hydroxyapatite (FeOxHA) nanoparticles reported here differ from those reported before by their advantage of homogeneity and simple preparation; moreover, the presence of carboxymethyldextran (CMD), together with hydroxyapatite (HA), allows access to the cellular membrane, which makes our magnetic apatite unique. These nanoparticles combine magnetic behavior, Raman label ability and the property of interaction with the cellular membrane; they therefore represent an interesting material for structural differentiation of the cell membrane. It was observed by Raman spectroscopy, scanning electron microscopy (SEM) and fluorescence microscopy that FeOxHA adheres to the plasma membrane and does not penetrate the membrane. These insights make the nanoparticles a promising material for magnetic cell sorting, e.g. in microfluidic device applications.

The molecular mechanisms of plant plasma membrane intrinsic proteins trafficking and stress response.

PubMed

Wang, Xing; Zhang, Ji-long; Feng, Xiu-xiu; Li, Hong-jie; Zhang, Gen-fa

2017-04-20

Plasma membrane intrinsic proteins (PIPs) are plant channel proteins located on the plasma membrane. PIPs transfer water, CO 2 and small uncharged solutes through the plasma membrane. PIPs have high selectivity to substrates, suggestive of a central role in maintaining cellular water balance. The expression, activity and localization of PIPs are regulated at the transcriptional and post-translational levels, and also affected by environmental factors. Numerous studies indicate that the expression patterns and localizations of PIPs can change in response to abiotic stresses. In this review, we summarize the mechanisms of PIP trafficking, transcriptional and post-translational regulations, and abiotic stress responses. Moreover, we also discuss the current research trends and future directions on PIPs.

Membrane raft association is a determinant of plasma membrane localization.

PubMed

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

2014-06-10

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

Organization of lipids in fiber-cell plasma membranes of the eye lens.

PubMed

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

2017-03-01

The plasma membrane together with the cytoskeleton forms the only supramolecular structure of the matured fiber cell which accounts for mostly all fiber cell lipids. The purpose of this review is to inform researchers about the importance of the lipid bilayer portion of the lens fiber cell plasma membranes in the maintaining lens homeostasis, and thus protecting against cataract development. Copyright © 2016 Elsevier Ltd. All rights reserved.

Photosynthesis Activates Plasma Membrane H+-ATPase via Sugar Accumulation.

PubMed

Okumura, Masaki; Inoue, Shin-Ichiro; Kuwata, Keiko; Kinoshita, Toshinori

2016-05-01

Plant plasma membrane H(+)-ATPase acts as a primary transporter via proton pumping and regulates diverse physiological responses by controlling secondary solute transport, pH homeostasis, and membrane potential. Phosphorylation of the penultimate threonine and the subsequent binding of 14-3-3 proteins in the carboxyl terminus of the enzyme are required for H(+)-ATPase activation. We showed previously that photosynthesis induces phosphorylation of the penultimate threonine in the nonvascular bryophyte Marchantia polymorpha However, (1) whether this response is conserved in vascular plants and (2) the process by which photosynthesis regulates H(+)-ATPase phosphorylation at the plasma membrane remain unresolved issues. Here, we report that photosynthesis induced the phosphorylation and activation of H(+)-ATPase in Arabidopsis (Arabidopsis thaliana) leaves via sugar accumulation. Light reversibly phosphorylated leaf H(+)-ATPase, and this process was inhibited by pharmacological and genetic suppression of photosynthesis. Immunohistochemical and biochemical analyses indicated that light-induced phosphorylation of H(+)-ATPase occurred autonomously in mesophyll cells. We also show that the phosphorylation status of H(+)-ATPase and photosynthetic sugar accumulation in leaves were positively correlated and that sugar treatment promoted phosphorylation. Furthermore, light-induced phosphorylation of H(+)-ATPase was strongly suppressed in a double mutant defective in ADP-glucose pyrophosphorylase and triose phosphate/phosphate translocator (adg1-1 tpt-2); these mutations strongly inhibited endogenous sugar accumulation. Overall, we show that photosynthesis activated H(+)-ATPase via sugar production in the mesophyll cells of vascular plants. Our work provides new insight into signaling from chloroplasts to the plasma membrane ion transport mechanism. © 2016 American Society of Plant Biologists. All Rights Reserved.

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 Road not Taken: Less Traveled Roads from the TGN to the Plasma Membrane

PubMed Central

Spang, Anne

2015-01-01

The trans-Golgi network functions in the distribution of cargo into different transport vesicles that are destined to endosomes, lysosomes and the plasma membrane. Over the years, it has become clear that more than one transport pathway promotes plasma membrane localization of proteins. In spite of the importance of temporal and spatial control of protein localization at the plasma membrane, the regulation of sorting into and the formation of different transport containers are still poorly understood. In this review different transport pathways, with a special emphasis on exomer-dependent transport, and concepts of regulation and sorting at the TGN are discussed. PMID:25764365

The Road not Taken: Less Traveled Roads from the TGN to the Plasma Membrane.

PubMed

Spang, Anne

2015-03-10

The trans-Golgi network functions in the distribution of cargo into different transport vesicles that are destined to endosomes, lysosomes and the plasma membrane. Over the years, it has become clear that more than one transport pathway promotes plasma membrane localization of proteins. In spite of the importance of temporal and spatial control of protein localization at the plasma membrane, the regulation of sorting into and the formation of different transport containers are still poorly understood. In this review different transport pathways, with a special emphasis on exomer-dependent transport, and concepts of regulation and sorting at the TGN are discussed.

In vitro sealing of iatrogenic fetal membrane defects by a collagen plug imbued with fibrinogen and plasma.

PubMed

Engels, A C; Hoylaerts, M F; Endo, M; Loyen, S; Verbist, G; Manodoro, S; DeKoninck, P; Richter, J; Deprest, J A

2013-02-01

We aimed to demonstrate local thrombin generation by fetal membranes, as well as its ability to generate fibrin from fibrinogen concentrate. Furthermore, we aimed to investigate the efficacy of collagen plugs, soaked with plasma and fibrinogen, to seal iatrogenic fetal membrane defects. Thrombin generation by homogenized fetal membranes was measured by calibrated automated thrombography. To identify the coagulation caused by an iatrogenic membrane defect, we analyzed fibrin formation by optical densitometry, upon various concentrations of fibrinogen. The ability of a collagen plug soaked with fibrinogen and plasma was tested in an ex vivo model for its ability to seal an iatrogenic fetal membrane defect. Fetal membrane homogenates potently induced thrombin generation in amniotic fluid and diluted plasma. Upon the addition of fibrinogen concentrate, potent fibrin formation was triggered. Measured by densiometry, fibrin formation was optimal at 1250 µg/mL fibrinogen in combination with 4% plasma. A collagen plug soaked with fibrinogen and plasma sealed an iatrogenic membrane defect about 35% better than collagen plugs without these additives (P = 0.037). These in vitro experiments suggest that the addition of fibrinogen and plasma may enhance the sealing efficacy of collagen plugs in closing iatrogenic fetal membrane defects. © 2013 John Wiley & Sons, Ltd.

Dynamic organization of myristoylated Src in the live cell plasma membrane

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

Smith, Adam W.; Huang, Hector H.; Endres, Nicholas F.

The spatial organization of lipid-anchored proteins in the plasma membrane directly influences cell signaling, but measuring such organization in situ is experimentally challenging. The canonical oncogene, c-Src, is a lipid anchored protein that plays a key role in integrin-mediated signal transduction within focal adhesions and cell–cell junctions. Because of its activity in specific plasma membrane regions, structural motifs within the protein have been hypothesized to play an important role in its subcellular localization. This study used a combination of time-resolved fluorescence fluctuation spectroscopy and super-resolution microscopy to quantify the dynamic organization of c-Src in live cell membranes. Pulsed-interleaved excitation fluorescencemore » cross-correlation spectroscopy (PIE–FCCS) showed that a small fraction of c-Src transiently sorts into membrane clusters that are several times larger than the monomers. Photoactivated localization microscopy (PALM) confirmed that c-Src partitions into clusters with low probability and showed that the characteristic size of the clusters is 10–80 nm. Finally, time-resolved fluorescence anisotropy measurements were used to quantify the rotational mobility of c-Src to determine how it interacts with its local environment. Altogether, these results build a quantitative description of the mobility and clustering behavior of the c-Src nonreceptor tyrosine kinase in the live cell plasma membrane.« less

Dynamic organization of myristoylated Src in the live cell plasma membrane

DOE PAGES

Smith, Adam W.; Huang, Hector H.; Endres, Nicholas F.; ...

2016-01-15

The spatial organization of lipid-anchored proteins in the plasma membrane directly influences cell signaling, but measuring such organization in situ is experimentally challenging. The canonical oncogene, c-Src, is a lipid anchored protein that plays a key role in integrin-mediated signal transduction within focal adhesions and cell–cell junctions. Because of its activity in specific plasma membrane regions, structural motifs within the protein have been hypothesized to play an important role in its subcellular localization. This study used a combination of time-resolved fluorescence fluctuation spectroscopy and super-resolution microscopy to quantify the dynamic organization of c-Src in live cell membranes. Pulsed-interleaved excitation fluorescencemore » cross-correlation spectroscopy (PIE–FCCS) showed that a small fraction of c-Src transiently sorts into membrane clusters that are several times larger than the monomers. Photoactivated localization microscopy (PALM) confirmed that c-Src partitions into clusters with low probability and showed that the characteristic size of the clusters is 10–80 nm. Finally, time-resolved fluorescence anisotropy measurements were used to quantify the rotational mobility of c-Src to determine how it interacts with its local environment. Altogether, these results build a quantitative description of the mobility and clustering behavior of the c-Src nonreceptor tyrosine kinase in the live cell plasma membrane.« less

Rapid, directed transport of DC-SIGN clusters in the plasma membrane

PubMed Central

Liu, Ping; Weinreb, Violetta; Ridilla, Marc; Betts, Laurie; Patel, Pratik; de Silva, Aravinda M.; Thompson, Nancy L.; Jacobson, Ken

2017-01-01

C-type lectins, including dendritic cell–specific intercellular adhesion molecule-3–grabbing nonintegrin (DC-SIGN), are all-purpose pathogen receptors that exist in nanoclusters in plasma membranes of dendritic cells. A small fraction of these clusters, obvious from the videos, can undergo rapid, directed transport in the plane of the plasma membrane at average speeds of more than 1 μm/s in both dendritic cells and MX DC-SIGN murine fibroblasts ectopically expressing DC-SIGN. Surprisingly, instantaneous speeds can be considerably greater. In MX DC-SIGN cells, many cluster trajectories are colinear with microtubules that reside close to the ventral membrane, and the microtubule-depolymerizing drug, nocodazole, markedly reduced the areal density of directed movement trajectories, suggesting a microtubule motor–driven transport mechanism; by contrast, latrunculin A, which affects the actin network, did not depress this movement. Rapid, retrograde movement of DC-SIGN may be an efficient mechanism for bringing bound pathogen on the leading edge and projections of dendritic cells to the perinuclear region for internalization and processing. Dengue virus bound to DC-SIGN on dendritic projections was rapidly transported toward the cell center. The existence of this movement within the plasma membrane points to an unexpected lateral transport mechanism in mammalian cells and challenges our current concepts of cortex-membrane interactions. PMID:29134199

Effect of fluorine substitution on the interaction of lipophilic ions with the plasma membrane of mammalian cells.

PubMed Central

Kürschner, M; Nielsen, K; von Langen, J R; Schenk, W A; Zimmermann, U; Sukhorukov, V L

2000-01-01

The effects of the anionic tungsten carbonyl complex [W(CO)(5)SC(6)H(5)](-) and its fluorinated analog [W(CO)(5)SC(6)F(5)](-) on the electrical properties of the plasma membrane of mouse myeloma cells were studied by the single-cell electrorotation technique. At micromolar concentrations, both compounds gave rise to an additional antifield peak in the rotational spectra of cells, indicating that the plasma membrane displayed a strong dielectric dispersion. This means that both tungsten derivatives act as lipophilic ions that are able to introduce large amounts of mobile charges into the plasma membrane. The analysis of the rotational spectra allowed the evaluation not only of the passive electric properties of the plasma membrane and cytoplasm, but also of the ion transport parameters, such as the surface concentration, partition coefficient, and translocation rate constant of the lipophilic anions dissolved in the plasma membrane. Comparison of the membrane transport parameters for the two anions showed that the fluorine-substituted analog was more lipophilic, but its translocation across the plasma membrane was slower by at least one order of magnitude than that of the parent hydrogenated anion. PMID:10969010

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

PubMed

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

2015-07-07

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

The Electroporation as a Tool for Studying the Role of Plasma Membrane in the Mechanism of Cytotoxicity of Bisphosphonates and Menadione.

PubMed

Šilkūnas, Mantas; Saulė, Rita; Batiuškaitė, Danutė; Saulis, Gintautas

2016-10-01

In this study, the role of the cell plasma membrane as a barrier in the mechanism of the cytotoxicity of nitrogen-containing bisphosphonates and menadione was studied, and the possibility of increasing the efficiency of bisphosphonates and menadione (vitamin K 3 ) as chemotherapeutic agents by permeabilizing the cell plasma membrane has been investigated in vitro. The plasma membrane barrier was reduced by electropermeabilization with the pulse of strong electric field. Two membrane-impermeant bisphosphonates with different hydrophilicities were chosen as study objects: ibandronate and pamidronate. For the comparison, an amphiphilic vitamin K 3 , which is able to cross the cell membrane, was studied as well. The impact of nitrogen-containing bisphosphonates and vitamin K 3 on MH-22A cells viability was evaluated for the case of long (9 days) and short (20 min) exposure. When cells were cultured in the medium with vitamin K 3 for 9-10 days, it exhibited toxicity of 50 % over the control at 6.2 µM for mouse hepatoma MH-22A cells. Ibandronate and pamidronate were capable of reducing drastically the cell viability only in the case of long 9-days incubation and at high concentrations (~20 µM for pamidronate and over 100 µM for ibandronate). Single, square-wave electric pulse with the duration of 100 µs and the field strength of 2 kV/cm was used to electroporate mouse hepatoma MH-22A cells in vitro. The results obtained here showed that the combination of the exposure of cells to membrane-impermeable bisphosphonates pamidronate and ibandronate with electropermeabilization of the cell plasma membrane did not increase their cytotoxicity. In the case of membrane-permeable vitamin K 3 , cell electropermeabilization did increase vitamin K 3 killing efficiency. However, this increase was not substantial, within the range of 20-30 % depending on the duration of the exposure. Electropermeabilization improved cytotoxic effect of vitamin K 3 but not of pamidronate

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

PubMed

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

2018-01-01

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

Membrane tension regulates clathrin-coated pit dynamics

NASA Astrophysics Data System (ADS)

Liu, Allen

2014-03-01

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

Dynamic complexity: plant receptor complexes at the plasma membrane.

PubMed

Burkart, Rebecca C; Stahl, Yvonne

2017-12-01

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

The connection of cytoskeletal network with plasma membrane and the cell wall

PubMed Central

Liu, Zengyu; Persson, Staffan; Zhang, Yi

2015-01-01

The cell wall provides external support of the plant cells, while the cytoskeletons including the microtubules and the actin filaments constitute an internal framework. The cytoskeletons contribute to the cell wall biosynthesis by spatially and temporarily regulating the transportation and deposition of cell wall components. This tight control is achieved by the dynamic behavior of the cytoskeletons, but also through the tethering of these structures to the plasma membrane. This tethering may also extend beyond the plasma membrane and impact on the cell wall, possibly in the form of a feedback loop. In this review, we discuss the linking components between the cytoskeletons and the plasma membrane, and/or the cell wall. We also discuss the prospective roles of these components in cell wall biosynthesis and modifications, and aim to provide a platform for further studies in this field. PMID:25693826

NAD+/NADH and/or CoQ/CoQH2 ratios from plasma membrane electron transport may determine ceramide and sphingosine-1-phosphate levels accompanying G1 arrest and apoptosis.

PubMed

De Luca, Thomas; Morré, Dorothy M; Zhao, Haiyun; Morré, D James

2005-01-01

To elucidate possible biochemical links between growth arrest from antiproliferative chemotherapeutic agents and apoptosis, our work has focused on agents (EGCg, capsaicin, cis platinum, adriamycin, anti-tumor sulfonylureas, phenoxodiol) that target tNOX. tNOX is a cancer-specific cell surface NADH oxidase (ECTO-NOX protein), that functions in cancer cells as the terminal oxidase for plasma membrane electron transport. When tNOX is active, coenzyme Q(10) (ubiquinone) of the plasma membrane is oxidized and NADH is oxidized at the cytosolic surface of the plasma membrane. However, when tNOX is inhibited and plasma membrane electron transport is diminished, both reduced coenzyme Q(10) (ubiquinol) and NADH would be expected to accumulate. To relate inhibition of plasma membrane redox to increased ceramide levels and arrest of cell proliferation in G(1) and apoptosis, we show that neutral sphingomyelinase, a major contributor to plasma membrane ceramide, is inhibited by reduced glutathione and ubiquinone. Ubiquinol is without effect or stimulates. In contrast, sphingosine kinase, which generates anti-apoptotic sphingosine-1-phosphate, is stimulated by ubiquinone but inhibited by ubiquinol and NADH. Thus, the quinone and pyridine nucleotide products of plasma membrane redox, ubiquinone and ubiquinol, as well as NAD(+) and NADH, may directly modulate in a reciprocal manner two key plasma membrane enzymes, sphingomyelinase and sphingosine kinase, potentially leading to G(1) arrest (increase in ceramide) and apoptosis (loss of sphingosine-1-phosphate). As such, the findings provide potential links between coenzyme Q(10)-mediated plasma membrane electron transport and the anticancer action of several clinically-relevant anticancer agents.

Ionic protein-lipid interaction at the plasma membrane: what can the charge do?

PubMed

Li, Lunyi; Shi, Xiaoshan; Guo, Xingdong; Li, Hua; Xu, Chenqi

2014-03-01

Phospholipids are the major components of cell membranes, but they have functional roles beyond forming lipid bilayers. In particular, acidic phospholipids form microdomains in the plasma membrane and can ionically interact with proteins via polybasic sequences, which can have functional consequences for the protein. The list of proteins regulated by ionic protein-lipid interaction has been quickly expanding, and now includes membrane proteins, cytoplasmic soluble proteins, and viral proteins. Here we review how acidic phospholipids in the plasma membrane regulate protein structure and function via ionic interactions, and how Ca(2+) regulates ionic protein-lipid interactions via direct and indirect mechanisms. Copyright © 2014 Elsevier Ltd. All rights reserved.

Modular assembly of synthetic proteins that span the plasma membrane in mammalian cells.

PubMed

Qudrat, Anam; Truong, Kevin

2016-12-09

To achieve synthetic control over how a cell responds to other cells or the extracellular environment, it is important to reliably engineer proteins that can traffic and span the plasma membrane. Using a modular approach to assemble proteins, we identified the minimum necessary components required to engineer such membrane-spanning proteins with predictable orientation in mammalian cells. While a transmembrane domain (TM) fused to the N-terminus of a protein is sufficient to traffic it to the endoplasmic reticulum (ER), an additional signal peptidase cleavage site downstream of this TM enhanced sorting out of the ER. Next, a second TM in the synthetic protein helped anchor and accumulate the membrane-spanning protein on the plasma membrane. The orientation of the components of the synthetic protein were determined through measuring intracellular Ca 2+ signaling using the R-GECO biosensor and through measuring extracellular quenching of yellow fluorescent protein variants by saturating acidic and salt conditions. This work forms the basis of engineering novel proteins that span the plasma membrane to potentially control intracellular responses to extracellular conditions.

Phospholipid composition of the plasma membrane of the green alga, Hydrodictyon africanum.

PubMed Central

Bailey, D S; Northcote, D H

1976-01-01

A plasma-membrane fraction was isolated from the alga Hydrodictyon africanum by micro-dissection and its phospholipid components were analysed. Phosphatidylcholine was the major phospholipid of the preparation. Both phosphatidylserine and diphosphatidylglycerol were enriched in the fraction compared with the whole cell, but the relative amount of phosphatidylglycerol present was less than that in the whole cell. Phosphatidylinositol was absent from the plasma-membrane preparation. Images PLATE 1 PLATE 2 PMID:182144

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

Calmodulin-stimulated Ca(2+)-ATPases in the vacuolar and plasma membranes in cauliflower.

PubMed

Askerlund, P

1997-07-01

The subcellular locations of Ca(2+)-ATPases in the membranes of cauliflower (Brassica oleracea L.) inflorescences were investigated. After continuous sucrose gradient centrifugation a 111-kD calmodulin (CaM)-stimulated and caM-binding Ca(2+)-ATPase (BCA1; P. Askerlund [1996] Plant Physiol 110: 913-922; S. Malmström, P. Askerlund, M.G. Plamgren [1997] FEBS Lett 400: 324-328) comigrated with vacuolar membrane markers, whereas a 116-kD caM-binding Ca(2+)-ATPase co-migrated with a marker for the plasma membrane. The 116 kD Ca(2+)-ATPase was enriched in plasma membranes obtained by aqueous two-phase partitioning, which is in agreement with a plasma membrane location of this Ca(2+)-ATPase. Countercurrent distribution of a low-density intracellular membrane fraction in an aqueous two-phase system resulted in the separation of the endoplasmic reticulum and vacuolar membranes. The 111-kD Ca(2+)-ATPase co-migrated with a vacuolar membrane marker after countercurrent distribution but not with markers for the endoplasmic reticulum. A vacuolar membrane location of the 111-kD Ca(2+)-AtPase was further supported by experiments with isolated vacuoles from cauliflower: (a) Immunoblotting with an antibody against the 111-kD Ca(2+)-ATPase showed that it was associated with the vacuoles, and (b) ATP-dependent Ca2+ uptake by the intact vacuoles was found to be CaM stimulated and partly protonophore insensitive.

Influence of plasma modification on hygienic properties of textile fabrics with nonporous membrane coating

NASA Astrophysics Data System (ADS)

Voznesensky, E. F.; Ibragimov, R. G.; Vishnevskaya, O. V.; Sisoev, V. A.; Lutfullina, G. G.; Tihonova, N. V.

2017-11-01

The work investigated the possibility of using plasma modification to improve the hygienic properties of textile materials with nonporous membrane coating to improve vapor-, air-permeability and water-resistant. Determined that, after plasma modification changes degree of supramolecular orderliness of the polymers nonporous membrane coating and the base fabric.

Isothermal titration calorimetric studies on the interaction of the major bovine seminal plasma protein, PDC-109 with phospholipid membranes.

PubMed

Anbazhagan, V; Sankhala, Rajeshwer S; Singh, Bhanu Pratap; Swamy, Musti J

2011-01-01

The interaction of the major bovine seminal plasma protein, PDC-109 with lipid membranes was investigated by isothermal titration calorimetry. Binding of the protein to model membranes made up of diacyl phospholipids was found to be endothermic, with positive values of binding enthalpy and entropy, and could be analyzed in terms of a single type of binding sites on the protein. Enthalpies and entropies for binding to diacylphosphatidylcholine membranes increased with increase in temperature, although a clear-cut linear dependence was not observed. The entropically driven binding process indicates that hydrophobic interactions play a major role in the overall binding process. Binding of PDC-109 with dimyristoylphosphatidylcholine membranes containing 25 mol% cholesterol showed an initial increase in the association constant as well as enthalpy and entropy of binding with increase in temperature, whereas the values decreased with further increase in temperature. The affinity of PDC-109 for phosphatidylcholine increased at higher pH, which is physiologically relevant in view of the basic nature of the seminal plasma. Binding of PDC-109 to Lyso-PC could be best analysed in terms of two types of binding interactions, a high affinity interaction with Lyso-PC micelles and a low-affinity interaction with the monomeric lipid. Enthalpy-entropy compensation was observed for the interaction of PDC-109 with phospholipid membranes, suggesting that water structure plays an important role in the binding process.

Isothermal Titration Calorimetric Studies on the Interaction of the Major Bovine Seminal Plasma Protein, PDC-109 with Phospholipid Membranes

PubMed Central

Anbazhagan, V.; Sankhala, Rajeshwer S.; Singh, Bhanu Pratap; Swamy, Musti J.

2011-01-01

The interaction of the major bovine seminal plasma protein, PDC-109 with lipid membranes was investigated by isothermal titration calorimetry. Binding of the protein to model membranes made up of diacyl phospholipids was found to be endothermic, with positive values of binding enthalpy and entropy, and could be analyzed in terms of a single type of binding sites on the protein. Enthalpies and entropies for binding to diacylphosphatidylcholine membranes increased with increase in temperature, although a clear-cut linear dependence was not observed. The entropically driven binding process indicates that hydrophobic interactions play a major role in the overall binding process. Binding of PDC-109 with dimyristoylphosphatidylcholine membranes containing 25 mol% cholesterol showed an initial increase in the association constant as well as enthalpy and entropy of binding with increase in temperature, whereas the values decreased with further increase in temperature. The affinity of PDC-109 for phosphatidylcholine increased at higher pH, which is physiologically relevant in view of the basic nature of the seminal plasma. Binding of PDC-109 to Lyso-PC could be best analysed in terms of two types of binding interactions, a high affinity interaction with Lyso-PC micelles and a low-affinity interaction with the monomeric lipid. Enthalpy-entropy compensation was observed for the interaction of PDC-109 with phospholipid membranes, suggesting that water structure plays an important role in the binding process. PMID:22022488

Gravity perception requires statoliths settled on specific plasma membrane areas in characean rhizoids and protonemata.

PubMed

Braun, Markus

2002-05-01

The noninvasive infrared laser micromanipulation technique (optical tweezers, optical trapping) and centrifugation were used to study susception and perception, the early events in the gravitropic pathway of tip-growing characean rhizoids and protonemata. Reorientation of the growth direction in both cell types was only initiated when at least 2-3 statoliths settled on specific areas of the plasma membrane. This statolith-sensitive plasma membrane area is confined to the statolith region (10-35 microns behind the tip) in positively gravitropic rhizoids, whereas in negatively gravitropic protonemata, this area is limited to the apical plasma membrane (0-10 microns). Statolith sedimentation towards the sensitive plasma membrane areas is mediated by the concerted action of actin and gravity. The process of sedimentation, the pure physical movement, of statoliths is not sufficient to initiate graviresponses in both cell types. It is concluded that specific statolith-sensitive plasma membrane areas play a crucial role in the signal transduction pathway of gravitropism. These areas may represent the primary sites for gravity perception and may transform the information derived from the gravity-induced statolith sedimentation into physiological signals which trigger the molecular mechanisms of the opposite graviresponses in characean rhizoids and protonemata.

Preparation of synaptic plasma membrane and postsynaptic density proteins using a discontinuous sucrose gradient.

PubMed

Bermejo, Marie Kristel; Milenkovic, Marija; Salahpour, Ali; Ramsey, Amy J

2014-09-03

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in the late 1960's, proteins associated with the synaptic plasma membrane can be isolated by ultracentrifugation on a sucrose density gradient. Once synaptic membranes are isolated, the macromolecular complex known as the post-synaptic density can be subsequently isolated due to its detergent insolubility. The techniques used to isolate synaptic plasma membranes and post-synaptic density proteins remain essentially the same after 40 years, and are widely used in current neuroscience research. This article details the fractionation of proteins associated with the synaptic plasma membrane and post-synaptic density using a discontinuous sucrose gradient. Resulting protein preparations are suitable for western blotting or 2D DIGE analysis.

Analysis of plasma membrane phosphoinositides from fusogenic carrot cells

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

Wheeler, J.J.; Boss, W.F.

1987-04-01

Phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP/sub 2/) were found to be associated with the plasma membrane-rich fractions isolated by aqueous polymer two-phase partitioning from fusogenic cells. They represented at least 5% and 0.7% of the total inositol-labeled lipids in the plasma membrane-rich fractions, respectively, and were present in a ratio of about 7:1 (PIP:PIP/sub 2/). In addition, two unidentified inositol-labeled compounds, which together were approximately 3% of the inositol-labeled lipids, were found predominantly in the plasma membrane-rich fractions and migrated between PIP/sub 2/ and PIP. The R/sub f/s of these compounds were approximately 0.31 and 0.34 in the solventmore » system CHCl/sub 3/:MeOH:15N NH/sub 4/OH:H/sub 2/O (90:90:7:22) using LK5 plates presoaked in 1% potassium oxalate. These compounds incorporated /sup 32/P/sub i/, (/sup 3/H)inositol and were hydrolyzed in mild base. These data suggested that they were glycero-phospholipids. Although the compounds did not comigrate with lysoPIP obtained from bovine brain (R/sub f/ approx. 0.35), when endogenous PIP was hydrolyzed to lysoPIP, the breakdown product migrated in the region of the unidentified inositol lipids.« less

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

PubMed Central

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

2011-01-01

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

Glucose-independent inhibition of yeast plasma-membrane H+-ATPase by calmodulin antagonists.