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Sample records for plasma membrane aquaporins

  1. Elevated cAMP increases aquaporin-3 plasma membrane diffusion.

    PubMed

    Marlar, Saw; Arnspang, Eva C; Koffman, Jennifer S; Løcke, Else-Merete; Christensen, Birgitte M; Nejsum, Lene N

    2014-03-15

    Regulated urine concentration takes place in the renal collecting duct upon arginine vasopressin (AVP) stimulation, where subapical vesicles containing aquaporin-2 (AQP2) are inserted into the apical membrane instantly increasing water reabsorption and urine concentration. The reabsorped water exits via basolateral AQP3 and AQP4. Upon long-term stimulation with AVP or during thirst, expression levels of both AQP2 and AQP3 are increased; however, there is so far no evidence for short-term AVP regulation of AQP3 or AQP4. To facilitate the increase in transepithelial water transport, AQP3 may be short-term regulated via changes in protein-protein interactions, incorporation into lipid rafts, and/or changes in steady-state turnover, which could result in changes in the diffusion behavior of AQP3. Thus we measured AQP3 diffusion coefficients upon stimulation with the AVP mimic forskolin to reveal if AQP3 could be short-term regulated by AVP. k-Space image correlation spectroscopy (kICS) analysis of time-lapse image sequences of basolateral enhanced green fluorescent protein-tagged AQP3 (AQP3-EGFP) revealed that the forskolin-mediated elevation of cAMP increased the diffusion coefficient by 58% from 0.0147 ± 0.0082 μm(2)/s (control) to 0.0232 ± 0.0085 μm(2)/s (forskolin, P < 0.05). Quantum dot-conjugated antibody labeling also revealed a significant increase in AQP3 diffusion upon forskolin treatment by 44% [0.0104 ± 0.0040 μm(2)/s (control) vs. 0.0150 ± 0.0016 μm(2)/s (forskolin, P < 0.05)]. Immunoelectron microscopy showed no obvious difference in AQP3-EGFP expression levels or localization in the plasma membrane upon forskolin stimulation. Thus AQP3-EGFP diffusion is altered upon increased cAMP, which may correspond to basolateral adaptations in response to the increased apical water readsorption.

  2. Expression patterns of genes encoding plasma membrane aquaporins during fruit development in cucumber (Cucumis sativus L.).

    PubMed

    Shi, Jin; Wang, Jinfang; Li, Ren; Li, Dianbo; Xu, Fengfeng; Sun, Qianqian; Zhao, Bin; Mao, Ai-Jun; Guo, Yang-Dong

    2015-11-01

    Aquaporins are membrane channels precisely regulating water movement through cell membranes in most living organisms. Despite the advances in the physiology of fruit development, their participation during fruit development in cucumber still barely understood. In this paper, the expressions of 12 genes encoding plasma membrane intrinsic proteins (PIPs) were analyzed during cucumber fruit development in our work. Based on the homology search with known PIPs from rice, Arabidopsis and strawberry, 12 cucumber PIP genes subfamily members were identified. Cellular localization assays indicated that CsPIPs were localized in the plasma membrane. The qRT-PCR analysis of CsPIPs showed that 12 CsPIPs were differentially expressed during fruit development. These results suggest that 12 genes encoding plasma membrane intrinsic proteins (CsPIPs) play very important roles in cucumber life cycle and the data generated will be helpful in understanding their precise roles during fruit development in cucumber.

  3. Association between water and carbon dioxide transport in leaf plasma membranes: assessing the role of aquaporins.

    PubMed

    Zhao, Manchun; Tan, Hwei-Ting; Scharwies, Johannes; Levin, Kara; Evans, John R; Tyerman, Stephen D

    2017-06-01

    The role of some aquaporins as CO2 permeable channels has been controversial. Low CO2 permeability of plant membranes has been criticized because of unstirred layers and other limitations. Here we measured both water and CO2 permeability (Pos , PCO2 ) using stopped flow on plasma membrane vesicles (pmv) isolated from Pisum sativum (pea) and Arabidopsis thaliana leaves. We excluded the chemical limitation of carbonic anhydrase (CA) in the vesicle acidification technique for PCO2 using different temperatures and CA concentrations. Unstirred layers were excluded based on small vesicle size and the positive correlation between vesicle diameter and PCO2 . We observed high aquaporin activity (Pos 0.06 to 0.22 cm s(-1) ) for pea pmv based on all the criteria for their function using inhibitors and temperature dependence. Inhibitors of Pos did not alter PCO2 . PCO2 ranged from 0.001 to 0.012 cm s(-1) (mean 0.0079 + 0.0007 cm s(-1) ) with activation energy of 30.2 kJ mol(-1) . Intrinsic variation between pmv batches from normally grown or stressed plants revealed a weak (R(2)  = 0.27) positive linear correlation between Pos and PCO2 . Despite the low PCO2 , aquaporins may facilitate CO2 transport across plasma membranes, but probably via a different pathway than for water. © 2016 John Wiley & Sons Ltd.

  4. Plant plasma membrane aquaporins in natural vesicles as potential stabilizers and carriers of glucosinolates.

    PubMed

    Martínez-Ballesta, Maria Del Carmen; Pérez-Sánchez, Horacio; Moreno, Diego A; Carvajal, Micaela

    2016-07-01

    Their biodegradable nature and ability to target cells make biological vesicles potential nanocarriers for bioactives delivery. In this work, the interaction between proteoliposomes enriched in aquaporins derived from broccoli plants and the glucosinolates was evaluated. The vesicles were stored at different temperatures and their integrity was studied. Determination of glucosinolates, showed that indolic glucosinolates were more sensitive to degradation in aqueous solution than aliphatic glucosinolates. Glucoraphanin was stabilized by leaf and root proteoliposomes at 25°C through their interaction with aquaporins. An extensive hydrogen bond network, including different aquaporin residues, and hydrophobic interactions, as a consequence of the interaction between the linear alkane chain of glucoraphanin and Glu31 and Leu34 protein residues, were established as the main stabilizing elements. Combined our results showed that plasma membrane vesicles from leaf and root tissues of broccoli plants may be considered as suitable carriers for glucosinolate which stabilization can be potentially attributed to aquaporins. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. 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.

  6. Plasma Membrane Aquaporins Play a Significant Role during Recovery from Water Deficit1

    PubMed Central

    Martre, Pierre; Morillon, Raphaël; Barrieu, François; North, Gretchen B.; Nobel, Park S.; Chrispeels, Maarten J.

    2002-01-01

    The role of plasma membrane aquaporins (PIPs) in water relations of Arabidopsis was studied by examining plants with reduced expression of PIP1 and PIP2 aquaporins, produced by crossing two different antisense lines. Compared with controls, the double antisense (dAS) plants had reduced amounts of PIP1 and PIP2 aquaporins, and the osmotic hydraulic conductivity of isolated root and leaf protoplasts was reduced 5- to 30-fold. The dAS plants had a 3-fold decrease in the root hydraulic conductivity expressed on a root dry mass basis, but a compensating 2.5-fold increase in the root to leaf dry mass ratio. The leaf hydraulic conductance expressed on a leaf area basis was similar for the dAS compared with the control plants. As a result, the hydraulic conductance of the whole plant was unchanged. Under sufficient and under water-deficient conditions, stomatal conductance, transpiration rate, plant hydraulic conductance, leaf water potential, osmotic pressure, and turgor pressure were similar for the dAS compared with the control plants. However, after 4 d of rewatering following 8 d of drying, the control plants recovered their hydraulic conductance and their transpiration rates faster than the dAS plants. Moreover, after rewatering, the leaf water potential was significantly higher for the control than for the dAS plants. From these results, we conclude that the PIPs play an important role in the recovery of Arabidopsis from the water-deficient condition. PMID:12481094

  7. Characterization of protein phosphatase 2A acting on phosphorylated plasma membrane aquaporin of tulip petals.

    PubMed

    Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2004-05-01

    A protein phosphatase holo-type enzyme (38, 65, and 75 kDa) preparation and a free catalytic subunit (38 kDa) purified from tulip petals were characterized as protein phosphatase 2A (PP2A) by immunological and biochemical approaches. The plasma membrane containing the putative plasma membrane aquaporin (PM-AQP) was prepared from tulip petals, phosphorylated in vitro, and used as the substrate for both of the purified PP2A preparations. Although both preparations dephosphorylated the phosphorylated PM-AQP at 20 degrees C, only the holo-type enzyme preparation acted at 5 degrees C on the phosphorylated PM-AQP with higher substrate specificity, suggesting that regulatory subunits are required for low temperature-dependent dephosphorylation of PM-AQP in tulip petals.

  8. Plasma Membrane Aquaporins Are Involved in Winter Embolism Recovery in Walnut Tree1

    PubMed Central

    Sakr, Soulaiman; Alves, Georges; Morillon, Raphaël; Maurel, Karine; Decourteix, Mélanie; Guilliot, Agnès; Fleurat-Lessard, Pierrette; Julien, Jean-Louis; Chrispeels, Maarten J.

    2003-01-01

    In perennial plants, freeze-thaw cycles during the winter months can induce the formation of air bubbles in xylem vessels, leading to changes in their hydraulic conductivity. Refilling of embolized xylem vessels requires an osmotic force that is created by the accumulation of soluble sugars in the vessels. Low water potential leads to water movement from the parenchyma cells into the xylem vessels. The water flux gives rise to a positive pressure essential for the recovery of xylem hydraulic conductivity. We investigated the possible role of plasma membrane aquaporins in winter embolism recovery in walnut (Juglans regia). First, we established that xylem parenchyma starch is converted to sucrose in the winter months. Then, from a xylem-derived cDNA library, we isolated two PIP2 aquaporin genes (JrPIP2,1 and JrPIP2,2) that encode nearly identical proteins. The water channel activity of the JrPIP2,1 protein was demonstrated by its expression in Xenopus laevis oocytes. The expression of the two PIP2 isoforms was investigated throughout the autumn-winter period. In the winter period, high levels of PIP2 mRNA and corresponding protein occurred simultaneously with the rise in sucrose. Furthermore, immunolocalization studies in the winter period show that PIP2 aquaporins were mainly localized in vessel-associated cells, which play a major role in controlling solute flux between parenchyma cells and xylem vessels. Taken together, our data suggest that PIP2 aquaporins could play a role in water transport between xylem parenchyma cells and embolized vessels. PMID:14526109

  9. Phosphorylation of plasma membrane aquaporin regulates temperature-dependent opening of tulip petals.

    PubMed

    Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2004-05-01

    The opening and closing of tulip petals was reproduced in the dark by changing the temperature from 5 degrees C to 20 degrees C for opening and 20 degrees C to 5 degrees C for closing. The opening process was accompanied by (3)H(2)O transport through the stem from the incubation medium to the petals. A Ca(2+)-channel blocker and a Ca(2+)-chelator inhibited petal opening and (3)H(2)O transport. Several proteins in the isolated plasma membrane fraction were phosphorylated in the presence of 25 micro M Ca(2+) at 20 degrees C. The 31-kDa protein that was phosphorylated, was suggested immunologically as the putative plasma membrane aquaporin (PM-AQP). This phosphorylated PM-AQP clearly reacted with the anti-phospho-Ser. In-gel assay revealed the presence of a 45-kDa Ca(2+)-dependent protein kinase in the isolated plasma membrane. Phosphorylation of the putative PM-AQP was thought to activate the water channel composed of PM-AQP. Dephosphorylation of the phosphorylated PM-AQP was also observed during petal closing at 5 degrees C, suggesting the inactivation of the water channel.

  10. 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.

  11. Mutual Interactions between Aquaporins and Membrane Components

    PubMed Central

    Martínez-Ballesta, Maria del Carmen; Carvajal, Micaela

    2016-01-01

    In recent years, a number of studies have been focused on the structural evaluation of protein complexes in order to get mechanistic insights into how proteins communicate at the molecular level within the cell. Specific sites of protein-aquaporin interaction have been evaluated and new forms of regulation of aquaporins described, based on these associations. Heterotetramerizations of aquaporin isoforms are considered as novel regulatory mechanisms for plasma membrane (PIPs) and tonoplast (TIPs) proteins, influencing their intrinsic permeability and trafficking dynamics in the adaptive response to changing environmental conditions. However, protein–protein interaction is an extensive theme that is difficult to tackle and new methodologies are being used to study the physical interactions involved. Bimolecular fluorescence complementation and the identification of cross-linked peptides based on tandem mass spectra, that are complementary to other methodologies such as heterologous expression, co-precipitation assays or confocal fluorescence microscopy, are discussed in this review. The chemical composition and the physical characteristics of the lipid bilayer also influence many aspects of membrane aquaporins, including their functionality. The molecular driving forces stabilizing the positions of the lipids around aquaporins could define their activity, thereby altering the conformational properties. Therefore, an integrative approach to the relevance of the membrane-aquaporin interaction to different processes related to plant cell physiology is provided. Finally, it is described how the interactions between aquaporins and copolymer matrixes or biological compounds offer an opportunity for the functional incorporation of aquaporins into new biotechnological advances. PMID:27625676

  12. Mutual Interactions between Aquaporins and Membrane Components.

    PubMed

    Martínez-Ballesta, Maria Del Carmen; Carvajal, Micaela

    2016-01-01

    In recent years, a number of studies have been focused on the structural evaluation of protein complexes in order to get mechanistic insights into how proteins communicate at the molecular level within the cell. Specific sites of protein-aquaporin interaction have been evaluated and new forms of regulation of aquaporins described, based on these associations. Heterotetramerizations of aquaporin isoforms are considered as novel regulatory mechanisms for plasma membrane (PIPs) and tonoplast (TIPs) proteins, influencing their intrinsic permeability and trafficking dynamics in the adaptive response to changing environmental conditions. However, protein-protein interaction is an extensive theme that is difficult to tackle and new methodologies are being used to study the physical interactions involved. Bimolecular fluorescence complementation and the identification of cross-linked peptides based on tandem mass spectra, that are complementary to other methodologies such as heterologous expression, co-precipitation assays or confocal fluorescence microscopy, are discussed in this review. The chemical composition and the physical characteristics of the lipid bilayer also influence many aspects of membrane aquaporins, including their functionality. The molecular driving forces stabilizing the positions of the lipids around aquaporins could define their activity, thereby altering the conformational properties. Therefore, an integrative approach to the relevance of the membrane-aquaporin interaction to different processes related to plant cell physiology is provided. Finally, it is described how the interactions between aquaporins and copolymer matrixes or biological compounds offer an opportunity for the functional incorporation of aquaporins into new biotechnological advances.

  13. Specific aquaporins facilitate Nox-produced hydrogen peroxide transport through plasma membrane in leukaemia cells.

    PubMed

    Vieceli Dalla Sega, Francesco; Zambonin, Laura; Fiorentini, Diana; Rizzo, Benedetta; Caliceti, Cristiana; Landi, Laura; Hrelia, Silvana; Prata, Cecilia

    2014-04-01

    In the last decade, the generation and the role of reactive oxygen species (ROS), particularly hydrogen peroxide, in cell signalling transduction pathways have been intensively studied, and it is now clear that an increase of ROS level affects cellular growth and proliferation pathways related to cancer development. Hydrogen peroxide (H2O2) has been long thought to permeate biological membranes by simple diffusion since recent evidence challenged this notion disclosing the role of aquaporin water channels (AQP) in mediating H2O2 transport across plasma membranes. We previously demonstrated that NAD(P)H oxidase (Nox)-generated ROS sustain glucose uptake and cellular proliferation in leukaemia cells. The aim of this study was to assess whether specific AQP isoforms can channel Nox-produced H2O2 across the plasma membrane of leukaemia cells affecting downstream pathways linked to cell proliferation. In this work, we demonstrate that AQP inhibition caused a decrease in intracellular ROS accumulation in leukaemia cells both when H2O2 was produced by Nox enzymes and when it was exogenously added. Furthermore, AQP8 overexpression or silencing resulted to modulate VEGF capacity of triggering an H2O2 intracellular level increase or decrease, respectively. Finally, we report that AQP8 is capable of increasing H2O2-induced phosphorylation of both PI3K and p38 MAPK and that AQP8 expression affected positively cell proliferation. Taken together, the results here reported indicate that AQP8 is able to modulate H2O2 transport through the plasma membrane affecting redox signalling linked to leukaemia cell proliferation. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Identification and characterization of plasma membrane aquaporins isolated from fiber cells of Calotropis procera

    PubMed Central

    Aslam, Usman; Khatoon, Asia; Cheema, Hafiza Masooma Naseer; Bashir, Aftab

    2013-01-01

    Calotropis procera, commonly known as “milkweed”, possesses long seed trichomes for seed dispersal and has the ability to survive under harsh conditions such as drought and salinity. Aquaporins are water channel proteins expressed in all land plants, divided into five subfamilies plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), NOD26-like proteins (NIPs), small basic intrinsic proteins (SIPs), and the unfamiliar X intrinsic proteins (XIPs). PIPs constitute the largest group of water channel proteins that are involved in different developmental and regulatory mechanisms including water permeability, cell elongation, and stomata opening. Aquaporins are also involved in abiotic stress tolerance and cell expansion mechanisms, but their role in seed trichomes (fiber cells) has never been investigated. A large number of clones isolated from C. procera fiber cDNA library showed sequence homology to PIPs. Both expressed sequence tags (ESTs) and real-time polymerase chain reaction (PCR) studies revealed that the transcript abundance of this gene family in fiber cells of C. procera is greater than that of cotton. Full-length cDNAs of CpPIP1 and CpPIP2 were isolated from C. procera fiber cDNA library and used for constructing plant expression vectors under constitutive (2×35S) and trichome-specific (GhLTP3) promoters. Transgenic tobacco plants were developed via Agrobacterium-mediated transformation. The phenotypic characteristics of the plants were observed after confirming the integration of transgene in plants. It was observed that CpPIP2 expression cassette under 2×35S and GhLTP3 promoter enhanced the numbers of stem and leave trichomes. However, 2×35S::CpPIP2 has a more amplified effect on trichome density and length than GhLTP3::CpPIP2 and other PIP constructs. These findings imply the role of C. procera PIP aquaporins in fiber cell elongation. The PIPs-derived cell expansion mechanism may be exploited through transgenic approaches

  15. Identification and characterization of plasma membrane aquaporins isolated from fiber cells of Calotropis procera.

    PubMed

    Aslam, Usman; Khatoon, Asia; Cheema, Hafiza Masooma Naseer; Bashir, Aftab

    2013-07-01

    Calotropis procera, commonly known as "milkweed", possesses long seed trichomes for seed dispersal and has the ability to survive under harsh conditions such as drought and salinity. Aquaporins are water channel proteins expressed in all land plants, divided into five subfamilies plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), NOD26-like proteins (NIPs), small basic intrinsic proteins (SIPs), and the unfamiliar X intrinsic proteins (XIPs). PIPs constitute the largest group of water channel proteins that are involved in different developmental and regulatory mechanisms including water permeability, cell elongation, and stomata opening. Aquaporins are also involved in abiotic stress tolerance and cell expansion mechanisms, but their role in seed trichomes (fiber cells) has never been investigated. A large number of clones isolated from C. procera fiber cDNA library showed sequence homology to PIPs. Both expressed sequence tags (ESTs) and real-time polymerase chain reaction (PCR) studies revealed that the transcript abundance of this gene family in fiber cells of C. procera is greater than that of cotton. Full-length cDNAs of CpPIP1 and CpPIP2 were isolated from C. procera fiber cDNA library and used for constructing plant expression vectors under constitutive (2×35S) and trichome-specific (GhLTP3) promoters. Transgenic tobacco plants were developed via Agrobacterium-mediated transformation. The phenotypic characteristics of the plants were observed after confirming the integration of transgene in plants. It was observed that CpPIP2 expression cassette under 2×35S and GhLTP3 promoter enhanced the numbers of stem and leave trichomes. However, 2×35S::CpPIP2 has a more amplified effect on trichome density and length than GhLTP3::CpPIP2 and other PIP constructs. These findings imply the role of C. procera PIP aquaporins in fiber cell elongation. The PIPs-derived cell expansion mechanism may be exploited through transgenic approaches for

  16. Short-term control of maize cell and root water permeability through plasma membrane aquaporin isoforms.

    PubMed

    Hachez, Charles; Veselov, Dmitry; Ye, Qing; Reinhardt, Hagen; Knipfer, Thorsten; Fricke, Wieland; Chaumont, François

    2012-01-01

    Although it is widely accepted that aquaporins are involved in the regulation of root water uptake, the role of specific isoforms in this process is poorly understood. The mRNA expression and protein level of specific plasma membrane intrinsic proteins (PIPs) were analysed in Zea mays in relation to cell and root hydraulic conductivity. Plants were analysed during the day/night period, under different growth conditions (aeroponics/hydroponics) and in response to short-term osmotic stress applied through polyethylene glycol (PEG). Higher protein levels of ZmPIP1;2, ZmPIP2;1/2;2, ZmPIP2;5 and ZmPIP2;6 during the day coincided with a higher water permeability of root cortex cells during the day compared with night period. Similarly, plants which were grown under aeroponic conditions and which developed a hypodermis ('exodermis') with Casparian bands, effectively forcing more water along a membranous uptake path across roots, showed increased levels of ZmPIP2;5 and ZmPIP1;2 in the rhizodermis and exodermis. When PEG was added to the root medium (2-8 h), expression of PIPs and cell water permeability in roots increased. These data support a role of specific PIP isoforms, in particular ZmPIP1;2 and ZmPIP2;5, in regulating root water uptake and cortex cell hydraulic conductivity in maize.

  17. Sucrose-induced Receptor Kinase SIRK1 Regulates a Plasma Membrane Aquaporin in Arabidopsis*

    PubMed Central

    Wu, Xu Na; Sanchez Rodriguez, Clara; Pertl-Obermeyer, Heidi; Obermeyer, Gerhard; Schulze, Waltraud X.

    2013-01-01

    The transmembrane receptor kinase family is the largest protein kinase family in Arabidopsis, and it contains the highest fraction of proteins with yet uncharacterized functions. Here, we present functions of SIRK1, a receptor kinase that was previously identified with rapid transient phosphorylation after sucrose resupply to sucrose-starved seedlings. SIRK1 was found to be an active kinase with increasing activity in the presence of an external sucrose supply. In sirk1 T-DNA insertional mutants, the sucrose-induced phosphorylation patterns of several membrane proteins were strongly reduced; in particular, pore-gating phosphorylation sites in aquaporins were affected. SIRK1-GFP fusions were found to directly interact with aquaporins in affinity pull-down experiments on microsomal membrane vesicles. Furthermore, protoplast swelling assays of sirk1 mutants and SIRK1-GFP expressing lines confirmed a direct functional interaction of receptor kinase SIRK1 and aquaporins as substrates for phosphorylation. A lack of SIRK1 expression resulted in the failure of mutant protoplasts to control water channel activity upon changes in external sucrose concentrations. We propose that SIRK1 is involved in the regulation of sucrose-specific osmotic responses through direct interaction with and activation of an aquaporin via phosphorylation and that the duration of this response is controlled by phosphorylation-dependent receptor internalization. PMID:23820729

  18. Heterologous Expression of Tulip Petal Plasma Membrane Aquaporins in Pichia pastoris for Water Channel Analysis▿

    PubMed Central

    Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2009-01-01

    Water channels formed by aquaporins (AQPs) play an important role in the control of water homeostasis in individual cells and in multicellular organisms. Plasma membrane intrinsic proteins (PIPs) constitute a subclass of plant AQPs. TgPIP2;1 and TgPIP2;2 from tulip petals are members of the PIP family. In this study, we overexpressed TgPIP2;1 and TgPIP2;2 in Pichia pastoris and monitored their water channel activity (WCA) either by an in vivo spheroplast-bursting assay performed after hypo-osmotic shock or by growth assay. Osmolarity, pH, and inhibitors of AQPs, protein kinases (PKs), and protein phosphatases (PPs) affect the WCA of heterologous AQPs in this expression system. The WCA of TgPIP2;2-expressing spheroplasts was affected by inhibitors of PKs and PPs, which indicates that the water channel of this homologue is regulated by phosphorylation in P. pastoris. From the results reported herein, we suggest that P. pastoris can be employed as a heterologous expression system to assay the WCA of PIPs and to monitor the AQP-mediated channel gating mechanism, and it can be developed to screen inhibitors/effectors of PIPs. PMID:19251885

  19. Heterologous expression of tulip petal plasma membrane aquaporins in Pichia pastoris for water channel analysis.

    PubMed

    Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2009-05-01

    Water channels formed by aquaporins (AQPs) play an important role in the control of water homeostasis in individual cells and in multicellular organisms. Plasma membrane intrinsic proteins (PIPs) constitute a subclass of plant AQPs. TgPIP2;1 and TgPIP2;2 from tulip petals are members of the PIP family. In this study, we overexpressed TgPIP2;1 and TgPIP2;2 in Pichia pastoris and monitored their water channel activity (WCA) either by an in vivo spheroplast-bursting assay performed after hypo-osmotic shock or by growth assay. Osmolarity, pH, and inhibitors of AQPs, protein kinases (PKs), and protein phosphatases (PPs) affect the WCA of heterologous AQPs in this expression system. The WCA of TgPIP2;2-expressing spheroplasts was affected by inhibitors of PKs and PPs, which indicates that the water channel of this homologue is regulated by phosphorylation in P. pastoris. From the results reported herein, we suggest that P. pastoris can be employed as a heterologous expression system to assay the WCA of PIPs and to monitor the AQP-mediated channel gating mechanism, and it can be developed to screen inhibitors/effectors of PIPs.

  20. Ammonia induces aquaporin-4 rearrangement in the plasma membrane of cultured astrocytes.

    PubMed

    Bodega, Guillermo; Suárez, Isabel; López-Fernández, Luis A; García, María I; Köber, Mariana; Penedo, Marcos; Luna, Mónica; Juárez, Silvia; Ciordia, Sergio; Oria, Marc; Córdoba, Joan; Fernández, Benjamín

    2012-12-01

    Aquaporin-4 (AQP4) is a water channel protein mainly located in the astroglial plasma membrane, the precise function of which in the brain edema that accompanies hepatic encephalopathy (HE) is unclear. Since ammonia is the main pathogenic agent in HE, its effect on AQP4 expression and distribution in confluent primary astroglial cultures was examined via their exposure to ammonium chloride (1, 3 and 5 mM) for 5 and 10 days. Ammonia induced the general inhibition of AQP4 mRNA synthesis except in the 1 mM/5 day treatment. However, the AQP4 protein content measured was dependent on the method of analysis; an apparent increase was recorded in treated cells in in-cell Western assays, while an apparent reduction was seen with the classic Western blot method, perhaps due to differences in AQP4 aggregation. Ammonia might therefore induce the formation of insoluble AQP4 aggregates in the astroglial plasma membrane. The finding of AQP4 in the pellet of classic Western blot samples, plus data obtained via confocal microscopy, atomic force microscopy (using immunolabeled cells with gold nanoparticles) and scanning electron microscopy, all corroborate this hypothesis. The effect of ammonia on AQP4 seems not to be due to any osmotic effect; identical osmotic stress induced by glutamine and salt had no significant effect on the AQP4 content. AQP4 functional analysis (subjecting astrocytes to a hypo-osmotic medium and using flow cytometry to measure cell size) demonstrated a smaller water influx in ammonia-treated astrocytes suggesting that AQP4 aggregates are representative of an inactive status; however, more confirmatory studies are required to fully understand the functional status of AQP4 aggregates. The present results suggest that ammonia affects AQP4 expression and distribution, and that astrocytes change their expression of AQP4 mRNA as well as the aggregation status of the ensuing protein depending on the ammonia concentration and duration of exposure. Copyright © 2012

  1. Characterization of four plasma membrane aquaporins in tulip petals: a putative homolog is regulated by phosphorylation.

    PubMed

    Azad, Abul Kalam; Katsuhara, Maki; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2008-08-01

    We suggested previously that temperature-dependent tulip (Tulipa gesneriana) petal movement that is concomitant with water transport is regulated by reversible phosphorylation of an unidentified plasma membrane intrinsic protein (PIP). In this study, four full-length cDNAs of PIPs from tulip petals were identified and cloned. Two PIPs, namely TgPIP1;1 and TgPIP1;2, are members of the PIP1 subfamily, and the remaining two PIPs, namely TgPIP2;1 and TgPIP2;2, belong to the PIP2 subfamily of aquaporins and were named according to the nomenclature of PIP genes in plants. Of these four homologs, only TgPIP2;2 displayed significant water channel activity in the heterologous expression assay using Xenopus laevis oocytes. The water channel activity of this functional isoform was abolished by mercury and was affected by inhibitors of protein kinase and protein phosphatase. Using a site-directed mutagenesis approach to substitute several serine residues with alanine, and assessing water channel activity using the methylotrophic yeast Pichia pastoris expression assay, we showed that Ser35, Ser116 and Ser274 are the putative phosphorylation sites of TgPIP2;2. Real-time reverse transcription-PCR analysis revealed that the transcript levels of TgPIP1;1 and TgPIP1;2 in tulip petals, stems, leaves, bulbs and roots are very low when compared with those of TgPIP2;1 and TgPIP2;2. The transcript level of TgPIP2;1 is negligible in roots, and TgPIP2;2 is ubiquitously expressed in all organs with significant transcript levels. From the data reported herein, we suggest that TgPIP2;2 might be modulated by phosphorylation and dephosphorylation for regulating water channel activity, and may play a role in transcellular water transport in all tulip organs.

  2. Maize plasma membrane aquaporin ZmPIP2;5, but not ZmPIP1;2, facilitates transmembrane diffusion of hydrogen peroxide.

    PubMed

    Bienert, Gerd P; Heinen, Robert B; Berny, Marie C; Chaumont, François

    2014-01-01

    Plant aquaporins play important roles in transmembrane water transport processes, but some also facilitate the diffusion of other small uncharged solutes ranging from gases to metalloids. Recent evidence suggests that the transmembrane movement of hydrogen peroxide, an intra- and intercellular multifunctional signaling and defense compound, can be regulated by aquaporins. We addressed the question whether maize aquaporins belonging to the plasma membrane intrinsic protein (PIP) subfamily facilitate hydrogen peroxide diffusion using heterologous expression in the yeast Saccharomyces cerevisiae. We showed that ZmPIP proteins belonging to the PIP1 and PIP2 groups were significantly expressed in yeast cells only after codon optimization of their cDNA. In accordance with previous localization studies in oocytes and plants, ZmPIP1;2 was mainly retained in intracellular membranes, while ZmPIP2;5 was localized to the plasma membrane. However, upon co-expression with ZmPIP2;5, ZmPIP1;2 was re-localized to the plasma membrane. Using a non-functional plasma membrane-localized ZmPIP2;5 mutant to deliver ZmPIP1;2 to the plasma membrane, we demonstrated that, in contrast to wild type ZmPIP2;5, ZmPIP1;2 was not permeable to hydrogen peroxide. Our study further highlighted the fact that, when using the yeast system, which is widely employed to study substrates for plant aquaporins and other transporters, although positive transport assay results allow direct conclusions to be drawn regarding solute permeability, negative results require additional control experiments to show that the protein is expressed and localized correctly before concluding on the lack of transport activity. © 2013.

  3. Virus-induced plasma membrane aquaporin PsPIP2;1 silencing inhibits plant water transport of Pisum sativum.

    PubMed

    Song, Juanjuan; Ye, Guoliang; Qian, Zhengjiang; Ye, Qing

    2016-12-01

    Aquaporins (AQPs) are known to facilitate water transport across cell membranes, but the role of a single AQP in regulating plant water transport, particularly in plants other than Arabidopsis remains largely unexplored. In the present study, a virus-induced gene silencing (VIGS) technique was employed to suppress the expression of a specific plasma membrane aquaporin PsPIP2;1 of Pea plants (Pisum sativum), and subsequent effects of the gene suppression on root hydraulic conductivity (Lpr), leaf hydraulic conductivity (K leaf ), root cell hydraulic conductivity (Lprc), and leaf cell hydraulic conductivity (Lplc) were investigated, using hydroponically grown Pea plants. Compared with control plants, VIGS-PsPIP2;1 plants displayed a significant suppression of PsPIP2;1 in both roots and leaves, while the expression of other four PIP isoforms (PsPIP1;1, PsPIP1;2, PsPIP2;2, and PsPIP2;3) that were simultaneously monitored were not altered. As a consequence, significant declines in water transport of VIGS-PsPIP2;1 plants were observed at both organ and cell levels, i.e., as compared to control plants, Lpr and K leaf were reduced by 29 %, and Lprc and Lplc were reduced by 20 and 29 %, respectively. Our results demonstrate that PsPIP2;1 alone contributes substantially to root and leaf water transport in Pea plants, and highlight VIGS a useful tool for investigating the role of a single AQP in regulating plant water transport.

  4. Tonoplast- and Plasma Membrane-Localized Aquaporin-Family Transporters in Blue Hydrangea Sepals of Aluminum Hyperaccumulating Plant

    PubMed Central

    Negishi, Takashi; Oshima, Kenshiro; Hattori, Masahira; Kanai, Masatake; Mano, Shoji; Nishimura, Mikio; Yoshida, Kumi

    2012-01-01

    Hydrangea (Hydrangea macrophylla) is tolerant of acidic soils in which toxicity generally arises from the presence of the soluble aluminum (Al) ion. When hydrangea is cultivated in acidic soil, its resulting blue sepal color is caused by the Al complex formation of anthocyanin. The concentration of vacuolar Al in blue sepal cells can reach levels in excess of approximately 15 mM, suggesting the existence of an Al-transport and/or storage system. However, until now, no Al transporter has been identified in Al hyperaccumulating plants, animals or microorganisms. To identify the transporter being responsible for Al hyperaccumulation, we prepared a cDNA library from blue sepals according to the sepal maturation stage, and then selected candidate genes using a microarray analysis and an in silico study. Here, we identified the vacuolar and plasma membrane-localized Al transporters genes vacuolar Al transporter (VALT) and plasma membrane Al transporter 1 (PALT1), respectively, which are both members of the aquaporin family. The localization of each protein was confirmed by the transient co-expression of the genes. Reverse transcription-PCR and immunoblotting results indicated that VALT and PALT1 are highly expressed in sepal tissue. The overexpression of VALT and PALT1 in Arabidopsis thaliana conferred Al-tolerance and Al-sensitivity, respectively. PMID:22952644

  5. Ectopic expression of Arabidopsis thaliana plasma membrane intrinsic protein 2 aquaporins in lily pollen increases the plasma membrane water permeability of grain but not of tube protoplasts

    PubMed Central

    Sommer, Aniela; Geist, Birgit; Da Ines, Olivier; Gehwolf, Renate; Schäffner, Anton R.; Obermeyer, Gerhard

    2010-01-01

    Summary To investigate the role of aquaporin-mediated water transport during pollen grain germination and tube growth, Arabidopsis thaliana plasma membrane intrinsic proteins (PIPs) were expressed in pollen of Lilium longiflorum (lily). Successful expression of AtPIPs in particle-bombarded lily pollen grains was monitored by co-expression with fluorescent proteins and single-cell RT-PCR, and by measuring the water permeability coefficient (Pos) in swelling assays using protoplasts prepared from transformed pollen grains and tubes. Expression of AtPIP1;1 and AtPIP1;2 in pollen grains resulted in Pos values similar to those measured in nontransformed pollen grain protoplasts (6.65 ± 2.41 μm s−1), whereas expression of AtPIP2 significantly increased Pos (AtPIP2;1, 13.79 ± 6.38; AtPIP2;2, 10.16 ± 3.30 μm s−1). Transformation with combinations of AtPIP1 and AtPIP2 did not further enhance Pos. Native pollen tube protoplasts showed higher Pos values (13.23 ± 4.14 μm s−1) than pollen grain protoplasts but expression of AtPIP2;1 (18.85 ± 7.60 μm s−1) did not significantly increase their Pos values. Expression of none of the tested PIPs had any effect on pollen tube growth rates. The ectopic expression of AtPIP2s in lily pollen increased the water permeability of the plasma membrane in pollen grains, but not in pollen tubes. The measured endogenous water permeability does not limit water uptake during tube growth, but has to be regulated to prevent tube bursting. PMID:18761636

  6. Solanaceae XIPs are plasma membrane aquaporins that facilitate the transport of many uncharged substrates.

    PubMed

    Bienert, Gerd Patrick; Bienert, Manuela Désirée; Jahn, Thomas Paul; Boutry, Marc; Chaumont, François

    2011-04-01

    Major intrinsic proteins (MIPs) transport water and uncharged solutes across membranes in all kingdoms of life. Recently, an uncharacterized MIP subfamily was identified in the genomes of plants and fungi and named X Intrinsic Proteins (XIPs). Here, we describe the genetic features, localization, expression, and functions of a group of Solanaceae XIPs. XIP cDNA and gDNA were cloned from tobacco, potato, tomato, and morning glory. A conserved sequence motif in the first intron of Solanaceae XIPs initiates an RNA-processing mechanism that results in two splice variants (α and β). When transiently or stably expressed in tobacco plants, yellow fluorescent protein-tagged NtXIP1;1α and NtXIP1;1β were both localized in the plasma membrane. Transgenic tobacco lines expressing NtXIP1;1-promoter-GUS constructs and RT-PCR studies showed that NtXIP1;1 was expressed in all organs. The NtXIP1;1 promoter was mainly active in cell layers facing the environment in all above-ground tissues. Heterologous expression of Solanaceae XIPs in Xenopus laevis oocytes and various Saccharomyces cerevisiae mutants demonstrated that these isoforms facilitate the transport of bulky solutes, such as glycerol, urea, and boric acid. In contrast, permeability for water was undetectable. These data suggest that XIPs function in the transport of uncharged solutes across the cell plasma membrane in specific plant tissues, including at the interface between the environment and external cell layers.

  7. Differential responses of plasma membrane aquaporins in mediating water transport of cucumber seedlings under osmotic and salt stresses.

    PubMed

    Qian, Zheng-Jiang; Song, Juan-Juan; Chaumont, François; Ye, Qing

    2015-03-01

    It has long been recognized that inhibition of plant water transport by either osmotic stress or salinity is mediated by aquaporins (AQPs), but the function and regulation of AQPs are highly variable among distinct isoforms and across different species. In this study, cucumber seedlings were subjected to polyethylene glycol (PEG) or NaCl stress for duration of 2 h or 24 h. The 2 h treatment with PEG or NaCl had non-significant effect on the expression of plasma membrane AQP (CsPIPs) in roots, indicating the decrease in hydraulic conductivity of roots (Lpr ) and root cells (Lprc ) measured in these conditions were due to changes in AQP activity. After both 2 h and 24 h PEG or NaCl exposure, the decrease in hydraulic conductivity of leaves (Kleaf ) and leaf cells (Lplc ) could be attributed to a down-regulation of the two most highly expressed isoforms, CsPIP1;2 and CsPIP2;4. In roots, both Lpr and Lprc were further reduced after 24 h PEG exposure, but partially recovered after 24 h NaCl treatment, which were consistent with changes in the expression of CsPIP genes. Overall, the results demonstrated differential responses of CsPIPs in mediating water transport of cucumber seedlings, and the regulatory mechanisms differed according to applied stresses, stress durations and specific organs. © 2014 John Wiley & Sons Ltd.

  8. Effect of overexpression of radish plasma membrane aquaporins on water-use efficiency, photosynthesis and growth of Eucalyptus trees.

    PubMed

    Tsuchihira, Ayako; Hanba, Yuko T; Kato, Naoki; Doi, Tomonori; Kawazu, Tetsu; Maeshima, Masayoshi

    2010-03-01

    Eucalyptus is a diverse genus of flowering trees with more than 700 genotypic species which are mostly native to Australia. We selected 19 wild provenances of Eucalyptus camaldulensis grown in Australia, compared their growth rate and drought tolerance and determined the protein levels of plasma membrane aquaporins (PIPs). There was a positive relationship between the drought tolerance and PIP content. PIPs are divided into two subgroups, PIP1 and PIP2. Most members of the PIP2 subgroup, but not PIP1 subgroup, exhibit water channel activity. We introduced two radish (Raphanus sativus L.) PIPs, RsPIP1;1 and RsPIP2;1, into a hybrid clone of Eucalyptus grandis and Eucalyptus urophylla to examine the effect of their overexpression. Expression of these genes was confirmed by real-time polymerase chain reaction (PCR) and the protein accumulation of RsPIP2;1 by immunoblotting. Drought tolerance was not enhanced in transgenic lines of either gene. However, one transgenic line expressing RsPIP2;1 showed high photosynthesis activity and growth rate under normal growth conditions. For RsPIP1;1-transformed lines, the RsPIP1;1 protein did not accumulate, and the abundance of endogenous PIP1 and PIP2 was decreased. The endogenous PIP1 and PIP2 genes were suppressed in these lines. Therefore, the decreased levels of PIP1 and PIP2 protein may be due to co-suppression of the PIP genes and/or high turnover of PIP proteins. RsPIP1;1-expressing lines gave low values of photosynthesis and growth compared with the control. These results suggest that down-regulation of PIP1 and PIP2 causes serious damage and that up-regulation of PIP2 improves the photosynthetic activity and growth of Eucalyptus trees.

  9. Aquaporin, forward osmosis and biomimetic membranes.

    PubMed

    Kocherginsky, Nikolai

    2013-12-01

    Aquaporin attracted attention not only of physiologists and biophysicists, but also of chemical engineers. Here we critically analyze a paper describing aquaporin-based artificial membranes, suggested for forward osmosis-based water purification (Wang et al. 2012, Small 8, pp. 1185-1190). Related papers published later by the same group are also discussed. We indicate recently developed general approach to describe membrane transport, membrane permeability and selectivity, which is applicable for forward osmosis. In addition, we also mention our papers describing simple nitrocellulose-based membranes, which have selective aqueous channels without proteins, but successfully imitate many properties of biomembranes.

  10. Regulation of plasma membrane aquaporins by inoculation with a Bacillus megaterium strain in maize (Zea mays L.) plants under unstressed and salt-stressed conditions.

    PubMed

    Marulanda, Adriana; Azcón, Rosario; Chaumont, François; Ruiz-Lozano, Juan Manuel; Aroca, Ricardo

    2010-07-01

    It is documented that some plant-growth-promoting rhizobacteria (PGPR) enhance plant salt tolerance. However, as to how PGPR may influence two crucial components of plant salt tolerance such as, root hydraulic characteristics and aquaporin regulation has been almost unexplored. Here, maize (Zea mays L.) plants were inoculated with a Bacillus megaterium strain previously isolated from a degraded soil and characterized as PGPR. Inoculated plants were found to exhibit higher root hydraulic conductance (L) values under both unstressed and salt-stressed conditions. These higher L values in inoculated plants correlated with higher plasma membrane type two (PIP2) aquaporin amount in their roots under salt-stressed conditions. Also, ZmPIP1;1 protein amount under salt-stressed conditions was higher in inoculated leaves than in non-inoculated ones. Hence, the different regulation of PIP aquaporin expression and abundance by the inoculation with the B. megaterium strain could be one of the causes of the different salt response in terms of root growth, necrotic leaf area, leaf relative water content and L by the inoculation treatment.

  11. Aquaporins

    PubMed Central

    2012-01-01

    While it is well known that a balanced level of hydration is fundamental for healthy skin, the physiological mechanisms underlying the control of hydration, particularly in the epidermis, are yet to be fully elucidated. Over the past 10 years, much research has been carried out to understand the nature and regulation of the water gradient that exists across the layers of the epidermis. Of central importance is the role played by membrane-bound pores called aquaporins, which facilitate the passage of water and, in some cases, small molecules such as glycerol. This paper provides an overview of the principal aquaporin present in the epidermis, aquaporin 3, and how the level of hydration of the epidermis is correlated to endogenous levels of glycerol and to the distribution of aquaporin 3 channels. The role of aquaporin 3 in skin diseases is considered along with possible clinical implications of aquaporin 3 modulation. PMID:22798977

  12. Loop B serine of a plasma membrane aquaporin type PIP2 but not PIP1 plays a key role in pH sensing.

    PubMed

    Yaneff, Agustín; Sigaut, Lorena; Gómez, Natalia; Aliaga Fandiño, Cecilia; Alleva, Karina; Pietrasanta, Lía Isabel; Amodeo, Gabriela

    2016-11-01

    In the plant kingdom, the plasma membrane intrinsic aquaporins (PIPs) constitute a highly conserved group of water channels with the capacity of rapidly adjusting the water permeability (Pf) of a cell by a gating response. Most evidence regarding this mechanism was obtained by different biophysical approaches including the crystallization of a Spinaca olaracea PIP2 aquaporin (SoPIP2;1) in an open and close conformation. A close state seems to prevail under certain stimuli such as cytosolic pH decrease, intracellular Ca(2+) concentration increase and dephosphorylation of specific serines. In this work we decided to address whether the state of phosphorylation of a loop B serine - highly conserved in all PIPs - combined with cytosolic acidification can jointly affect the gating response. To achieve this goal we generated loop B serine mutants of two PIP types of Fragaria×ananassa (FaPIP2;1S121A and FaPIP1;1S131A) in order to simulate a dephosphorylated state and characterize their behavior in terms of Pf and pH sensitivities. The response was tested for different co-expressions of PIPs (homo and heterotetramers combining wild-type and mutant PIPs) in Xenopus oocytes. Our results show that loop B serine phosphorylation status affects pH gating of FaPIP2;1 but not of FaPIP1;1 by changing its sensitivity to more alkaline pHs. Therefore, we propose that a counterpoint of different regulatory mechanisms - heterotetramerization, serine phosphorylation status and pH sensitivity - affect aquaporin gating thus ruling the Pf of a membrane that expresses PIPs when fast responses are mandatory.

  13. A fruit-specific plasma membrane aquaporin subtype PIP1;1 is regulated during strawberry (Fragaria x ananassa) fruit ripening.

    PubMed

    Mut, Paula; Bustamante, Claudia; Martínez, Gustavo; Alleva, Karina; Sutka, Moira; Civello, Marcos; Amodeo, Gabriela

    2008-04-01

    Despite the advances in the physiology of fruit ripening, the role and contribution of water pathways are still barely considered. Our aim was therefore to characterize aquaporins, proteins that render the molecular basis for putative regulatory mechanisms in water transport. We focused our work on strawberry (Fragaria xananassa) fruit, a non-climacteric fruit of special interest because of its forced brief commercial shelf life. A full-length cDNA was isolated with high homology with plasma membrane (PM) intrinsic proteins (named FaPIP1;1), showing a profile with high expression in fruit, less in ovaries and no detection at all in other parts. Its cellular localization was confirmed at the PM. As reported in other plasma membrane intrinsic proteins subtype 1 (PIP1s), when expressing the protein in Xenopus leavis oocytes, FaPIP1;1 shows low water permeability values that only increased when it is coexpressed with a plasma membrane intrinsic protein subtype 2. Northern blotting using total RNA shows that its expression increases during fruit ripening. Moreover, functional characterization of isolated PM vesicles from red stage fruit unequivocally demonstrates the presence of active water channels, i.e. high water permeability values and a low Arrhenius activation energy, both evidences of water transport mediated by proteins. Interestingly, as many ripening-related strawberry genes, the expression pattern of FaPIP1;1 was also repressed by the presence of auxins. We therefore report a fruit specific PIP1 aquaporin with an accumulation pattern tightly associated to auxins and to the ripening process that might be responsible for increasing water permeability at the level of the PM in ripe fruit.

  14. Recombinant Production of Human Aquaporin-1 to an Exceptional High Membrane Density in Saccharomyces cerevisiae

    PubMed Central

    Bomholt, Julie; Hélix-Nielsen, Claus; Scharff-Poulsen, Peter; Pedersen, Per Amstrup

    2013-01-01

    In the present paper we explored the capacity of yeast Saccharomyces cerevisiae as host for heterologous expression of human Aquaporin-1. Aquaporin-1 cDNA was expressed from a galactose inducible promoter situated on a plasmid with an adjustable copy number. Human Aquaporin-1 was C-terminally tagged with yeast enhanced GFP for quantification of functional expression, determination of sub-cellular localization, estimation of in vivo folding efficiency and establishment of a purification protocol. Aquaporin-1 was found to constitute 8.5 percent of total membrane protein content after expression at 15°C in a yeast host over-producing the Gal4p transcriptional activator and growth in amino acid supplemented minimal medium. In-gel fluorescence combined with western blotting showed that low accumulation of correctly folded recombinant Aquaporin-1 at 30°C was due to in vivo mal-folding. Reduction of the expression temperature to 15°C almost completely prevented Aquaporin-1 mal-folding. Bioimaging of live yeast cells revealed that recombinant Aquaporin-1 accumulated in the yeast plasma membrane. A detergent screen for solubilization revealed that CYMAL-5 was superior in solubilizing recombinant Aquaporin-1 and generated a monodisperse protein preparation. A single Ni-affinity chromatography step was used to obtain almost pure Aquaporin-1. Recombinant Aquaporin-1 produced in S. cerevisiae was not N-glycosylated in contrast to the protein found in human erythrocytes. PMID:23409185

  15. Neuromyelitis Optica Immunoglobulin G present in sera from neuromyelitis optica patients affects aquaporin-4 expression and water permeability of the astrocyte plasma membrane.

    PubMed

    Melamud, Luciana; Fernández, Juan M; Rivarola, Valeria; Di Giusto, Gisela; Ford, Paula; Villa, Andrés; Capurro, Claudia

    2012-06-01

    NMO-IgG autoantibody selectively binds to aquaporin-4 (AQP4), the most abundant water channel in the central nervous system and is now considered a useful serum biomarker of neuromyelitis optica (NMO). A series of clinical and pathological observations suggests that NMO-IgG may play a central role in NMO physiopathology. The current study evaluated, in well-differentiated astrocytes cultures, the consequences of NMO-IgG binding on the expression pattern of AQP4 and on plasma membrane water permeability. To avoid or to facilitate AQP4 down-regulation, cells were exposed to inactivated sera in two different situations (1 hr at 4°C or 12 hr at 37°C). AQP4 expression was detected by immunofluorescence studies using a polyclonal anti-AQP4 or a human anti-IgG antibody, and the water permeability coefficient was evaluated by a videomicroscopy technique. Our results showed that, at low temperatures, cell exposure to either control or NMO-IgG sera does not affect either AQP4 expression or plasma membrane water permeability, indicating that the simple binding of NMO-IgG does not affect the water channel's activity. However, at 37°C, long-term exposure to NMO-IgG induced a loss of human IgG signal from the plasma membrane along with M1-AQP4 isoform removal and a significant reduction of water permeability. These results suggest that binding of NMO-IgG to cell membranes expressing AQP4 is a specific mechanism that may account for at least part of the pathogenic process. Copyright © 2012 Wiley Periodicals, Inc.

  16. The role of plasma membrane intrinsic protein aquaporins in water transport through roots: diurnal and drought stress responses reveal different strategies between isohydric and anisohydric cultivars of grapevine.

    PubMed

    Vandeleur, Rebecca K; Mayo, Gwenda; Shelden, Megan C; Gilliham, Matthew; Kaiser, Brent N; Tyerman, Stephen D

    2009-01-01

    We report physiological and anatomical characteristics of water transport across roots grown in soil of two cultivars of grapevine (Vitis vinifera) differing in response to water stress (Grenache, isohydric; Chardonnay, anisohydric). Both cultivars have similar root hydraulic conductances (Lo; normalized to root dry weight) that change diurnally. There is a positive correlation between Lo and transpiration. Under water stress, both cultivars have reduced minimum daily Lo (predawn) attributed to the development of apoplastic barriers. Water-stressed and well-watered Chardonnay had the same diurnal change in amplitude of Lo, while water-stressed Grenache showed a reduction in daily amplitude compared with well-watered plants. Hydraulic conductivity of root cortex cells (Lpcell) doubles in Chardonnay but remains unchanged in Grenache. Of the two most highly expressed plasma membrane intrinsic protein (PIP) aquaporins in roots (VvPIP1;1 and VvPIP2;2), only VvPIP2;2 functions as a water channel in Xenopus laevis oocytes. VvPIP1;1 interacts with VvPIP2;2 to induce 3-fold higher water permeability. These two aquaporins are colocated in the root from in situ hybridization and immunolocalization of VvPIP1 and VvPIP2 subfamily members. They occur in root tip, exodermis, root cortex (detected up to 30 mm), and stele. VvPIP2;2 mRNA does not change diurnally or with water stress, in contrast to VvPIP1;1, in which expression reflects the differences in Lo and Lpcell between cultivars in their responses to water stress and rewatering. VvPIP1;1 may regulate water transport across roots such that transpirational demand is matched by root water transport capacity. This occurs on a diurnal basis and in response to water stress that corresponds to the difference in drought tolerance between the cultivars.

  17. Transgenic banana plants overexpressing a native plasma membrane aquaporin MusaPIP1;2 display high tolerance levels to different abiotic stresses.

    PubMed

    Sreedharan, Shareena; Shekhawat, Upendra K S; Ganapathi, Thumballi R

    2013-10-01

    Water transport across cellular membranes is regulated by a family of water channel proteins known as aquaporins (AQPs). As most abiotic stresses like suboptimal temperatures, drought or salinity result in cellular dehydration, it is imperative to study the cause-effect relationship between AQPs and the cellular consequences of abiotic stress stimuli. Although plant cells have a high isoform diversity of AQPs, the individual and integrated roles of individual AQPs in optimal and suboptimal physiological conditions remain unclear. Herein, we have identified a plasma membrane intrinsic protein gene (MusaPIP1;2) from banana and characterized it by overexpression in transgenic banana plants. Cellular localization assay performed using MusaPIP1;2::GFP fusion protein indicated that MusaPIP1;2 translocated to plasma membrane in transformed banana cells. Transgenic banana plants overexpressing MusaPIP1;2 constitutively displayed better abiotic stress survival characteristics. The transgenic lines had lower malondialdehyde levels, elevated proline and relative water content and higher photosynthetic efficiency as compared to equivalent controls under different abiotic stress conditions. Greenhouse-maintained hardened transgenic plants showed faster recovery towards normal growth and development after cessation of abiotic stress stimuli, thereby underlining the importance of these plants in actual environmental conditions wherein the stress stimuli is often transient but severe. Further, transgenic plants where the overexpression of MusaPIP1;2 was made conditional by tagging it with a stress-inducible native dehydrin promoter also showed similar stress tolerance characteristics in in vitro and in vivo assays. Plants developed in this study could potentially enable banana cultivation in areas where adverse environmental conditions hitherto preclude commercial banana cultivation.

  18. Dual regulation of root hydraulic conductivity and plasma membrane aquaporins by plant nitrate accumulation and high-affinity nitrate transporter NRT2.1.

    PubMed

    Li, Guowei; Tillard, Pascal; Gojon, Alain; Maurel, Christophe

    2016-04-01

    The water status and mineral nutrition of plants critically determine their growth and development. Nitrate (NO3(-)), the primary nitrogen source of higher plants, is known to impact the water transport capacity of roots (root hydraulic conductivity, Lpr). To explore the effects and mode of action of NO3(-) on Lpr, we used an extended set of NO3(-) transport (nrt1.1, nrt1.2, nrt1.5 and nrt2.1), signaling (nrt1.1 and nrt2.1) and metabolism (nia) mutants in Arabidopsis, grown under various NO3(-) conditions. First, a strong positive relationship between Lpr and NO3(-) accumulation, in shoots rather than in roots, was revealed. Secondly, a specific 30% reduction of Lpr in nrt2.1 plants unraveled a major role for the high-affinity NO3(-) transporter NRT2.1 in increasing Lpr These results indicate that NO3(-)signaling rather than nitrogen assimilation products governs Lpr in Arabidopsis. Quantitative real-time reverse transcription-PCR and enzyme-linked immunosorbent assays (ELISAs) were used to investigate the effects of NO3(-) availability on plasma membrane aquaporin (plasma membrane intrinsic protein; PIP) expression. Whereas PIP regulation mostly occurs at the post-translational level in wild-type plants, a regulation of PIPs at both the transcriptional and translational levels was uncovered in nrt2.1 plants. In conclusion, this work reveals that control of Arabidopsis Lpr and PIP functions by NO3(-) involves novel shoot to root signaling and NRT2.1-dependent functions.

  19. Aquaporins and membrane diffusion of CO2 in living organisms.

    PubMed

    Kaldenhoff, Ralf; Kai, Lei; Uehlein, Norbert

    2014-05-01

    Determination of CO2 diffusion rates in living cells revealed inconsistencies with existing models about the mechanisms of membrane gas transport. Mainly, these discrepancies exist in the determined CO2 diffusion rates of bio-membranes, which were orders of magnitudes below those for pure lipid bilayers or theoretical considerations as well as in the observation that membrane insertion of specific aquaporins was rescuing high CO2 transport rates. This effect was confirmed by functional aquaporin protein analysis in heterologous expression systems as well as in bacteria, plants and partly in mammals. This review summarizes the arguments in favor of and against aquaporin facilitated membrane diffusion of CO2 and reports about its importance for the physiology of living organisms. Most likely, the aquaporin tetramer forming an additional fifth pore is required for CO2 diffusion facilitation. Aquaporin tetramer formation, membrane integration and disintegration could provide a mechanism for regulation of cellular CO2 exchange. The physiological importance of aquaporin mediated CO2 membrane diffusion could be shown for plants and cyanobacteria and partly for mammals. Taking the mentioned results into account, consequences for our current picture of cell membrane transport emerge. It appears that in some or many instances, membranes might not be as permeable as it was suggested by current bio-membrane models, opening an additional way of controlling the cellular influx or efflux of volatile substances like CO2. This article is part of a Special Issue entitled Aquaporins. © 2013.

  20. Aquaporins: water channel proteins of the cell membrane.

    PubMed

    Takata, Kuniaki; Matsuzaki, Toshiyuki; Tajika, Yuki

    2004-01-01

    Aquaporins (AQP) are integral membrane proteins that serve as channels in the transfer of water, and in some cases, small solutes across the membrane. They are conserved in bacteria, plants, and animals. Structural analyses of the molecules have revealed the presence of a pore in the center of each aquaporin molecule. In mammalian cells, more than 10 isoforms (AQP0-AQP10) have been identified so far. They are differentially expressed in many types of cells and tissues in the body. AQP0 is abundant in the lens. AQP1 is found in the blood vessels, kidney proximal tubules, eye, and ear. AQP2 is expressed in the kidney collecting ducts, where it shuttles between the intracellular storage sites and the plasma membrane under the control of antidiuretic hormone (ADH). Mutations of AQP2 result in diabetes insipidus. AQP3 is present in the kidney collecting ducts, epidermis, urinary, respiratory, and digestive tracts. AQP3 in organs other than the kidney may be involved in the supply of water to them. AQP4 is present in the brain astrocytes, eye, ear, skeletal muscle, stomach parietal cells, and kidney collecting ducts. AQP5 is in the secretory cells such as salivary, lacrimal, and sweat glands. AQP5 is also expressed in the ear and eye. AQP6 is localized intracellular vesicles in the kidney collecting duct cells. AQP7 is expressed in the adipocytes, testis, and kidney. AQP8 is expressed in the kidney, testis, and liver. AQP9 is present in the liver and leukocytes. AQP10 is expressed in the intestine. The diverse and characteristic distribution of aquaporins in the body suggests their important and specific roles in each organ.

  1. Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes.

    PubMed

    Bienert, Gerd P; Møller, Anders L B; Kristiansen, Kim A; Schulz, Alexander; Møller, Ian M; Schjoerring, Jan K; Jahn, Thomas P

    2007-01-12

    The metabolism of aerobic organisms continuously produces reactive oxygen species. Although potentially toxic, these compounds also function in signaling. One important feature of signaling compounds is their ability to move between different compartments, e.g. to cross membranes. Here we present evidence that aquaporins can channel hydrogen peroxide (H2O2). Twenty-four aquaporins from plants and mammals were screened in five yeast strains differing in sensitivity toward oxidative stress. Expression of human AQP8 and plant Arabidopsis TIP1;1 and TIP1;2 in yeast decreased growth and survival in the presence of H2O2. Further evidence for aquaporin-mediated H2O2 diffusion was obtained by a fluorescence assay with intact yeast cells using an intracellular reactive oxygen species-sensitive fluorescent dye. Application of silver ions (Ag+), which block aquaporin-mediated water diffusion in a fast kinetics swelling assay, also reversed both the aquaporin-dependent growth repression and the H2O2-induced fluorescence. Our results present the first molecular genetic evidence for the diffusion of H2O2 through specific members of the aquaporin family.

  2. Aquaporin-Based Biomimetic Polymeric Membranes: Approaches and Challenges

    PubMed Central

    Habel, Joachim; Hansen, Michael; Kynde, Søren; Larsen, Nanna; Midtgaard, Søren Roi; Jensen, Grethe Vestergaard; Bomholt, Julie; Ogbonna, Anayo; Almdal, Kristoffer; Schulz, Alexander; Hélix-Nielsen, Claus

    2015-01-01

    In recent years, aquaporin biomimetic membranes (ABMs) for water separation have gained considerable interest. Although the first ABMs are commercially available, there are still many challenges associated with further ABM development. Here, we discuss the interplay of the main components of ABMs: aquaporin proteins (AQPs), block copolymers for AQP reconstitution, and polymer-based supporting structures. First, we briefly cover challenges and review recent developments in understanding the interplay between AQP and block copolymers. Second, we review some experimental characterization methods for investigating AQP incorporation including freeze-fracture transmission electron microscopy, fluorescence correlation spectroscopy, stopped-flow light scattering, and small-angle X-ray scattering. Third, we focus on recent efforts in embedding reconstituted AQPs in membrane designs that are based on conventional thin film interfacial polymerization techniques. Finally, we describe some new developments in interfacial polymerization using polyhedral oligomeric silsesquioxane cages for increasing the physical and chemical durability of thin film composite membranes. PMID:26264033

  3. Aquaporin-Based Biomimetic Polymeric Membranes: Approaches and Challenges.

    PubMed

    Habel, Joachim; Hansen, Michael; Kynde, Søren; Larsen, Nanna; Midtgaard, Søren Roi; Jensen, Grethe Vestergaard; Bomholt, Julie; Ogbonna, Anayo; Almdal, Kristoffer; Schulz, Alexander; Hélix-Nielsen, Claus

    2015-07-31

    In recent years, aquaporin biomimetic membranes (ABMs) for water separation have gained considerable interest. Although the first ABMs are commercially available, there are still many challenges associated with further ABM development. Here, we discuss the interplay of the main components of ABMs: aquaporin proteins (AQPs), block copolymers for AQP reconstitution, and polymer-based supporting structures. First, we briefly cover challenges and review recent developments in understanding the interplay between AQP and block copolymers. Second, we review some experimental characterization methods for investigating AQP incorporation including freeze-fracture transmission electron microscopy, fluorescence correlation spectroscopy, stopped-flow light scattering, and small-angle X-ray scattering. Third, we focus on recent efforts in embedding reconstituted AQPs in membrane designs that are based on conventional thin film interfacial polymerization techniques. Finally, we describe some new developments in interfacial polymerization using polyhedral oligomeric silsesquioxane cages for increasing the physical and chemical durability of thin film composite membranes.

  4. Tonoplast (BvTIP1;2) and plasma membrane (BvPIP2;1) aquaporins show different mechanosensitive properties.

    PubMed

    Goldman, Ramiro P; Jozefkowicz, Cintia; Canessa Fortuna, Agustina; Sutka, Moira; Alleva, Karina; Ozu, Marcelo

    2017-06-01

    Previous works proposed that aquaporins behave as mechanosensitive channels. However, principal issues about mechanosensitivity of aquaporins are not known. In this work, we characterized the mechanosensitive properties of the water channels BvTIP1;2 (TIP1) and BvPIP2;1 (PIP2) from red beet (Beta vulgaris). We simultaneously measured the mechanical behavior and the water transport rates during the osmotic response of emptied-out oocytes expressing TIP1 or PIP2. Our results indicate that TIP1 is a mechanosensitive aquaporin, whereas PIP2 is not. We found that a single exponential function between the osmotic permeability coefficient and the volumetric elastic modulus governs the mechanosensitivity of TIP1. Finally, homology modeling analysis indicates that putative residues involved in mechanosensitivity show different quantity and distribution in TIP1 and PIP2. © 2017 Federation of European Biochemical Societies.

  5. The Role of Plasma Membrane Intrinsic Protein Aquaporins in Water Transport through Roots: Diurnal and Drought Stress Responses Reveal Different Strategies between Isohydric and Anisohydric Cultivars of Grapevine1[OA

    PubMed Central

    Vandeleur, Rebecca K.; Mayo, Gwenda; Shelden, Megan C.; Gilliham, Matthew; Kaiser, Brent N.; Tyerman, Stephen D.

    2009-01-01

    We report physiological and anatomical characteristics of water transport across roots grown in soil of two cultivars of grapevine (Vitis vinifera) differing in response to water stress (Grenache, isohydric; Chardonnay, anisohydric). Both cultivars have similar root hydraulic conductances (Lo; normalized to root dry weight) that change diurnally. There is a positive correlation between Lo and transpiration. Under water stress, both cultivars have reduced minimum daily Lo (predawn) attributed to the development of apoplastic barriers. Water-stressed and well-watered Chardonnay had the same diurnal change in amplitude of Lo, while water-stressed Grenache showed a reduction in daily amplitude compared with well-watered plants. Hydraulic conductivity of root cortex cells (Lpcell) doubles in Chardonnay but remains unchanged in Grenache. Of the two most highly expressed plasma membrane intrinsic protein (PIP) aquaporins in roots (VvPIP1;1 and VvPIP2;2), only VvPIP2;2 functions as a water channel in Xenopus laevis oocytes. VvPIP1;1 interacts with VvPIP2;2 to induce 3-fold higher water permeability. These two aquaporins are colocated in the root from in situ hybridization and immunolocalization of VvPIP1 and VvPIP2 subfamily members. They occur in root tip, exodermis, root cortex (detected up to 30 mm), and stele. VvPIP2;2 mRNA does not change diurnally or with water stress, in contrast to VvPIP1;1, in which expression reflects the differences in Lo and Lpcell between cultivars in their responses to water stress and rewatering. VvPIP1;1 may regulate water transport across roots such that transpirational demand is matched by root water transport capacity. This occurs on a diurnal basis and in response to water stress that corresponds to the difference in drought tolerance between the cultivars. PMID:18987216

  6. The Interactions of Aquaporins and Mineral Nutrients in Higher Plants.

    PubMed

    Wang, Min; Ding, Lei; Gao, Limin; Li, Yingrui; Shen, Qirong; Guo, Shiwei

    2016-07-29

    Aquaporins, major intrinsic proteins (MIPs) present in the plasma and intracellular membranes, facilitate the transport of small neutral molecules across cell membranes in higher plants. Recently, progress has been made in understanding the mechanisms of aquaporin subcellular localization, transport selectivity, and gating properties. Although the role of aquaporins in maintaining the plant water status has been addressed, the interactions between plant aquaporins and mineral nutrients remain largely unknown. This review highlights the roles of various aquaporin orthologues in mineral nutrient uptake and transport, as well as the regulatory effects of mineral nutrients on aquaporin expression and activity, and an integrated link between aquaporins and mineral nutrient metabolism was identified.

  7. The Interactions of Aquaporins and Mineral Nutrients in Higher Plants

    PubMed Central

    Wang, Min; Ding, Lei; Gao, Limin; Li, Yingrui; Shen, Qirong; Guo, Shiwei

    2016-01-01

    Aquaporins, major intrinsic proteins (MIPs) present in the plasma and intracellular membranes, facilitate the transport of small neutral molecules across cell membranes in higher plants. Recently, progress has been made in understanding the mechanisms of aquaporin subcellular localization, transport selectivity, and gating properties. Although the role of aquaporins in maintaining the plant water status has been addressed, the interactions between plant aquaporins and mineral nutrients remain largely unknown. This review highlights the roles of various aquaporin orthologues in mineral nutrient uptake and transport, as well as the regulatory effects of mineral nutrients on aquaporin expression and activity, and an integrated link between aquaporins and mineral nutrient metabolism was identified. PMID:27483251

  8. Mass Spectrometry of Membrane Proteins: A Focus on Aquaporins

    PubMed Central

    Schey, Kevin L.; Grey, Angus C.; Nicklay, Joshua J.

    2015-01-01

    Membrane proteins are abundant, critically important biomolecules that conduct essential functions in all cells and are the targets of a significant number of therapeutic drugs. However, the analysis of their expression, modification, protein–protein interactions, and structure by mass spectrometry has lagged behind similar studies of soluble proteins. Here we review the limitations to analysis of integral membrane and membrane-associated proteins and highlight advances in sample preparation and mass spectrometry methods that have led to the successful analysis of this protein class. Advances in the analysis of membrane protein posttranslational modification, protein–protein interaction, protein structure, and tissue distributions by imaging mass spectrometry are discussed. Furthermore, we focus our discussion on the application of mass spectrometry for the analysis of aquaporins as a prototypical integral membrane protein and how advances in analytical methods have revealed new biological insights into the structure and function of this family of proteins. PMID:23394619

  9. Mass spectrometry of membrane proteins: a focus on aquaporins.

    PubMed

    Schey, Kevin L; Grey, Angus C; Nicklay, Joshua J

    2013-06-04

    Membrane proteins are abundant, critically important biomolecules that conduct essential functions in all cells and are the targets of a significant number of therapeutic drugs. However, the analysis of their expression, modification, protein-protein interactions, and structure by mass spectrometry has lagged behind similar studies of soluble proteins. Here we review the limitations to analysis of integral membrane and membrane-associated proteins and highlight advances in sample preparation and mass spectrometry methods that have led to the successful analysis of this protein class. Advances in the analysis of membrane protein posttranslational modification, protein-protein interaction, protein structure, and tissue distributions by imaging mass spectrometry are discussed. Furthermore, we focus our discussion on the application of mass spectrometry for the analysis of aquaporins as a prototypical integral membrane protein and how advances in analytical methods have revealed new biological insights into the structure and function of this family of proteins.

  10. Preparation of supported lipid membranes for aquaporin Z incorporation.

    PubMed

    Li, Xuesong; Wang, Rong; Tang, Chuyang; Vararattanavech, Ardcharaporn; Zhao, Yang; Torres, Jaume; Fane, Tony

    2012-06-01

    There has been a recent surge of interest to mimic the performance of natural cellular membranes by incorporating water channel proteins-aquaporins (AQPs) into various ultrathin films for water filtration applications. To make biomimetic membranes one of the most crucial steps is preparing a defect-free platform for AQPs incorporation on a suitable substrate. In this study two methods were used to prepare supported lipid membranes on NF membrane surfaces under a benign pH condition of 7.8. One method was direct vesicle fusion on a hydrophilic membrane NF-270; the other was vesicle fusion facilitated by hydraulic pressure on a modified hydrophilic NF-270 membrane whose surface has been spin-coated with positively charged lipids. Experiments revealed that the supported lipid membrane without AQPs prepared by the spin coating plus vesicle fusion had a much lower defect density than that prepared by vesicle fusion alone. It appears that the surface roughness and charge are the main factors determining the quality of the supported lipid membrane. Aquaporin Z (AqpZ) proteins were successfully incorporated into 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes and its permeability was measured by the stopped-flow experimental procedure. However, after the proteoliposomes have been fused onto the modified substrate, the AqpZ function in the resultant membrane was not observed and AFM images showed distinct aggregations of unfused proteoliposomes or AqpZ proteins on the substrate surface. It is speculated that the inhibition of AqpZ function may be caused by the low lipid mobility on the NF membrane surface. Further investigations to evaluate and optimize the structure-performance relationship are required.

  11. Can Stabilization and Inhibition of Aquaporins Contribute to Future Development of Biomimetic Membranes?

    PubMed Central

    To, Janet; Torres, Jaume

    2015-01-01

    In recent years, the use of biomimetic membranes that incorporate membrane proteins, i.e., biomimetic-hybrid membranes, has increased almost exponentially. Key membrane proteins in these systems have been aquaporins, which selectively permeabilize cellular membranes to water. Aquaporins may be incorporated into synthetic lipid bilayers or to more stable structures made of block copolymers or solid-state nanopores. However, translocation of aquaporins to these alien environments has adverse consequences in terms of performance and stability. Aquaporins incorporated in biomimetic membranes for use in water purification and desalination should also withstand the harsh environment that may prevail in these conditions, such as high pressure, and presence of salt or other chemicals. In this respect, modified aquaporins that can be adapted to these new environments should be developed. Another challenge is that biomimetic membranes that incorporate high densities of aquaporin should be defect-free, and this can only be efficiently ascertained with the availability of completely inactive mutants that behave otherwise like the wild type aquaporin, or with effective non-toxic water channel inhibitors that are so far inexistent. In this review, we describe approaches that can potentially be used to overcome these challenges. PMID:26266425

  12. The grape aquaporin VvSIP1 transports water across the ER membrane.

    PubMed

    Noronha, Henrique; Agasse, Alice; Martins, Ana Paula; Berny, Marie C; Gomes, Dulceneia; Zarrouk, Olfa; Thiebaud, Pierre; Delrot, Serge; Soveral, Graça; Chaumont, François; Gerós, Hernâni

    2014-03-01

    Water diffusion through biological membranes is facilitated by aquaporins, members of the widespread major intrinsic proteins (MIPs). In the present study, the localization, expression, and functional characterization of a small basic intrinsic protein (SIP) from the grapevine were assessed. VvSIP1 was expressed in leaves and berries from field-grown vines, and in leaves and stems from in vitro plantlets, but not in roots. When expressed in tobacco mesophyll cells and in Saccharomyces cerevisiae, fluorescent-tagged VvSIP1 was localized at the endoplasmic reticulum (ER). Stopped-flow spectroscopy showed that VvSIP1-enriched ER membrane vesicles from yeast exhibited higher water permeability and lower activation energy for water transport than control vesicles, indicating the involvement of protein-mediated water diffusion. This aquaporin was able to transport water but not glycerol, urea, sorbitol, glucose, or inositol. VvSIP1 expression in Xenopus oocytes failed to increase the water permeability of the plasma membrane. VvSIP1-His-tag was solubilized and purified to homogeneity from yeast ER membranes and the reconstitution of the purified protein in phosphatidylethanolamine liposomes confirmed its water channel activity. To provide further insights into gene function, the expression of VvSIP1 in mature grapes was studied when vines were cultivated in different field conditions, but its transcript levels did not increase significantly in water-stressed plants and western-exposed berries. However, the expression of the aquaporin genes VvSIP1, VvPIP2;2, and VvTIP1;1 was up-regulated by heat in cultured cells.

  13. Characterization of plant aquaporins.

    PubMed

    Kaldenhoff, Ralf; Bertl, Adam; Otto, Beate; Moshelion, Menachem; Uehlein, Norbert

    2007-01-01

    Plants have been reported to contain a large set of aquaporins (38 for Arabidopsis), which has been divided into four subfamilies on the basis of similarities in their amino acid sequences. They belong to the large superfamily of major intrinsic proteins (MIP), which was the basis for the nomenclature PIP, TIP, and NIP, also indicating the subcellular localization plasma membrane, tonoplast, and nodule of the respective founding member. The fourth subfamily of small and basic intrinsic proteins is not well characterized so far. The increasing number of reports dealing with various aspects of plant aquaporins is starting to advance our understanding of aquaporin biology in plants. Fundamental questions include: what is the basic function of the different plant aquaporins, what is their primary substrate, and what is the consequence of function/malfunction of a particular aquaporin for the overall function of the plant? Biochemical and biophysical techniques can be employed to get information on the basic functional characteristics of plant aquaporins. An impressive set of techniques has been used to study aquaporin function on molecular, subcellular, and cellular levels in plants, as well as in heterologous expression systems. The physiological role of aquaporins in plants is much less well understood, but reports unraveling the physiological role of aquaporins, mainly employing genetic techniques and functional measurement on the whole plant level, are emerging. The goal of this chapter is to give an overview on the applied methods, together with some exemplary findings.

  14. Aquaporins in clinical medicine.

    PubMed

    Verkman, A S

    2012-01-01

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

  15. Boric acid and salinity effects on maize roots. Response of aquaporins ZmPIP1 and ZmPIP2, and plasma membrane H+-ATPase, in relation to water and nutrient uptake.

    PubMed

    Martinez-Ballesta, Maria del Carmen; Bastías, Elizabeth; Zhu, Chuanfeng; Schäffner, Anton R; González-Moro, Begoña; González-Murua, Carmen; Carvajal, Micaela

    2008-04-01

    Under saline conditions, an optimal cell water balance, possibly mediated by aquaporins, is important to maintain the whole-plant water status. Furthermore, excessive accumulation of boric acid in the soil solution can be observed in saline soils. In this work, the interaction between salinity and excess boron with respect to the root hydraulic conductance (L(0)), abundance of aquaporins (ZmPIP1 and ZmPIP2), ATPase activity and root sap nutrient content, in the highly boron- and salt-tolerant Zea mays L. cv. amylacea, was evaluated. A downregulation of root ZmPIP1 and ZmPIP2 aquaporin contents were observed in NaCl-treated plants in agreement with the L(0) measurements. However, in the H3BO3-treated plants differences in the ZmPIP1 and ZmPIP2 abundance were observed. The ATPase activity was related directly to the amount of ATPase protein and Na+ concentration in the roots, for which an increase in NaCl- and H3BO3+ NaCl-treated plants was observed with respect to untreated and H3BO3-treated plants. Although nutrient imbalance may result from the effect of salinity or H3BO3 alone, an ameliorative effect was observed when both treatments were applied together. In conclusion, our results suggest that under salt stress, the activity of specific membrane components can be influenced directly by boric acid, regulating the functions of certain aquaporin isoforms and ATPase as possible components of the salinity tolerance mechanism.

  16. The osmopressor response is linked to upregulation of aquaporin-1 tyrosine phosphorylation on red blood cell membranes.

    PubMed

    Chu, You Hsiang; Hsu, Yu-Juei; Lee, Herng Sheng; Ho, Shung-Tai; Tung, Che-Se; Tseng, Ching-Jiunn; Li, Min-Hui; Lin, Tso-Chou; Lu, Chih-Cherng

    2013-07-01

    Studies in patients with an impaired efferent baroreflex led us to discover that ingesting water induces a robust increase in blood pressure and vascular resistance. This response was also present in healthy subjects with intact baroreflexes, described as osmopressor response. This study was to discover the physiology of the osmopressor response by determining functional activation of the aquaporin-1 water channel receptor on red blood cell membranes in young healthy subjects. In a randomized, controlled, crossover fashion, 22 young healthy subjects (age, 19-27 years) ingested either 500 or 50 mL of water. Heart rate, blood pressure, cardiac index, and total peripheral vascular resistance were measured using a Finometer hemodynamic monitor. Blood sampling was performed at 5 minutes before and at 25 and 50 minutes after either the water ingestion or control session. Immunoblotting for aquaporin-1 tyrosine phosphorylation was performed before and after subjects ingested either 500 or 50 mL of water. At 25 minutes after the ingestion of 500 mL of water, total peripheral resistance increased significantly, and plasma osmolality decreased. Functional expression of aquaporin-1 tyrosine phosphorylation on red blood cell membranes increased significantly at 25 and 50 minutes after subjects ingested 500 mL of water compared with that before water ingestion. This study concludes that water ingestion produces upregulation of aquaporin-1 tyrosine phosphorylation on red blood cell, which presents as a novel biological marker that occurs simultaneously with the osmopressor response.

  17. Aquaporins: New Targets for Cancer Therapy.

    PubMed

    Wang, Liping; Zhang, Yixiang; Wu, Xiongzhi; Yu, Guohua

    2016-12-01

    Aquaporins are a family of integral membrane proteins that are expressed in all living organisms and play vital roles in transcellular and transepithelial water movement. Cell viability and motility are critical for progression of cancer. Cell survival requires the suitable concentration of water and solutes. The balance is largely maintained by aquaporins whose major function is the transport of water and small solutes across the plasma membrane. The important role of aquaporins has received more and more attention in the recent years. A number of recent studies have revealed that aquaporins may be involved in cell migration and angiogenesis. This review will highlight the expression of aquaporins in different malignant neoplasms. Remarkably, we will summarize the influence of drugs on aquaporins, not only the traditional Chinese medicine but also the Western medicine. Therapeutic targeting of aquaporins may thus be advantageous for blocking the mechanism common for a number of key cancer phenotypes. © The Author(s) 2015.

  18. Key roles of aquaporins in tumor biology.

    PubMed

    Papadopoulos, Marios C; Saadoun, Samira

    2015-10-01

    Aquaporins are protein channels that facilitate the flow of water across plasma cell membranes in response to osmotic gradients. This review summarizes the evidence that aquaporins play key roles in tumor biology including tumor-associated edema, tumor cell migration, tumor proliferation and tumor angiogenesis. Aquaporin inhibitors may thus be a novel class of anti-tumor agents. However, attempts to produce small molecule aquaporin inhibitors have been largely unsuccessful. Recently, monoclonal human IgG antibodies against extracellular aquaporin-4 domains have become available and could be engineered to kill aquaporin-4 over-expressing cells in the malignant brain tumor glioblastoma. We conclude this review by discussing future directions in aquaporin tumor research. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Novel fatty acid acylation of lens integral membrane protein aquaporin-0.

    PubMed

    Schey, Kevin L; Gutierrez, Danielle B; Wang, Zhen; Wei, Junhua; Grey, Angus C

    2010-11-16

    Fatty acid acylation of proteins is a well-studied co- or posttranslational modification typically conferring membrane trafficking signals or membrane anchoring properties to proteins. Commonly observed examples of protein acylation include N-terminal myristoylation and palmitoylation of cysteine residues. In the present study, direct tissue profiling mass spectrometry of bovine and human lens sections revealed an abundant signal tentatively assigned as a lipid-modified form of aquaporin-0. LC/MS/MS proteomic analysis of hydrophobic tryptic peptides from lens membrane proteins revealed both N-terminal and C-terminal peptides modified by 238 and 264 Da which were subsequently assigned by accurate mass measurement as palmitoylation and oleoylation, respectively. Specific sites of modification were the N-terminal methionine residue and lysine 238 revealing, for the first time, an oleic acid modification via an amide linkage to a lysine residue. The specific fatty acids involved reflect their abundance in the lens fiber cell plasma membrane. Imaging mass spectrometry indicated abundant acylated AQP0 in the inner cortical region of both bovine and human lenses and acylated truncation products in the lens nucleus. Additional analyses revealed that the lipid-modified forms partitioned exclusively to a detergent-resistant membrane fraction, suggesting a role in membrane domain targeting.

  20. Submission to GenBank of the Plasma membrane intrinsic protein (PIP) Subfamily in Cotton – GenBank Accession No. GU998827-GU998830 and GenBank Accession TPA;inferential No. BK007045-BK007052

    USDA-ARS?s Scientific Manuscript database

    The plasma membrane intrinsic proteins (PIP) are one of the five aquaporin protein subfamilies. Aquaporin proteins are known to facilitate water transport through biological membranes. In order to identify NIP aquaporin gene candidates in cotton (Gossypium hirsutum L.), in silico and molecular clon...

  1. cAMP regulated membrane diffusion of a green fluorescent protein-aquaporin 2 chimera.

    PubMed Central

    Umenishi, F; Verbavatz, J M; Verkman, A S

    2000-01-01

    To study the membrane mobility of aquaporin water channels, clones of stably transfected LLC-PK1 cells were isolated with plasma membrane expression of GFP-AQP1 and GFP-AQP2, in which the green fluorescent protein (GFP) was fused upstream and in-frame to each aquaporin (AQP). The GFP fusion did not affect AQP tetrameric association or water transport function. GFP-AQP lateral mobility was measured by irreversibly bleaching a spot (diameter 0.8 microm) on the membrane with an Argon laser beam (488 nm) and following the fluorescence recovery into the bleached area resulting from GFP translational diffusion. In cells expressing GFP-AQP1, fluorescence recovered to >96% of its initial level with t(1/2) of 38 +/- 2 s (23 degrees C) and 21 +/- 1 s (37 degrees C), giving diffusion coefficients (D) of 5.3 and 9.3 x 10(-11) cm(2)/s. GFP-AQP1 diffusion was abolished by paraformaldehyde fixation, slowed >50-fold by the cholesterol-binding agent filipin, but not affected by cAMP agonists. In cells expressing GFP-AQP2, fluorescence recovered to >98% with D of 5.7 and 9.0 x 10(-11) cm(2)/s at 23 degrees C and 37 degrees C. In contrast to results for GFP-AQP1, the cAMP agonist forskolin slowed GFP-AQP2 mobility by up to tenfold. The cAMP slowing was blocked by actin filament disruption with cytochalasin D, by K(+)-depletion in combination with hypotonic shock, and by mutation of the protein kinase A phosphorylation consensus site (S256A) at the AQP2 C-terminus. These results indicate unregulated diffusion of AQP1 in membranes, but regulated AQP2 diffusion that was dependent on phosphorylation at serine 256, and an intact actin cytoskeleton and clathrin coated pit. The cAMP-induced immobilization of phosphorylated AQP2 provides evidence for AQP2-protein interactions that may be important for retention of AQP2 in specialized membrane domains for efficient membrane recycling. PMID:10653816

  2. Reversible, Temperature-Dependent Supramolecular Assembly of Aquaporin-4 Orthogonal Arrays in Live Cell Membranes

    PubMed Central

    Crane, Jonathan M.; Verkman, A.S.

    2009-01-01

    Abstract The shorter “M23” isoform of the glial cell water channel aquaporin-4 (AQP4) assembles into orthogonal arrays of particles (OAPs) in cell plasma membranes, whereas the full-length “M1” isoform does not. N-terminal residues are responsible for OAP formation by AQP4-M23 and for blocking of OAP formation in AQP4-M1. In investigating differences in OAP formation by certain N-terminus mutants of AQP4, as measured by freeze-fracture electron microscopy versus live-cell imaging, we discovered reversible, temperature-dependent OAP assembly of certain weakly associating AQP4 mutants. Single-particle tracking of quantum-dot-labeled AQP4 in live cells and total internal reflection fluorescence microscopy showed >80% of M23 in OAPs at 10–50°C compared to <10% of M1. However, OAP formation by N-terminus cysteine-substitution mutants of M1, which probe palmitoylation-regulated OAP assembly, was strongly temperature-dependent, increasing from <10% at 37°C to >70% at 10°C for the double mutant M1-C13A/C17A. OAP assembly by this mutant, but not by native M23, could also be modulated by reducing its membrane density. Exposure of native M1 and single cysteine mutants to 2-bromopalmitate confirmed the presence of regulated OAP assembly by S-palmitoylation. Kinetic studies showed rapid and reversible OAP formation during cooling and OAP disassembly during heating. Our results provide what to our knowledge is the first information on the energetics of AQP4 OAP assembly in plasma membranes. PMID:19948131

  3. The role of aquaporins in the anti-glioblastoma capacity of the cold plasma-stimulated medium

    NASA Astrophysics Data System (ADS)

    Yan, Dayun; Xiao, Haijie; Zhu, Wei; Nourmohammadi, Niki; Zhang, Lijie Grace; Bian, Ka; Keidar, Michael

    2017-02-01

    The cold atmospheric plasma (CAP) is a promising novel anti-cancer method. Our previous study showed that the cold plasma-stimulated medium (PSM) exerted remarkable anti-cancer effect as effectively as the direct CAP treatment did. H2O2 has been identified as a key anti-cancer substance in PSM. However, the mechanisms underlying intracellular H2O2 regulation by cancer cells is largely unknown. Aquaporins (AQPs) are the confirmed membrane channels of H2O2. In this study, we first demonstrated that the anti-glioblastoma capacity of PSM could be inhibited by silencing the expression of AQP8 in glioblastoma cells (U87MG) or using the aquaporins-blocker silver atoms. This discovery illustrates the key intermediate role of AQPs in the toxicity of PSM on cancer cells. Because the expression of AQPs varies significantly among different cancer cell lines, this study may facilitate the understanding on the diverse responses of cancer cells to PSM or the direct CAP treatment.

  4. A preliminary study of aquaporin 1 immunolocalization in chronic subdural hematoma membranes.

    PubMed

    Basaldella, Luca; Perin, Alessandro; Orvieto, Enrico; Marton, Elisabetta; Itskevich, David; Dei Tos, Angelo Paolo; Longatti, Pierluigi

    2010-07-01

    Aquaporin 1 (AQP1) is a molecular water channel expressed in many anatomical locations, particularly in epithelial barriers specialized in water transport. The aim of this study was to investigate AQP1 expression in chronic subdural hematoma (CSDH) membranes. In this preliminary study, 11 patients with CSDH underwent burr hole craniectomy and drainage. Membrane specimens were stained with a monoclonal antibody targeting AQP1 for immunohistochemical analysis. The endothelial cells of the sinusoid capillaries of the outer membranes exhibited an elevated immunoreactivity to AQP1 antibody compared to the staining intensity of specimens from the inner membrane and normal dura. These findings suggest that the outer membrane might be the source of the increased fluid accumulation responsible for chronic hematoma enlargement.

  5. Super-resolution imaging of aquaporin-4 orthogonal arrays of particles in cell membranes.

    PubMed

    Rossi, Andrea; Moritz, Tobias J; Ratelade, Julien; Verkman, A S

    2012-09-15

    Aquaporin-4 (AQP4) is a water channel expressed in astrocytes, skeletal muscle and epithelial cells that forms supramolecular aggregates in plasma membranes called orthogonal arrays of particles (OAPs). AQP4 is expressed as a short isoform (M23) that forms large OAPs, and a long isoform (M1) that does not form OAPs by itself but can mingle with M23 to form relatively small OAPs. AQP4 OAPs were imaged with ~20 nm spatial precision by photoactivation localization microscopy (PALM) in cells expressing chimeras of M1- or M23-AQP4 with photoactivatable fluorescent proteins. Native AQP4 was imaged by direct stochastic optical reconstruction microscopy (dSTORM) using a primary anti-AQP4 antibody and fluorescent secondary antibodies. We found that OAP area increased from 1878±747 to 3647±958 nm(2) with decreasing M1:M23 ratio from 1:1 to 1:3, and became elongated. Two-color dSTORM indicated that M1 and M23 co-assemble in OAPs with a M1-enriched periphery surrounding a M23-enriched core. Native AQP4 in astrocytes formed OAPs with an area of 2142±829 nm(2), which increased to 5137±1119 nm(2) with 2-bromopalmitate. PALM of AQP4 OAPs in live cells showed slow diffusion (average ~10(-12) cm(2)/s) and reorganization. OAP area was not altered by anti-AQP4 IgG autoantibodies (NMO-IgG) that cause the neurological disease neuromyelitis optica. Super-resolution imaging allowed elucidation of novel nanoscale structural and dynamic features of OAPs.

  6. Super-resolution imaging of aquaporin-4 orthogonal arrays of particles in cell membranes

    PubMed Central

    Rossi, Andrea; Moritz, Tobias J.; Ratelade, Julien; Verkman, A. S.

    2012-01-01

    Summary Aquaporin-4 (AQP4) is a water channel expressed in astrocytes, skeletal muscle and epithelial cells that forms supramolecular aggregates in plasma membranes called orthogonal arrays of particles (OAPs). AQP4 is expressed as a short isoform (M23) that forms large OAPs, and a long isoform (M1) that does not form OAPs by itself but can mingle with M23 to form relatively small OAPs. AQP4 OAPs were imaged with ∼20 nm spatial precision by photoactivation localization microscopy (PALM) in cells expressing chimeras of M1- or M23-AQP4 with photoactivatable fluorescent proteins. Native AQP4 was imaged by direct stochastic optical reconstruction microscopy (dSTORM) using a primary anti-AQP4 antibody and fluorescent secondary antibodies. We found that OAP area increased from 1878±747 to 3647±958 nm2 with decreasing M1:M23 ratio from 1:1 to 1:3, and became elongated. Two-color dSTORM indicated that M1 and M23 co-assemble in OAPs with a M1-enriched periphery surrounding a M23-enriched core. Native AQP4 in astrocytes formed OAPs with an area of 2142±829 nm2, which increased to 5137±1119 nm2 with 2-bromopalmitate. PALM of AQP4 OAPs in live cells showed slow diffusion (average ∼10−12 cm2/s) and reorganization. OAP area was not altered by anti-AQP4 IgG autoantibodies (NMO-IgG) that cause the neurological disease neuromyelitis optica. Super-resolution imaging allowed elucidation of novel nanoscale structural and dynamic features of OAPs. PMID:22718347

  7. Effects of proteoliposome composition and draw solution types on separation performance of aquaporin-based proteoliposomes: implications for seawater desalination using aquaporin-based biomimetic membranes.

    PubMed

    Zhao, Yang; Vararattanavech, Ardcharaporn; Li, Xuesong; Hélixnielsen, Claus; Vissing, Thomas; Torres, Jaume; Wang, Rong; Fane, Anthony G; Tang, Chuyang Y

    2013-02-05

    Aquaporins are a large family of water transport proteins in cell membranes. Their high water permeability and solute rejection make them potential building blocks for high-performance biomimetic membranes for desalination. In the current study, proteoliposomes were prepared using AquaporinZ from Escherichia coli cells, and their separation properties were characterized by stopped-flow measurements. The current study systematically investigated the effect of proteoliposome composition (lipid type, protein-to-lipid ratio (PLR), and the addition of cholesterol) on water permeability and NaCl retention. Among the various lipids investigated, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)-based proteoliposomes were found to have excellent osmotic water permeability and NaCl reflection coefficient values. Increasing the PLR of DOPC proteoliposomes up to 1:200 increased their osmotic water permeability. However, further increase in the PLR reduced the osmotic water permeability probably due to the occurrence of defects in the proteoliposomes, whereas the addition of cholesterol improved their osmotic water permeation likely due to defects sealing. The current study also investigated the effect of major dissolved ions in seawater (e.g., Mg(2+) and SO(4)(2-)) on the stability of proteoliposomes, and design criteria for aquaporin-based biomimetic membranes are proposed in the context of desalination.

  8. Protein kinase A-regulated membrane trafficking of a green fluorescent protein-aquaporin 5 chimera in MDCK cells.

    PubMed

    Kosugi-Tanaka, Chisato; Li, Xuefei; Yao, Chenjuan; Akamatsu, Tetsuya; Kanamori, Norio; Hosoi, Kazuo

    2006-04-01

    The green fluorescent protein (GFP) of the jellyfish, Aeqorea victoria, was used as an autofluorescent tag to track the trafficking of aquaporin 5 (AQP5), an exocrine gland-type water channel. Two groups of chimeric proteins were constructed; one in which GFP was fused to the amino-terminus of AQP5 (GFP-AQP5) and the other, in which it was fused to the carboxyl terminus of it (AQP5-GFP). In each group, 2 chimeras were produced, a wild-type AQP5 with its normal sequence and a mutant AQP5 having a mutated amino acid at 259, i.e., GFP-AQP5-T259A and AQP5-GFP-T259A. They were used to transfect Madin-Darby canine kidney (MDCK) cells. The GFP-AQP5 chimera was localized in the intracellular vesicles, which trafficked to the plasma membrane in response to N(6), 2'-O-dibutyryladenosine 3', 5'-cyclic monophosphate (dbcAMP). Membrane trafficking was inhibited by N-[2-(p-bromocinnamylamino)ethyl]-5-isoquimolinesulfonamide (H-89) but not by palmitoyl-dl-carnitine chloride (PCC). In contrast, the AQP5-GFP chimera expressed in MDCK cells was localized constitutively on the plasma membrane. The cellular localization of the latter chimera was not affected by stimulation with dbcAMP in the presence or absence of H-89 or PCC. Replacement of Thr-259 with Ala-259 did not affect the dbcAMP-induced translocation of the chimeric protein, suggesting that phosphorylation of Thr-259 was not necessary for AQP5 trafficking under the present experimental conditions. Thus, the GFP-AQP5 chimera will be a useful tool to study AQP5 trafficking in vitro, whereas the constitutive membrane localization of the AQP5-GFP chimera suggests the importance of the carboxyl terminus of the AQP5 protein for its sorting, whether it is translocated to intracellular vesicles or to the plasma membrane.

  9. Impact of monoolein on aquaporin1-based supported lipid bilayer membranes

    PubMed Central

    Wang, Zhining; Wang, Xida; Ding, Wande; Wang, Miaoqi; Qi, Xin; Gao, Congjie

    2015-01-01

    Aquaporin (AQP) based biomimetic membranes have attracted considerable attention for their potential water purification applications. In this paper, AQP1 incorporated biomimetic membranes were prepared and characterized. The morphology and structure of the biomimetic membranes were characterized by in situ atomic force microscopy (AFM), infrared absorption spectroscopy, fluorescence microscopy, and contact angle measurements. The nanofiltration performance of the AQP1 incorporated membranes was investigated at 4 bar by using 2 g l−1 NaCl as feed solution. Lipid mobility plays an important role in the performance of the AQP1 incorporated supported lipid bilayer (SLB) membranes. We demonstrated that the lipid mobility is successfully tuned by the addition of monoolein (MO). Through in situ AFM and fluorescence recovery after photo-bleaching (FRAP) measurements, the membrane morphology and the molecular mobility were studied. The lipid mobility increased in the sequence DPPC < DPPC/MO (RMO = 5/5) < DOPC/MO (RMO = 5/5) < DOPC, which is consistent with the flux increment and salt rejection. This study may provide some useful insights for improving the water purification performance of biomimetic membranes. PMID:27877825

  10. Impact of monoolein on aquaporin1-based supported lipid bilayer membranes

    NASA Astrophysics Data System (ADS)

    Wang, Zhining; Wang, Xida; Ding, Wande; Wang, Miaoqi; Qi, Xin; Gao, Congjie

    2015-08-01

    Aquaporin (AQP) based biomimetic membranes have attracted considerable attention for their potential water purification applications. In this paper, AQP1 incorporated biomimetic membranes were prepared and characterized. The morphology and structure of the biomimetic membranes were characterized by in situ atomic force microscopy (AFM), infrared absorption spectroscopy, fluorescence microscopy, and contact angle measurements. The nanofiltration performance of the AQP1 incorporated membranes was investigated at 4 bar by using 2 g l-1 NaCl as feed solution. Lipid mobility plays an important role in the performance of the AQP1 incorporated supported lipid bilayer (SLB) membranes. We demonstrated that the lipid mobility is successfully tuned by the addition of monoolein (MO). Through in situ AFM and fluorescence recovery after photo-bleaching (FRAP) measurements, the membrane morphology and the molecular mobility were studied. The lipid mobility increased in the sequence DPPC < DPPC/MO (RMO = 5/5) < DOPC/MO (RMO = 5/5) < DOPC, which is consistent with the flux increment and salt rejection. This study may provide some useful insights for improving the water purification performance of biomimetic membranes.

  11. Identification of grapevine aquaporins and expression analysis in developing berries.

    PubMed

    Fouquet, Romain; Léon, Céline; Ollat, Nathalie; Barrieu, François

    2008-09-01

    Aquaporins are membrane water channels that play critical roles in controlling the water content of cells and tissues. In this work, nine full-length cDNAs encoding putative aquaporins were isolated from grape berry cDNA libraries. A phylogenetic analysis conducted with 28 aquaporin genes identified in the grapevine genome and previously characterized aquaporins from Arabidopsis indicates that three cDNAs encode putative tonoplast aquaporins (TIPs) whereas six cDNAs belong to the plasma membrane aquaporin subfamily (PIPs). Specific probes designed on the 3' untranslated regions of each cDNA were used for the preparation of cDNA macroarray filters and in situ hybridization experiments. Macroarray data indicate that expression levels of most TIP and PIP genes depend on grape berry developmental stages and point out to a global decrease of aquaporin gene expression during berry ripening. In young berries, high expression of aquaporin genes was preferentially observed in dividing and elongating cells and in cells involved in water and solutes transport. Taken together, the data provided in this paper indicate that aquaporins are implicated in various physiological aspects of grape berry development.

  12. Hypothermia increases aquaporin 4 (AQP4) plasma membrane abundance in human primary cortical astrocytes via a calcium/ transient receptor potential vanilloid 4 (TRPV4)- and calmodulin-mediated mechanism.

    PubMed

    Salman, Mootaz M; Kitchen, Philip; Woodroofe, M Nicola; Brown, James E; Bill, Roslyn M; Conner, Alex C; Conner, Matthew T

    2017-09-19

    Human aquaporin 4 (AQP4) is the primary water channel protein in brain astrocytes. Hypothermia is known to cause astrocyte swelling in culture, but the precise role of AQP4 in this process is unknown. Primary human cortical astrocytes were cultured under hypothermic (32°C) or normothermic (37°C) conditions. AQP4 transcript, total protein and surface localized protein were quantified using RT-qPCR, sandwich ELISA with whole cell lysates, or cell-surface biotinylation followed by ELISA analysis of the surface-localized protein, respectively. Four-hour mild hypothermic treatment increased the surface localization of AQP4 in human astrocytes to 155 ± 4% of normothermic controls, despite no change in total protein expression levels. The hypothermia-mediated increase in AQP4 surface abundance on human astrocytes was blocked using either calmodulin antagonist (trifluoperazine; TFP); TRPV4 antagonist, HC-067047 or calcium chelation using EGTA-AM. The TRPV4 agonist (GSK1016790A) mimicked the effect of hypothermia compared with untreated normothermic astrocytes. Hypothermia led to an increase in surface localization of AQP4 in human astrocytes through a mechanism likely dependent on the TRPV4 calcium channel and calmodulin activation. Understanding the effects of hypothermia on astrocytic AQP4 cell-surface expression may help develop new treatments for brain swelling based on an in-depth mechanistic understanding of AQP4 translocation. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  13. Criticality in Plasma Membranes

    NASA Astrophysics Data System (ADS)

    Machta, Ben; Sethna, James; Veatch, Sarah; Papanikolaou, Stefanos

    2010-03-01

    Recent work in giant plasma membrane vesicles (GPMVs) isolated from living cells demonstrated that they can be tuned with a single parameter (temperature) to criticality, not far from in vivo temperatures [1,2]. Criticality requires the fine-tuning of two parameters suggesting important biological function, and its presence resolves many of the paradoxes associated with putative lipid rafts. Here we present a minimal model of membrane inhomogeneities. We incorporate criticality using a conserved order parameter Ising model coupled to a simple actin cytoskeleton interacting through fields which act as point-like pinning sites. Using this model we make a host of experimentally testable predictions that are in line with recent published findings. At physiological temperatures we find inhomogeneities in the form of critical fluctuations with a length scale of roughly 20nm. Individual constituents making up these liquid domains are mobile, though they diffuse anomalously, but the correlated regions themselves can last as long as the cytoskeleton persists. We explain this by considering the effective long ranged interaction mediated by the Ising order parameter. In general we find Ising criticality organizes and spatially segregates membrane components by providing a channel for interaction over large distances. [1] Veatch et al., ACS Chem Biol. 2008 3(5):287-93 [2] Honerkamp-Smith, Veatch, and Keller, Biochim Biophys Acta. 2008 (in press)

  14. Highly permeable polymeric membranes based on the incorporation of the functional water channel protein Aquaporin Z

    PubMed Central

    Kumar, Manish; Grzelakowski, Mariusz; Zilles, Julie; Clark, Mark; Meier, Wolfgang

    2007-01-01

    The permeability and solute transport characteristics of amphiphilic triblock-polymer vesicles containing the bacterial water-channel protein Aquaporin Z (AqpZ) were investigated. The vesicles were made of a block copolymer with symmetric poly-(2-methyloxazoline)-poly-(dimethylsiloxane)-poly-(2-methyloxazoline) (PMOXA15-PDMS110-PMOXA15) repeat units. Light-scattering measurements on pure polymer vesicles subject to an outwardly directed salt gradient in a stopped-flow apparatus indicated that the polymer vesicles were highly impermeable. However, a large enhancement in water productivity (permeability per unit driving force) of up to ≈800 times that of pure polymer was observed when AqpZ was incorporated. The activation energy (Ea) of water transport for the protein-polymer vesicles (3.4 kcal/mol) corresponded to that reported for water-channel-mediated water transport in lipid membranes. The solute reflection coefficients of glucose, glycerol, salt, and urea were also calculated, and indicated that these solutes are completely rejected. The productivity of AqpZ-incorporated polymer membranes was at least an order of magnitude larger than values for existing salt-rejecting polymeric membranes. The approach followed here may lead to more productive and sustainable water treatment membranes, whereas the variable levels of permeability obtained with different concentrations of AqpZ may provide a key property for drug delivery applications. PMID:18077364

  15. Yeast-expressed human membrane protein aquaporin-1 yields excellent resolution of solid-state MAS NMR spectra.

    PubMed

    Emami, Sanaz; Fan, Ying; Munro, Rachel; Ladizhansky, Vladimir; Brown, Leonid S

    2013-02-01

    One of the biggest challenges in solid-state NMR studies of membrane proteins is to obtain a homogeneous natively folded sample giving high spectral resolution sufficient for structural studies. Eukaryotic membrane proteins are especially difficult and expensive targets in this respect. Methylotrophic yeast Pichia pastoris is a reliable producer of eukaryotic membrane proteins for crystallography and a promising economical source of isotopically labeled proteins for NMR. We show that eukaryotic membrane protein human aquaporin 1 can be doubly ((13)C/(15)N) isotopically labeled in this system and functionally reconstituted into phospholipids, giving excellent resolution of solid-state magic angle spinning NMR spectra.

  16. The aquaporin family of water channel proteins in clinical medicine.

    PubMed

    Lee, M D; King, L S; Agre, P

    1997-05-01

    The aquaporins are a family of membrane channel proteins that serve as selective pores through which water crosses the plasma membranes of many human tissues and cell types. The sites where aquaporins are expressed implicate these proteins in renal water reabsorption, cerebrospinal fluid secretion and reabsorption, generation of pulmonary secretions, aqueous humor secretion and reabsorption, lacrimation, and multiple other physiologic processes. Determination of the aquaporin gene sequences and their chromosomal locations has provided insight into the structure and pathophysiologic roles of these proteins, and primary and secondary involvement of aquaporins is becoming apparent in diverse clinical disorders. Aquaporin-1 (AQP1) is expressed in multiple tissues including red blood cells, and the Colton blood group antigens represent a polymorphism on the AQP1 protein. AQP2 is restricted to renal collecting ducts and has been linked to congenital nephrogenic diabetes insipidus in humans and to lithium-induced nephrogenic diabetes insipidus and fluid retention from congestive heart failure in rat models. Congenital cataracts result from mutations in the mouse gene encoding the lens homolog Aqp0 (Mip). The present understanding of aquaporin physiology is still incomplete; identification of additional members of the aquaporin family will affect future studies of multiple disorders of water distribution throughout the body. In some tissues, the aquaporins may participate in the transepithelial movement of fluid without being rate limiting, so aquaporins may be involved in clinical disorders without being causative. As outlined in this review, our challenge is to identify disease states in which aquaporins are involved, to define the aquaporins' roles mechanistically, and to search for ways to exploit this information therapeutically.

  17. Aquaporin-1 in the peritoneal membrane: Implications for water transport across capillaries and peritoneal dialysis.

    PubMed

    Devuyst, Olivier; Ni, Jie

    2006-08-01

    Peritoneal dialysis (PD) is an established mode of renal replacement therapy, based on the exchange of fluid and solutes between blood in peritoneal capillaries and a dialysate that has been introduced in the peritoneal cavity. The dialysis involves diffusive and convective transports and osmosis through the highly vascularized peritoneal membrane. Computer simulations predicted that the membrane contains ultrasmall pores (radius < 3 A) responsible for the transport of solute-free water across the capillary endothelium during crystalloid osmosis. The distribution of the water channel aquaporin-1 (AQP1), as well as its molecular structure ensuring an exquisite selectivity for water perfectly fit with the characteristics of the ultrasmall pore. Treatment with corticosteroids induces the expression of AQP1 in peritoneal capillaries and increases water permeability and ultrafiltration in rats, without affecting the osmotic gradient and the permeability for small solutes. Studies in knockout mice provided further evidence that osmotically-driven water transport across the peritoneal membrane is mediated by AQP1. AQP1 and endothelial NO synthase (eNOS) show a distinct regulation within the endothelium lining peritoneal capillaries. In acute peritonitis, the upregulation of eNOS and increased release of NO dissipate the osmotic gradient and result in ultrafiltration failure, despite the unchanged expression of AQP1. These data illustrate the potential of the peritoneal membrane to investigate the role and regulation of AQP1 in the endothelium. They also emphasize the critical role of AQP1 during peritoneal dialysis and suggest that manipulating AQP1 expression may be used to increase water permeability across the peritoneal membrane.

  18. Membrane domain specificity in the spatial distribution of aquaporins 5, 7, 9, and 11 in efferent ducts and epididymis of rats.

    PubMed

    Hermo, Louis; Schellenberg, Morgan; Liu, Lauren Ye; Dayanandan, Bama; Zhang, Tong; Mandato, Craig A; Smith, Charles E

    2008-12-01

    Water content within the epididymis of the male reproductive system is stringently regulated to promote sperm maturation. Several members of the aquaporin (AQP) family of water channel-forming integral membrane proteins have been identified in epididymal cells, but expression profiling for this epithelium is presently incomplete, and no AQP isoform has yet been identified on basolateral plasma membranes of these cells. In this study, we explored AQP expression by RT-PCR and light microscopy immunolocalizations using peroxidase and wide-field fluorescence techniques. The results indicate that several AQPs are coexpressed in the epididymis including AQP 5, 7, 9, and 11. Immunolocalizations suggested complex patterns in the spatial distribution of these AQPs. In principal cells, AQP 9 and 11 were present mainly on microvilli, whereas AQP 7 was localized primarily to lateral and then to basal plasma membranes in a region-specific manner. AQP 5 was also expressed regionally but was associated with membranes of endosomes. Additionally, AQPs were expressed by some but not all basal (AQP 7 and 11), clear (AQP 7 and 9), and halo (AQP 7 and 11) cells. These findings indicate unique associations of AQPs with specific membrane domains in a cell type- and region-specific manner within the epididymis of adult animals.

  19. Membrane Domain Specificity in the Spatial Distribution of Aquaporins 5, 7, 9, and 11 in Efferent Ducts and Epididymis of Rats

    PubMed Central

    Hermo, Louis; Schellenberg, Morgan; Liu, Lauren Ye; Dayanandan, Bama; Zhang, Tong; Mandato, Craig A.; Smith, Charles E.

    2008-01-01

    Water content within the epididymis of the male reproductive system is stringently regulated to promote sperm maturation. Several members of the aquaporin (AQP) family of water channel–forming integral membrane proteins have been identified in epididymal cells, but expression profiling for this epithelium is presently incomplete, and no AQP isoform has yet been identified on basolateral plasma membranes of these cells. In this study, we explored AQP expression by RT-PCR and light microscopy immunolocalizations using peroxidase and wide-field fluorescence techniques. The results indicate that several AQPs are coexpressed in the epididymis including AQP 5, 7, 9, and 11. Immunolocalizations suggested complex patterns in the spatial distribution of these AQPs. In principal cells, AQP 9 and 11 were present mainly on microvilli, whereas AQP 7 was localized primarily to lateral and then to basal plasma membranes in a region-specific manner. AQP 5 was also expressed regionally but was associated with membranes of endosomes. Additionally, AQPs were expressed by some but not all basal (AQP 7 and 11), clear (AQP 7 and 9), and halo (AQP 7 and 11) cells. These findings indicate unique associations of AQPs with specific membrane domains in a cell type– and region-specific manner within the epididymis of adult animals. (J Histochem Cytochem 56:1121–1135, 2008) PMID:18796408

  20. Small-Molecule Screening Identifies Modulators of Aquaporin-2 Trafficking

    PubMed Central

    Bogum, Jana; Faust, Dörte; Zühlke, Kerstin; Eichhorst, Jenny; Moutty, Marie C.; Furkert, Jens; Eldahshan, Adeeb; Neuenschwander, Martin; von Kries, Jens Peter; Wiesner, Burkhard; Trimpert, Christiane; Deen, Peter M.T.; Valenti, Giovanna; Rosenthal, Walter

    2013-01-01

    In the principal cells of the renal collecting duct, arginine vasopressin (AVP) stimulates the synthesis of cAMP, leading to signaling events that culminate in the phosphorylation of aquaporin-2 water channels and their redistribution from intracellular domains to the plasma membrane via vesicular trafficking. The molecular mechanisms that control aquaporin-2 trafficking and the consequent water reabsorption, however, are not completely understood. Here, we used a cell-based assay and automated immunofluorescence microscopy to screen 17,700 small molecules for inhibitors of the cAMP-dependent redistribution of aquaporin-2. This approach identified 17 inhibitors, including 4-acetyldiphyllin, a selective blocker of vacuolar H+-ATPase that increases the pH of intracellular vesicles and causes accumulation of aquaporin-2 in the Golgi compartment. Although 4-acetyldiphyllin did not inhibit forskolin-induced increases in cAMP formation and downstream activation of protein kinase A (PKA), it did prevent cAMP/PKA-dependent phosphorylation at serine 256 of aquaporin-2, which triggers the redistribution to the plasma membrane. It did not, however, prevent cAMP-induced changes to the phosphorylation status at serines 261 or 269. Last, we identified the fungicide fluconazole as an inhibitor of cAMP-mediated redistribution of aquaporin-2, but its target in this pathway remains unknown. In conclusion, our screening approach provides a method to begin dissecting molecular mechanisms underlying AVP-mediated water reabsorption, evidenced by our identification of 4-acetyldiphyllin as a modulator of aquaporin-2 trafficking. PMID:23559583

  1. Heteromerization of PIP aquaporins affects their intrinsic permeability

    PubMed Central

    Yaneff, Agustín; Sigaut, Lorena; Marquez, Mercedes; Alleva, Karina; Pietrasanta, Lía Isabel; Amodeo, Gabriela

    2014-01-01

    The plant aquaporin plasma membrane intrinsic proteins (PIP) subfamily represents one of the main gateways for water exchange at the plasma membrane (PM). A fraction of this subfamily, known as PIP1, does not reach the PM unless they are coexpressed with a PIP2 aquaporin. Although ubiquitous and abundantly expressed, the role and properties of PIP1 aquaporins have therefore remained masked. Here, we unravel how FaPIP1;1, a fruit-specific PIP1 aquaporin from Fragaria x ananassa, contributes to the modulation of membrane water permeability (Pf) and pH aquaporin regulation. Our approach was to combine an experimental and mathematical model design to test its activity without affecting its trafficking dynamics. We demonstrate that FaPIP1;1 has a high water channel activity when coexpressed as well as how PIP1–PIP2 affects gating sensitivity in terms of cytosolic acidification. PIP1–PIP2 random heterotetramerization not only allows FaPIP1;1 to arrive at the PM but also produces an enhancement of FaPIP2;1 activity. In this context, we propose that FaPIP1;1 is a key participant in the regulation of water movement across the membranes of cells expressing both aquaporins. PMID:24367080

  2. 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.

  3. Down-regulation of plasma intrinsic protein1 aquaporin in poplar trees is detrimental to recovery from embolism.

    PubMed

    Secchi, Francesca; Zwieniecki, Maciej A

    2014-04-01

    During their lifecycles, trees encounter multiple events of water stress that often result in embolism formation and temporal decreases in xylem transport capacity. The restoration of xylem transport capacity requires changes in cell metabolic activity and gene expression. Specifically, in poplar (Populus spp.), the formation of xylem embolisms leads to a clear up-regulation of plasma membrane protein1 (PIP1) aquaporin genes. To determine their role in poplar response to water stress, transgenic Populus tremula × Populus alba plants characterized by the strong down-regulation of multiple isoforms belonging to the PIP1 subfamily were used. Transgenic lines showed that they are more vulnerable to embolism, with 50% percent loss of conductance occurring 0.3 MPa earlier than in wild-type plants, and that they also have a reduced capacity to restore xylem conductance during recovery. Transgenic plants also show symptoms of a reduced capacity to control percent loss of conductance through stomatal conductance in response to drought, because they have a much narrower vulnerability safety margin. Finally, a delay in stomatal conductance recovery during the period of stress relief was observed. The presented results suggest that PIP1 genes are involved in the maintenance of xylem transport system capacity, in the promotion of recovery from stress, and in contribution to a plant's control of stomatal conductance under water stress.

  4. Liver plasma membranes: an effective method to analyze membrane proteome.

    PubMed

    Cao, Rui; Liang, Songping

    2012-01-01

    Plasma membrane proteins are critical for the maintenance of biological systems and represent important targets for the treatment of disease. The hydrophobicity and low abundance of plasma membrane proteins make them difficult to analyze. The protocols given here are the efficient isolation/digestion procedures for liver plasma membrane proteomic analysis. Both protocol for the isolation of plasma membranes and protocol for the in-gel digestion of gel-embedded plasma membrane proteins are presented. The later method allows the use of a high detergent concentration to achieve efficient solubilization of hydrophobic plasma membrane proteins while avoiding interference with the subsequent LC-MS/MS analysis.

  5. Membrane-associated aquaporin-1 facilitates osmotically driven water flux across the basolateral membrane of the thick ascending limb

    PubMed Central

    Cabral, Pablo D.

    2012-01-01

    The thick ascending limb of the loop of Henle (TAL) reabsorbs ∼30% of filtered NaCl but is impermeable to water. The observation that little water traverses the TAL indicates an absence of water channels at the apical membrane. Yet TAL cells swell when peritubular osmolality decreases indicating that water channels must be present in the basolateral side. Consequently, we hypothesized that the water channel aquaporin-1 (AQP1) facilitates water flux across the basolateral membrane of TALs. Western blotting revealed AQP1 expression in microdissected rat and mouse TALs. Double immunofluorescence showed that 95 ± 2% of tubules positive for the TAL-specific marker Tamm-Horsfall protein were also positive for AQP1 (n = 6). RT-PCR was used to demonstrate presence of AQP1 mRNA and the TAL-specific marker NKCC2 in microdissected TALs. Cell surface biotinylation assays showed that 23 ± 3% of the total pool of AQP1 was present at the TAL basolateral membrane (n = 7). To assess the functional importance of AQP1 in the basolateral membrane, we measured the rate of cell swelling initiated by decreasing peritubular osmolality as an indicator of water flux in microdissected TALs. Water flux was decreased by ∼50% in Aqp1 knockout mice compared with wild-types (4.0 ± 0.8 vs. 8.9 ± 1.7 fluorescent U/s, P < 0.02; n = 7). Furthermore, arginine vasopressin increased TAL AQP1 expression by 135 ± 17% (glycosylated) and 41 ± 11% (nonglycosylated; P < 0.01; n =5). We conclude that 1) the TAL expresses AQP1, 2) ∼23% of the total pool of AQP1 is localized to the basolateral membrane, 3) AQP1 mediates a significant portion of basolateral water flux, and 4) AQP1 is upregulated in TALs of rats infused with dDAVP. AQP1 could play an important role in regulation of TAL cell volume during changes in interstitial osmolality, such as during a high-salt diet or water deprivation. PMID:22674028

  6. 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.

  7. Aquaporin-1 in the peritoneal membrane: implications for peritoneal dialysis and endothelial cell function.

    PubMed

    Devuyst, Olivier; Ni, Jie; Verbavatz, Jean-Marc

    2005-09-01

    PD (peritoneal dialysis) is an established mode of renal replacement therapy, based on the exchange of fluid and solutes between blood in peritoneal capillaries and a dialysate that has been introduced into the peritoneal cavity. The dialysis process involves diffusive and convective transports and osmosis through the PM (peritoneal membrane). Computer simulations predicted that the PM contains ultrasmall pores (radius <3 A, 1 A=10(-10) m), responsible for up to 50% of UF (ultrafiltration), i.e. the osmotically driven water movement during PD. Several lines of evidence suggest that AQP1 (aquaporin-1) is the ultrasmall pore responsible for transcellular water permeability during PD. Treatment with corticosteroids induces the expression of AQP1 in the PM and improves water permeability and UF in rats without affecting the osmotic gradient and permeability for small solutes. Studies in knockout mice provided further evidence that osmotically driven water transport across the PM is mediated by AQP1. AQP1 and eNOS (endothelial nitric oxide synthase) show a distinct regulation within the endothelium lining the peritoneal capillaries. In acute peritonitis, the up-regulation of eNOS and increased release of nitric oxide dissipate the osmotic gradient and prevent UF, whereas AQP1 expression is unchanged. These results illustrate the usefulness of the PM to investigate the role and regulation of AQP1 in the endothelium. The results also emphasize the critical role of AQP1 during PD and suggest that manipulation of AQP1 expression may be used to increase water permeability across the PM.

  8. 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.

  9. 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.

  10. Plant plasma membrane protein extraction and solubilization for proteomic analysis.

    PubMed

    Santoni, Véronique

    2007-01-01

    The plasma membrane (PM) exists as the interface between the cytosol and the environment in all living cells and is one of the most complex and differentiated membrane. The identification and characterization of membrane proteins (either extrinsic or intrinsic) is a crucial challenge since many of these proteins are involved in essential cellular functions such as cell signaling, osmoregulation, nutrition, and metabolism. Methods to isolate PM fractions vary according to organisms, tissues, and cell type. This chapter emphasizes isolation, from the model plant Arabidopsis thaliana, of PM fractions from a microsomal membrane fraction by two-phase partitioning, a methodology that utilizes the different surface properties of membranes. PM proteins that do not span the lipid bilayer are generally well recovered after 2D gel electrophoresis. By contrast, the recovery of transmembrane proteins requires first the depletion of the PM fraction from soluble proteins, being either cytosolic contaminants or functionally associated proteins, and second, to the use of specific solubilization procedures. This chapter presents protocols to strip PM based on alkaline treatment of membranes and to solubilize hydrophobic proteins to increase their recovery on 2D gels. Aquaporins that are highly hydrophobic proteins are used to probe the relevance of the procedures.

  11. Thirsty business: cell, region, and membrane specificity of aquaporins in the testis, efferent ducts, and epididymis and factors regulating their expression.

    PubMed

    Hermo, Louis; Smith, Charles E

    2011-01-01

    Water content within the male reproductive tract is stringently regulated in order to promote sperm differentiation and maturation. Aquaporins (AQP) are a family of integral membrane proteins allowing the transcellular transport of water, gases, urea, glycerol, and ions. Past studies from our lab have revealed the following. In the testis, Sertoli cells express AQP 8, whereas germ cells express AQP 7. In the efferent ducts (ED), AQP 1, 9, and 10 localize to microvilli of nonciliated cells, in addition to a basolateral staining for AQP 1, whereas AQP 1 and 10 localize to ciliated cells. AQP 7 and 11 are expressed in the ED epithelium of young but not adult rats, suggesting suppression of translation as rats age. In the adult epididymis, AQP 1 appears in endothelial cells of vascular channels and myoid cells, whereas AQP 3 delineates basal cells. In principal cells, AQP 9 and 11 appear on microvilli, whereas AQP 7 localizes to lateral then to basal plasma membranes in a region-specific manner; AQP 7 also associates with myoid cells. AQP 5 is expressed in corpus and cauda regions. Additionally, several AQPs are expressed by some but not all basal (AQP 7, 11), clear (AQP 7, 9), and halo (AQP 7, 11) cells. Regulation studies reveal a role for estrogen, androgens, and lumicrine factors. These findings indicate unique associations of AQPs with specific membrane domains in a cell type- and region-specific manner within the EDs and epididymis, as well as complex regulation patterns of expression.

  12. Root aquaporins contribute to whole plant water fluxes under drought stress in rice (Oryza sativa L.).

    PubMed

    Grondin, Alexandre; Mauleon, Ramil; Vadez, Vincent; Henry, Amelia

    2016-02-01

    Aquaporin activity and root anatomy may affect root hydraulic properties under drought stress. To better understand the function of aquaporins in rice root water fluxes under drought, we studied the root hydraulic conductivity (Lpr) and root sap exudation rate (Sr) in the presence or absence of an aquaporin inhibitor (azide) under well-watered conditions and following drought stress in six diverse rice varieties. Varieties varied in Lpr and Sr under both conditions. The contribution of aquaporins to Lpr was generally high (up to 79% under well-watered conditions and 85% under drought stress) and differentially regulated under drought. Aquaporin contribution to Sr increased in most varieties after drought, suggesting a crucial role for aquaporins in osmotic water fluxes during drought and recovery. Furthermore, root plasma membrane aquaporin (PIP) expression and root anatomical properties were correlated with hydraulic traits. Three chromosome regions highly correlated with hydraulic traits of the OryzaSNP panel were identified, but did not co-locate with known aquaporins. These results therefore highlight the importance of aquaporins in the rice root radial water pathway, but emphasize the complex range of additional mechanisms related to root water fluxes and drought response.

  13. Aquaporin-11: A channel protein lacking apparent transport function expressed in brain

    PubMed Central

    Gorelick, Daniel A; Praetorius, Jeppe; Tsunenari, Takashi; Nielsen, Søren; Agre, Peter

    2006-01-01

    Background The aquaporins are a family of integral membrane proteins composed of two subfamilies: the orthodox aquaporins, which transport only water, and the aquaglyceroporins, which transport glycerol, urea, or other small solutes. Two recently described aquaporins, numbers 11 and 12, appear to be more distantly related to the other mammalian aquaporins and aquaglyceroporins. Results We report on the characterization of Aquaporin-11 (AQP11). AQP11 RNA and protein is found in multiple rat tissues, including kidney, liver, testes and brain. AQP11 has a unique distribution in brain, appearing in Purkinje cell dendrites, hippocampal neurons of CA1 and CA2, and cerebral cortical neurons. Immunofluorescent staining of Purkinje cells indicates that AQP11 is intracellular. Unlike other aquaporins, Xenopus oocytes expressing AQP11 in the plasma membrane failed to transport water, glycerol, urea, or ions. Conclusion AQP11 is functionally distinct from other proteins of the aquaporin superfamily and could represent a new aquaporin subfamily. Further studies are necessary to elucidate the role of AQP11 in the brain. PMID:16650285

  14. Membrane trafficking of aquaporin 1 is mediated by protein kinase C via microtubules and regulated by tonicity.

    PubMed

    Conner, Matthew T; Conner, Alex C; Brown, James E P; Bill, Roslyn M

    2010-02-09

    It is well-known that the rapid flow of water into and out of cells is controlled by membrane proteins called aquaporins (AQPs). However, the mechanisms that allow cells to quickly respond to a changing osmotic environment are less well established. Using GFP-AQP fusion proteins expressed in HEK293 cells, we demonstrate the reversible manipulation of cellular trafficking of AQP1. AQP1 trafficking was mediated by the tonicity of the cell environment in a specific PKC- and microtubule-dependent manner. This suggests that the increased level of water transport following osmotic change may be due a phosphorylation-dependent increase in the level of AQP1 trafficking resulting in membrane localization.

  15. Aquaporins in Brain Edema and Neuropathological Conditions

    PubMed Central

    Filippidis, Aristotelis S.; Carozza, Richard B.; Rekate, Harold L.

    2016-01-01

    The aquaporin (AQP) family of water channels are a group of small, membrane-spanning proteins that are vital for the rapid transport of water across the plasma membrane. These proteins are widely expressed, from tissues such as the renal epithelium and erythrocytes to the various cells of the central nervous system. This review will elucidate the basic structure and distribution of aquaporins and discuss the role of aquaporins in various neuropathologies. AQP1 and AQP4, the two primary aquaporin molecules of the central nervous system, regulate brain water and CSF movement and contribute to cytotoxic and vasogenic edema, where they control the size of the intracellular and extracellular fluid volumes, respectively. AQP4 expression is vital to the cellular migration and angiogenesis at the heart of tumor growth; AQP4 is central to dysfunctions in glutamate metabolism, synaptogenesis, and memory consolidation; and AQP1 and AQP4 adaptations have been seen in obstructive and non-obstructive hydrocephalus and may be therapeutic targets. PMID:28036023

  16. Fragment Screening of Human Aquaporin 1

    PubMed Central

    To, Janet; Torres, Jaume

    2016-01-01

    Aquaporins (AQPs) are membrane proteins that enable water transport across cellular plasma membranes in response to osmotic gradients. Phenotypic analyses have revealed important physiological roles for AQPs, and the potential for AQP water channel modulators in various disease states has been proposed. For example, AQP1 is overexpressed in tumor microvessels, and this correlates with higher metastatic potential and aggressiveness of the malignancy. Chemical modulators would help in identifying the precise contribution of water channel activity in these disease states. These inhibitors would also be important therapeutically, e.g., in anti-cancer treatment. This perceived importance contrasts with the lack of success of high-throughput screens (HTS) to identify effective and specific inhibitors of aquaporins. In this paper, we have screened a library of 1500 “fragments”, i.e., smaller than molecules used in HTS, against human aquaporin (hAQP1) using a thermal shift assay and surface plasmon resonance. Although these fragments may not inhibit their protein target, they bound to and stabilized hAQP1 (sub mM binding affinities (KD), with an temperature of aggregation shift ΔTagg of +4 to +50 °C) in a concentration-dependent fashion. Chemically expanded versions of these fragments should follow the determination of their binding site on the aquaporin surface. PMID:27023529

  17. Structural Analysis of Cloned Plasma Membrane Proteins by Freeze-Fracture Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Eskandari, Sepehr; Wright, Ernest M.; Kreman, Mike; Starace, Dorine M.; Zampighi, Guido A.

    1998-09-01

    We have used freeze-fracture electron microscopy to examine the oligomeric structure and molecular asymmetry of integral plasma membrane proteins. Recombinant plasma membrane proteins were functionally expressed in Xenopus laevis oocytes, and the dimensions of their freeze-fracture particles were analyzed. To characterize the freeze-fracture particles, we compared the particle cross-sectional area of proteins with α -helical transmembrane domains (opsin, aquaporin 1, and a connexin) with their area obtained from existing maps calculated from two-dimensional crystals. We show that the cross-sectional area of the freeze-fracture particles corresponds to the area of the transmembrane domain of the protein, and that the protein cross-sectional area varies linearly with the number membrane-spanning helices. On average, each helix occupies 1.40 ± 0.03 nm2. By using this information, we examined members from three classes of plasma membrane proteins: two ion channels, the cystic fibrosis transmembrane conductance regulator and connexin 50 hemi-channel; a water channel, the major intrinsic protein (the aquaporin 0); and a cotransporter, the Na+/glucose cotransporter. Our results suggest that the cystic fibrosis transmembrane conductance regulator is a dimer containing 25 ± 2 transmembrane helices, connexin 50 is a hexamer containing 24 ± 3 helices, the major intrinsic protein is a tetramer containing 24 ± 3 helices, and the Na+/glucose cotransporter is an asymmetrical monomer containing 15 ± 2 helices.

  18. Proteomic analysis of integral plasma membrane proteins.

    PubMed

    Zhao, Yingxin; Zhang, Wei; Kho, Yoonjung; Zhao, Yingming

    2004-04-01

    Efficient methods for profiling proteins integral to the plasma membrane are highly desirable for the identification of overexpressed proteins in disease cells. Such methods will aid in both understanding basic biological processes and discovering protein targets for the design of therapeutic monoclonal antibodies. Avoiding contamination by subcellular organelles and cytosolic proteins is crucial to the successful proteomic analysis of integral plasma membrane proteins. Here we report a biotin-directed affinity purification (BDAP) method for the preparation of integral plasma membrane proteins, which involves (1) biotinylation of cell surface membrane proteins in viable cells, (2) affinity enrichment using streptavidin beads, and (3) depletion of plasma membrane-associated cytosolic proteins by harsh washes with high-salt and high-pH buffers. The integral plasma membrane proteins are then extracted and subjected to SDS-PAGE separation and HPLC/MS/MS for protein identification. We used the BDAP method to prepare integral plasma membrane proteins from a human lung cancer cell line. Western blotting analysis showed that the preparation was almost completely devoid of actin, a major cytosolic protein. Nano-HPLC/MS/MS analysis of only 30 microg of protein extracted from the affinity-enriched integral plasma membrane preparation led to the identification of 898 unique proteins, of which 781 were annotated with regard to their plasma membrane localization. Among the annotated proteins, at least 526 (67.3%) were integral plasma membrane proteins. Notable among them were 62 prenylated proteins and 45 Ras family proteins. To our knowledge, this is the most comprehensive proteomic analysis of integral plasma membrane proteins in mammalian cells to date. Given the importance of integral membrane proteins for drug design, the described approach will expedite the characterization of plasma membrane subproteomes and the discovery of plasma membrane protein drug targets.

  19. Renal concentrating and diluting function in deficiency of specific aquaporin genes.

    PubMed

    Verkman, A S

    2002-01-01

    Aquaporins (AQP) are water-transporting proteins expressed in many fluid-transporting epithelia and endothelia. In kidney, AQP1 is expressed in plasma membranes of proximal tubule, thin descending limb of Henle and descending vasa recta, AQP2 in collecting duct luminal membrane, AQP3 and AQP4 in collecting duct basolateral membrane, AQP6 in intercalated cells, and AQP7 in the S3 segment of proximal tubule. Human mutations in AQP2 cause hereditary non-X-linked nephrogenic diabetes insipidus. Transgenic mice lacking the renal aquaporins have been useful in defining their role. Mice deficient in AQP1 are polyuric and unable to form a concentrated urine because of defective proximal tubule fluid absorption and countercurrent multiplication. Mice lacking AQP3 are markedly polyuric due to low water permeability across the cortical and outer medullary collecting duct. However, mice lacking AQP4, which is expressed mainly in inner medullary collecting duct, manifest only a mild defect in maximum urinary concentrating ability. The aquaporin null mice have normal urinary diluting ability. From many renal and extrarenal phenotype studies of aquaporin null mice, we conclude that aquaporins are important for rapid near-isosmolar transepithelial fluid absorption/secretion and for rapid vectorial water movement driven by osmotic gradients. The renal phenotype in aquaporin null mice suggests the utility of aquaporin blockers as novel aquaretic-diuretic agents. Copyright 2002 S. Karger AG, Basel

  20. Genome-wide identification, characterization, and expression profile of aquaporin gene family in flax (Linum usitatissimum)

    PubMed Central

    Shivaraj, S. M.; Deshmukh, Rupesh K.; Rai, Rhitu; Bélanger, Richard; Agrawal, Pawan K.; Dash, Prasanta K.

    2017-01-01

    Membrane intrinsic proteins (MIPs) form transmembrane channels and facilitate transport of myriad substrates across the cell membrane in many organisms. Majority of plant MIPs have water transporting ability and are commonly referred as aquaporins (AQPs). In the present study, we identified aquaporin coding genes in flax by genome-wide analysis, their structure, function and expression pattern by pan-genome exploration. Cross-genera phylogenetic analysis with known aquaporins from rice, arabidopsis, and poplar showed five subgroups of flax aquaporins representing 16 plasma membrane intrinsic proteins (PIPs), 17 tonoplast intrinsic proteins (TIPs), 13 NOD26-like intrinsic proteins (NIPs), 2 small basic intrinsic proteins (SIPs), and 3 uncharacterized intrinsic proteins (XIPs). Amongst aquaporins, PIPs contained hydrophilic aromatic arginine (ar/R) selective filter but TIP, NIP, SIP and XIP subfamilies mostly contained hydrophobic ar/R selective filter. Analysis of RNA-seq and microarray data revealed high expression of PIPs in multiple tissues, low expression of NIPs, and seed specific expression of TIP3 in flax. Exploration of aquaporin homologs in three closely related Linum species bienne, grandiflorum and leonii revealed presence of 49, 39 and 19 AQPs, respectively. The genome-wide identification of aquaporins, first in flax, provides insight to elucidate their physiological and developmental roles in flax. PMID:28447607

  1. Dynamic Changes in the Osmotic Water Permeability of Protoplast Plasma Membrane1[w

    PubMed Central

    Moshelion, Menachem; Moran, Nava; Chaumont, François

    2004-01-01

    The osmotic water permeability coefficient (Pf) of plasma membrane of maize (Zea mays) Black Mexican Sweet protoplasts changed dynamically during a hypoosmotic challenge, as revealed using a model-based computational approach. The best-fitting model had three free parameters: initial Pf, Pf rate-of-change (slopePf), and a delay, which were hypothesized to reflect changes in the number and/or activity of aquaporins in the plasma membrane. Remarkably, the swelling response was delayed 2 to 11 s after start of the noninstantaneous (but accounted for) bath flush. The Pf during the delay was ≤1 μm s−1. During the swelling period following the delay, Pf changed dynamically: within the first 15 s Pf either (1) increased gradually to approximately 8 μm s−1 (in the majority population of low-initial-Pf cells) or (2) increased abruptly to 10 to 20 μm s−1 and then decreased gradually to 3 to 6 μm s−1 (in the minority population of high-initial-Pf cells). We affirmed the validity of our computational approach by the ability to reproduce previously reported initial Pf values (including the absence of delay) in control experiments on Xenopus oocytes expressing the maize aquaporin ZmPIP2;5. Although mercury did not affect the Pf in swelling Black Mexican Sweet cells, phloretin, another aquaporin inhibitor, inhibited swelling in a predicted manner, prolonging the delay and slowing Pf increase, thereby confirming the hypothesis that Pf dynamics, delay included, reflected the varying activity of aquaporins. PMID:15310831

  2. Genome-wide analysis and expression profiling of the Solanum tuberosum aquaporins.

    PubMed

    Venkatesh, Jelli; Yu, Jae-Woong; Park, Se Won

    2013-12-01

    Aquaporins belongs to the major intrinsic proteins involved in the transcellular membrane transport of water and other small solutes. A comprehensive genome-wide search for the homologues of Solanum tuberosum major intrinsic protein (MIP) revealed 41 full-length potato aquaporin genes. All potato aquaporins are grouped into five subfamilies; plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), NOD26-like intrinsic proteins (NIPs), small basic intrinsic proteins (SIPs) and x-intrinsic proteins (XIPs). Functional predictions based on the aromatic/arginine (ar/R) selectivity filters and Froger's positions showed a remarkable difference in substrate transport specificity among subfamilies. The expression pattern of potato aquaporins, examined by qPCR analysis, showed distinct expression profiles in various organs and tuber developmental stages. Furthermore, qPCR analysis of potato plantlets, subjected to various abiotic stresses revealed the marked effect of stresses on expression levels of aquaporins. Taken together, the expression profiles of aquaporins imply that aquaporins play important roles in plant growth and development, in addition to maintaining water homeostasis in response to environmental stresses. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  3. Detecting aquaporin function and regulation

    NASA Astrophysics Data System (ADS)

    Madeira, Ana; Moura, Teresa; Soveral, Graça

    2016-02-01

    Water is the major component of cells and tissues throughout all forms of life. Fluxes of water and solutes through cell membranes and epithelia are essential for osmoregulation and energy homeostasis. Aquaporins are membrane channels expressed in almost every organism and involved in the bidirectional transfer of water and small solutes across cell membranes. Aquaporins have important biological roles and have been implicated in several pathophysiological conditions suggesting a great translational potential in aquaporin-based diagnostic and therapeutics. Detecting aquaporin function is critical for assessing regulation and screening for new activity modulators that can prompt the development of efficient medicines. Appropriate methods for functional analysis comprising suitable cell models and techniques to accurately evaluate water and solute membrane permeability are essential to validate aquaporin function and assess short-term regulation. The present review describes established assays commonly used to assess aquaporin function in cells and tissues, as well as the experimental biophysical strategies required to reveal functional regulation and identify modulators, the first step for aquaporin drug discovery.

  4. Fetal membranes as an interface between inflammation and metabolism: increased aquaporin 9 expression in the presence of spontaneous labor at term and chorioamnionitis.

    PubMed

    Mittal, Pooja; Romero, Roberto; Mazaki-Tovi, Shali; Tromp, Gerard; Tarca, Adi L; Kim, Yeon Mee; Chaiworapongsa, Tinnakorn; Kusanovic, Juan Pedro; Erez, Offer; Than, Nandor Gabor; Hassan, Sonia S

    2009-12-01

    Aquaporin 9 (AQP9) is a water channel protein characterized by its high permeability to nutrients such as lactate and glycerol, as well as urea and other small solutes. These unique properties of AQP9 suggest that this molecule may play a role in the modulation of nutrient flux through the fetal membranes in conditions associated with increased metabolic demand, such as spontaneous labor and inflammation. The objective of this study was to determine the expression of AQP9 in the chorioamniotic membranes from women with and without term labor, as well as those with preterm prelabor rupture of membranes (PPROM) with and without histologic chorioamnionitis. A cross-sectional study was performed which included patients in the following groups: (1) term not in labor (TNL; n = 14); (2) term, spontaneous labor (n = 14); and (3) PPROM with (n = 20) and without (n = 17) histologic chorioamnionitis. AQP9 mRNA expression in fetal membranes was quantified using quantitative real-time reverse transcription-polymerase chain reaction and analyzed with a linear model including gestational age as a covariate. (1) AQP9 mRNA expression was identified in all chorioamniotic membrane specimens; (2) AQP9 expression in fetal membranes was significantly higher in spontaneous term labor when compared with TNL (fold change 3.6; p = 0.01); and (3) Among patients with PPROM, the presence of histologic chorioamnionitis was associated with a higher expression of AQP9 in the chorioamniotic membranes compared with those from women without histologic chorioamnionitis (fold change 8.7; p < 0.001). Aquaporin 9 mRNA expression is higher in the fetal membranes from patients with spontaneous term labor and those with PPROM and histologic chorioamnionitis. These findings are novel, and suggest a role for aquaporin 9 in membrane-mediated transfer of nutrients to support the increased metabolic demands associated with the host immune response of the terminal pathway of parturition and histologic

  5. Physiology and pathophysiology of renal aquaporins.

    PubMed

    Kwon, T H; Hager, H; Nejsum, L N; Andersen, M L; Frøkiaer, J; Nielsen, S

    2001-05-01

    The discovery of aquaporin-1 (AQP1) by Agre and associates answered the longstanding biophysical question of how water specifically crosses biological membranes. In the kidney at least 7 aquaporins are expressed at distinct sites. AQP1 is extremely abundant in the proximal tubule and descending thin limb and is essential for urinary concentration. AQP2 is exclusively expressed in the principal cells of the connecting tubule and collecting duct and is the predominant vasopressin-regulated water channel. AQP3 and AQP4 are both present in the basolateral plasma membrane of collecting duct principal cells and represent exit pathways for water reabsorbed apically via AQP2. Studies in patients and transgenic mice have shown that both AQP2 and AQP3 are essential for urinary concentration. Three additional aquaporins are present in the kidney. AQP6 is present in intracellular vesicles in collecting duct intercalated cells and AQP8 are present intracellularly at low abundance in proximal tubules and collecting duct principal cells but the physiological function of these 2 channels remain undefined. AQP7 is abundant in the brush border of proximal tubule cells and is likely to be involved in proximal tubule water reabsorption. A series of studies have underscored crucial roles of aquaporins for regulation of renal water metabolism and hence body water balance. Moreover it has become clear that dysregulation of aquaporins, and especially AQP2 is critically involved in many water balance disorders. Lack of functional AQP2 is seen in primary forms of diabetes insipidus, and reduced expression and targeting is seen in several diseases associated with urinary concentrating defects such as acquired nephrogenic diabetes insipidus, postobstructive polyuria, as well as acute and chronic renal failure. In contrast, in conditions with water retention such as severe congestive heart failure, pregnancy and SIADH both AQP2 expression levels and apical plasma membrane targetting is

  6. In vivo silencing of aquaporin-1 by RNA interference inhibits angiogenesis in the chick embryo chorioallantoic membrane assay.

    PubMed

    Camerino, G M; Nicchia, G P; Dinardo, M M; Ribatti, D; Svelto, M; Frigeri, A

    2006-10-30

    Aquaporin-1 (AQP1) is a water channel protein mainly expressed in endothelial and epithelial cells of many tissues, including the vasculature where it serves to increase cell membrane water permeability. Previous studies in active multiple myeloma patients and in AQP1 KO mice indicated an involvement of AQP1 in physiological and tumor angiogenesis. To understand the physiological role of AQP1 in angiogenesis, we used a 21-nucleotide small interfering RNA duplexes (siRNA) to knockdown AQP1 in the chick embryo chorioallantoic membrane (CAM), a commonly used in vivo assay to study both angiogenic and angiostatic molecules. Chicken AQP1 sequence was identified and utilized to synthesize a siRNA directed to the AQP1 sequence. We then tested the efficiency of the siRNA in vitro, using an AQP1 transfected cell line. The level of AQP1 protein reduction obtained using siRNA was 98 % and 92 % after 1 and 2 day transfection respectively. RNA interference experiments were then performed in vivo by using the CAM assay. Results showed that after 4 days of treatment, AQP1 siRNA was able to strongly inhibit angiogenesis. This is the first study showing the in vivo use of RNA interference technique in the CAM assay. Our results strongly support the hypothesis that AQP1 could have a key role in physiological and pathological angiogenesis.

  7. 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.

  8. Determinants of AQP6 trafficking to intracellular sites versus the plasma membrane in transfected mammalian cells.

    PubMed

    Beitz, Eric; Liu, Kun; Ikeda, Masahiro; Guggino, William B; Agre, Peter; Yasui, Masato

    2006-02-01

    Most AQPs (aquaporins) function at the plasma membrane, however AQP6 is exclusively localized to membranes of intracellular vesicles in acid-secreting type-A intercalated cells of renal collecting ducts. The intracellular distribution indicates that AQP6 has a function distinct from trans-epithelial water movement. We show by mutational analyses and immunofluorescence that the N-terminus of AQP6 is a determinant for its intracellular localization. Presence or absence at the plasma membrane of AQP6 constructs was confirmed by electrophysiological methods. Addition of a GFP (green fluorescent protein) or a HA (haemagglutinin) epitope tag (GFP-AQP6 or HA-AQP6) to the N-terminus of AQP6, directed AQP6 to the plasma membranes of transfected Madin-Darby canine kidney cells. In contrast, addition of a GFP tag to the C-terminus (AQP6-GFP) caused the protein to remain intracellular, similar to untagged wild-type AQP6. Replacement of the N-terminus of AQP6 by that of AQP1 also directed AQP6 to the plasma membranes, whereas the N-terminus of AQP6 retained AQP1 in cytosolic sites. Our results suggest that the N-terminus of AQP6 is critical for trafficking of the protein to the intracellular sites. Moreover, our studies provide an approach for future identification of proteins involved in vesicle sorting in the acid-secreting type-A intercalated cells.

  9. The lipidation profile of aquaporin-0 correlates with the acyl composition of phosphoethanolamine lipids in lens membranes.

    PubMed

    Ismail, Vian S; Mosely, Jackie A; Tapodi, Antal; Quinlan, Roy A; Sanderson, John M

    2016-11-01

    The lens fiber major intrinsic protein (otherwise known as aquaporin-0 (AQP0), MIP26 and MP26) has been examined by mass spectrometry (MS) in order to determine the speciation of acyl modifications to the side chains of lysine residues and the N-terminal amino group. The speciation of acyl modifications to the side chain of one specific, highly conserved lysine residue (K238) and the N-terminal amino group of human and bovine AQP0 revealed, in decreasing order of abundance, oleoyl, palmitoyl, stearoyl, eicosenoyl, dihomo-γ-linolenoyl, palmitoleoyl and eicosadienoyl modifications. In the case of human AQP0, an arachidonoyl modification was also found at the N-terminus. The relative abundances of these modifications mirror the fatty acid composition of lens phosphatidylethanolamine lipids. This lipid class would be expected to be concentrated in the inner leaflet of the lens fiber membrane to which each of the potential AQP0 lipidation sites is proximal. Our data evidence a broad lipidation profile that is both species and site independent, suggesting a chemical-based ester aminolysis mechanism to explain such modifications. Copyright © 2016. Published by Elsevier B.V.

  10. Expression and localization of aquaporin-1 on the apical membrane of enterocytes in the small intestine of bottlenose dolphins.

    PubMed

    Suzuki, Miwa

    2010-02-01

    The small and large intestines are primary sites for water intake in mammals. To reveal how water is absorbed in the intestines of cetaceans, histological and molecular-biological studies were performed on the small intestine of the bottlenose dolphin, Tursiops truncatus. In histological studies using fresh specimens, obvious villi and deep crypts of Lieberkühn, lined by abundant enterocytes with microvilli and goblet cells, were observed in the mucosa. Expressions and immunolocalizations of aquaporin-1 (AQP1), a member of the water-selective channel termed AQP, were also investigated in the intestine. By reverse transcriptional polymerase chain reaction and rapid amplification of cDNA ends using RNA extracted from the dolphins' small intestines, the full length of mRNA for AQP1 was sequenced. The deductive amino acid sequence for an open reading frame showed high homologies with other mammals' AQP1, and water permeability of the protein was certified by cRNA injection to Xenopus oocytes. Immunohistochemistry showed AQP1 distribution on the apical membrane of the enterocytes, especially in the crypts. These data suggest that AQP1 is a channel protein responsible for water absorption in the small intestine of dolphins.

  11. 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.

  12. ISOLATION OF RAT LIVER PLASMA MEMBRANES

    PubMed Central

    Touster, Oscar; Aronson, N. N.; Dulaney, John T.; Hendrickson, Herman

    1970-01-01

    Nucleotide pyrophosphatase and phosphodiesterase I of rat liver have been found to be localized primarily in cell particulates highly enriched with respect to the most commonly accepted plasma membrane marker, 5'-nucleotidase, and therefore should themselves be assigned a plasma membrane localization. The observation that plasma membranes sediment in isotonic sucrose with both nuclear and microsomal fractions was exploited to obtain plasma membrane preparations from each fraction. Both preparations are similar in chemical and enzymic composition. Moreover, the preparative method developed in this study appears to give the best combination of yield, purity, and reproducibility available. The question of the possible identity of nucleotide pyrophosphatase and phosphodiesterase I is considered, and evidence is presented suggesting that these activities may be manifestations of the same enzyme. PMID:5497542

  13. 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.

  14. 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.

  15. Crystal Structure of an Ammonia-Permeable Aquaporin

    PubMed Central

    Kirscht, Andreas; Kaptan, Shreyas S.; Bienert, Gerd Patrick; Chaumont, François; Nissen, Poul; de Groot, Bert L.; Kjellbom, Per; Gourdon, Pontus; Johanson, Urban

    2016-01-01

    Aquaporins of the TIP subfamily (Tonoplast Intrinsic Proteins) have been suggested to facilitate permeation of water and ammonia across the vacuolar membrane of plants, allowing the vacuole to efficiently sequester ammonium ions and counteract cytosolic fluctuations of ammonia. Here, we report the structure determined at 1.18 Å resolution from twinned crystals of Arabidopsis thaliana aquaporin AtTIP2;1 and confirm water and ammonia permeability of the purified protein reconstituted in proteoliposomes as further substantiated by molecular dynamics simulations. The structure of AtTIP2;1 reveals an extended selectivity filter with the conserved arginine of the filter adopting a unique unpredicted position. The relatively wide pore and the polar nature of the selectivity filter clarify the ammonia permeability. By mutational studies, we show that the identified determinants in the extended selectivity filter region are sufficient to convert a strictly water-specific human aquaporin into an AtTIP2;1-like ammonia channel. A flexible histidine and a novel water-filled side pore are speculated to deprotonate ammonium ions, thereby possibly increasing permeation of ammonia. The molecular understanding of how aquaporins facilitate ammonia flux across membranes could potentially be used to modulate ammonia losses over the plasma membrane to the atmosphere, e.g., during photorespiration, and thereby to modify the nitrogen use efficiency of plants. PMID:27028365

  16. Crystal Structure of an Ammonia-Permeable Aquaporin.

    PubMed

    Kirscht, Andreas; Kaptan, Shreyas S; Bienert, Gerd Patrick; Chaumont, François; Nissen, Poul; de Groot, Bert L; Kjellbom, Per; Gourdon, Pontus; Johanson, Urban

    2016-03-01

    Aquaporins of the TIP subfamily (Tonoplast Intrinsic Proteins) have been suggested to facilitate permeation of water and ammonia across the vacuolar membrane of plants, allowing the vacuole to efficiently sequester ammonium ions and counteract cytosolic fluctuations of ammonia. Here, we report the structure determined at 1.18 Å resolution from twinned crystals of Arabidopsis thaliana aquaporin AtTIP2;1 and confirm water and ammonia permeability of the purified protein reconstituted in proteoliposomes as further substantiated by molecular dynamics simulations. The structure of AtTIP2;1 reveals an extended selectivity filter with the conserved arginine of the filter adopting a unique unpredicted position. The relatively wide pore and the polar nature of the selectivity filter clarify the ammonia permeability. By mutational studies, we show that the identified determinants in the extended selectivity filter region are sufficient to convert a strictly water-specific human aquaporin into an AtTIP2;1-like ammonia channel. A flexible histidine and a novel water-filled side pore are speculated to deprotonate ammonium ions, thereby possibly increasing permeation of ammonia. The molecular understanding of how aquaporins facilitate ammonia flux across membranes could potentially be used to modulate ammonia losses over the plasma membrane to the atmosphere, e.g., during photorespiration, and thereby to modify the nitrogen use efficiency of plants.

  17. Protein Homeostasis at the Plasma Membrane

    PubMed Central

    2014-01-01

    The plasma membrane (PM) and endocytic protein quality control (QC) in conjunction with the endosomal sorting machinery either repairs or targets conformationally damaged membrane proteins for lysosomal/vacuolar degradation. Here, we provide an overview of emerging aspects of the underlying mechanisms of PM QC that fulfill a critical role in preserving cellular protein homeostasis in health and diseases. PMID:24985330

  18. CLS meets the aquaporin family: clinical cases involving aquaporin systems.

    PubMed

    Gade, Wayne; Robinson, Brooke

    2006-01-01

    Virtually all human cells incorporate aquaporins, or water channel proteins, into their cell membrane. Indeed, many cells produce several aquaporins, each adapted for a specific physiologic function. Thus, it is not surprising that aquaporin malfunctions are associated with numerous important clinical conditions. This article describes the clinical aspects of malfunctions in aquaporins or their regulation. Although water can diffuse across biological membranes (osmosis) without the aid of a transport system, researchers had predicted for decades that rapid reabsorption by renal tubule cells must be aided by a channel or pore. Yet, not until the 1990s were the first members of the aquaporin (AQP) family identified. Led by Dr. Peter Agre, recipient of the 2003 Nobel Prize in Chemistry, researchers have since amassed an astounding amount of information about AQPs and their function. For example, the flow rate of water through AQP1 is an extraordinary three billion water molecules per second per aquaporin channel, while a relative trickle of water crosses the hydrophobic lipid bilayer of cell membranes devoid of AQPs. Our understanding of renal physiology and pathophysiology has advanced greatly as we account for the subtle implications of various AQP systems. For example, nephrogenic diabetes insipidus (NDI), the inability to produce concentrated urine, can result from several different malfunctions in the hormonally controlled AQP2 system. The list of diseases known to involve AQPs now includes: early onset of cataracts, Sjogren's syndrome, cerebral and pulmonary edemas, cirrhotic liver development of ascites, and congestive heart failure (CHF).

  19. Vasopressin and the Regulation of Aquaporin-2

    PubMed Central

    Wilson, Justin L.L.; Miranda, Carlos A.; Knepper, Mark A.

    2013-01-01

    Water excretion is regulated in large part through the regulation of the osmotic water permeability of the renal collecting duct epithelium. The water permeability is controlled by vasopressin through regulation of the water channel, aquaporin-2 (AQP2). Two processes contribute: 1) regulation of AQP2 trafficking to the apical plasma membrane; and 2) regulation of the total amount of the AQP2 protein in the cells. Regulation of AQP2 abundance is defective in several water balance disorders including many polyuric disorders and the syndrome of inappropriate antidiuresis (SIADH). Here we review vasopressin signaling in the renal collecting duct that is relevant to the two modes of water permeability regulation. PMID:23584881

  20. The aquaporin gene family of the ectomycorrhizal fungus Laccaria bicolor: lessons for symbiotic functions.

    PubMed

    Dietz, Sandra; von Bülow, Julia; Beitz, Eric; Nehls, Uwe

    2011-06-01

    Soil humidity and bulk water transport are essential for nutrient mobilization. Ectomycorrhizal fungi, bridging soil and fine roots of woody plants, are capable of modulating both by being integrated into water movement driven by plant transpiration and the nocturnal hydraulic lift. Aquaporins are integral membrane proteins that function as gradient-driven water and/or solute channels. Seven aquaporins were identified in the genome of the ectomycorrhizal basidiomycete Laccaria bicolor and their role in fungal transfer processes was analyzed. Heterologous expression in Xenopus laevis oocytes revealed relevant water permeabilities for three aquaporins. In fungal mycelia, expression of the corresponding genes was high compared with other members of the gene family, indicating the significance of the respective proteins for plasma membrane water permeability. As growth temperature and ectomycorrhiza formation modified gene expression profiles of these water-conducting aquaporins, specific roles in those aspects of fungal physiology are suggested. Two aquaporins, which were highly expressed in ectomycorrhizas, conferred plasma membrane ammonia permeability in yeast. This indicates that these proteins are an integral part of ectomycorrhizal fungus-based plant nitrogen nutrition in symbiosis.

  1. 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.

  2. 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.

  3. 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.

  4. All-trans-retinoic acid regulates aquaporin-3 expression and related cellular membrane permeability in the human amniotic environment.

    PubMed

    Prat, C; Bouvier, D; Comptour, A; Marceau, G; Belville, C; Clairefond, G; Blanc, P; Gallot, D; Blanchon, L; Sapin, V

    2015-08-01

    The aquaporins (AQP1, 3, 8, 9 and 11) are known to be expressed, and involved in the transport of water and small molecules through fetal membranes. To exert these crucial functions, these AQPs have to be finely regulated. All-trans-retinoic acid (atRA) was previously found to regulate some genes in this environment, raising the question of whether these AQPs were regulated by atRA. Explants, and primary and established amniotic cells were cultured to determine which AQP were transcriptionally modified by atRA, using the qRT-PCR strategy. Immunohistochemistry and glycerol uptake tests were used to determine the impact of atRA on AQP protein expression and function. Specific agonists of retinoic acid receptors were used to identify the molecular mechanisms of AQP promoter activation. A classical gene AQP promoter study was also used to identify DR5 retinoic acid receptor elements (RAREs). Beyond these AQPs, only one specific atRA-dependent increase in AQP3 transcripts and proteins level was established in amnion (not in chorion) and in related primary and established cells. We found three DR5-RAREs essential for inducing this transcriptional AQP3 through RARα. This transactivation of the AQP3 coding gene was functionally related to an increase of AQP3 permeability tests by a glycerol uptake assay. Our data support an atRA regulatory model of AQP3 expression leading to an increased cellular permeability in the epithelial amniotic environment. We cast new light on AF regulation in healthy pregnancy, and advance new hypotheses for obstetrical complications linked to impairment of the retinoic signaling pathway. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Aquaporins in Fetal Development.

    PubMed

    Martínez, Nora; Damiano, Alicia E

    2017-01-01

    Water homeostasis during fetal development is of crucial physiologic importance. The successful formation and development of the placenta is critical to maintain normal fetal growth and homeostasis. The expression of several aquaporins (AQPs ) was found from blastocyst stages to term placenta and fetal membranes. Therefore, AQPs are proposed to play important roles in normal pregnancy, fetal growth, and homeostasis of amniotic fluid volume, and water handling in other organs. However, the functional importance of AQPs in fetal development remains to be elucidated.

  6. Cisplatin cytotoxicity: DNA and plasma membrane targets.

    PubMed

    Rebillard, Amélie; Lagadic-Gossmann, Dominique; Dimanche-Boitrel, Marie-Thérèse

    2008-01-01

    Most current anticancer therapies induce tumor cell death through apoptosis where its specific involved pathways are poorly understood. For example, for many DNA-damaging agents, the specific biochemical lesions (DNA adducts) are associated with the induction of apoptosis via the mitochondria death pathway. However, several of these DNA-damaging agents like cisplatin induce apoptosis through plasma membrane disruption, triggering the Fas death receptor pathway. In this review, we focus on the role of early plasma membrane events in cisplatin-induced apoptosis. Special attention is given to changes in plasma membrane fluidity, inhibition of NHE1 exchanger, activation of acid sphingomyelinase and their consequences on the Fas death pathway in response to cisplatin.

  7. Two different effects of calcium on aquaporins in salinity-stressed pepper plants.

    PubMed

    Martínez-Ballesta, M Carmen; Cabañero, Francisco; Olmos, Enrique; Periago, Paula María; Maurel, Christophe; Carvajal, Micaela

    2008-06-01

    Two different effects of calcium were studied, respectively, in plasma membrane vesicles and in protoplasts isolated from roots of control pepper plants (Capsicum annuum L cv. California) or of plants treated with 50 mM NaCl, 10 mM CaCl(2) or 10 mM CaCl(2) + 50 mM NaCl. Under saline conditions, osmotic water permeability (P ( f )) values decreased in protoplasts and plasma membrane vesicles, and the same reduction was observed in the PIP1 aquaporin abundance, indicating inhibitory effects of NaCl on aquaporin functionality and protein abundance. The cytosolic Ca(2+) concentration, [Ca(2+)](cyt), was reduced by salinity, as observed by confocal microscope analysis. Two different actions of Ca(2+) were observed. On the one hand, increase in free cytosolic calcium concentrations associated with stress perception may lead to aquaporin closure. On the other hand, when critical requirements of Ca(2+) were reduced (by salinity), and extra-calcium would lead to an upregulation of aquaporins, indicating that a positive role of calcium at whole plant level combined with an inhibitory mechanism at aquaporin level may work in the regulation of pepper root water transport under salt stress. However, a link between these observations and other cell signalling in relation to water channel gating remains to be established.

  8. Progress on the application of aquaporins in Chinese medicine.

    PubMed

    Liang, Xing; Mao, Wei; Liu, Xu-Sheng

    2013-07-01

    Aquaporins are a group of membrane proteins, which are known as the passages of water molecules transforming through the biological membrane lipid bilayer and distributing in almost all of the organs and tissues of living creatures. Aquaporins play important roles in maintaining water balance and internal environment stability. As a new entry point, aquaporins are involved in the researches on water metabolism, physiological regulation and pathological essence in viscera-state more and more widely in recent years. The literature on traditional Chinese medical studies, which related to aquaporins and were published in the last decade, was reviewed and the progress on application of aquaporin in Chinese medicine was summarized in this paper.

  9. Phosphatase inhibition increases AQP2 accumulation in the rat IMCD apical plasma membrane.

    PubMed

    Ren, Huiwen; Yang, Baoxue; Ruiz, Joseph A; Efe, Orhan; Ilori, Titilayo O; Sands, Jeff M; Klein, Janet D

    2016-12-01

    Vasopressin triggers the phosphorylation and apical plasma membrane accumulation of aquaporin 2 (AQP2), and it plays an essential role in urine concentration. Vasopressin, acting through protein kinase A, phosphorylates AQP2. However, the phosphorylation state of AQP2 could also be affected by the action of protein phosphatases (PPs). Rat inner medullas (IM) were incubated with calyculin (PP1 and PP2A inhibitor, 50 nM) or tacrolimus (PP2B inhibitor, 100 nM). Calyculin did not affect total AQP2 protein abundance (by Western blot) but did significantly increase the abundances of pS256-AQP2 and pS264-AQP2. It did not change pS261-AQP2 or pS269-AQP2. Calyculin significantly enhanced the membrane accumulation (by biotinylation) of total AQP2, pS256-AQP2, and pS264-AQP2. Likewise, immunohistochemistry showed an increase in the apical plasma membrane association of pS256-AQP2 and pS264-AQP2 in calyculin-treated rat IM. Tacrolimus also did not change total AQP2 abundance but significantly increased the abundances of pS261-AQP2 and pS264-AQP2. In contrast to calyculin, tacrolimus did not change the amount of total AQP2 in the plasma membrane (by biotinylation and immunohistochemistry). Tacrolimus did increase the expression of pS264-AQP2 in the apical plasma membrane (by immunohistochemistry). In conclusion, PP1/PP2A regulates the phosphorylation and apical plasma membrane accumulation of AQP2 differently than PP2B. Serine-264 of AQP2 is a phosphorylation site that is regulated by both PP1/PP2A and PP2B. This dual regulatory pathway may suggest a previously unappreciated role for multiple phosphatases in the regulation of urine concentration. Copyright © 2016 the American Physiological Society.

  10. 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

  11. 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

  12. Plasma membrane regulates Ras signaling networks

    PubMed Central

    Chavan, Tanmay Sanjeev; Muratcioglu, Serena; Marszalek, Richard; Jang, Hyunbum; Keskin, Ozlem; Gursoy, Attila; Nussinov, Ruth; Gaponenko, Vadim

    2015-01-01

    Ras GTPases activate more than 20 signaling pathways, regulating such essential cellular functions as proliferation, survival, and migration. How Ras proteins control their signaling diversity is still a mystery. Several pieces of evidence suggest that the plasma membrane plays a critical role. Among these are: (1) selective recruitment of Ras and its effectors to particular localities allowing access to Ras regulators and effectors; (2) specific membrane-induced conformational changes promoting Ras functional diversity; and (3) oligomerization of membrane-anchored Ras to recruit and activate Raf. Taken together, the membrane does not only attract and retain Ras but also is a key regulator of Ras signaling. This can already be gleaned from the large variability in the sequences of Ras membrane targeting domains, suggesting that localization, environment and orientation are important factors in optimizing the function of Ras isoforms. PMID:27054048

  13. Plasma membrane regulates Ras signaling networks.

    PubMed

    Chavan, Tanmay Sanjeev; Muratcioglu, Serena; Marszalek, Richard; Jang, Hyunbum; Keskin, Ozlem; Gursoy, Attila; Nussinov, Ruth; Gaponenko, Vadim

    2015-01-01

    Ras GTPases activate more than 20 signaling pathways, regulating such essential cellular functions as proliferation, survival, and migration. How Ras proteins control their signaling diversity is still a mystery. Several pieces of evidence suggest that the plasma membrane plays a critical role. Among these are: (1) selective recruitment of Ras and its effectors to particular localities allowing access to Ras regulators and effectors; (2) specific membrane-induced conformational changes promoting Ras functional diversity; and (3) oligomerization of membrane-anchored Ras to recruit and activate Raf. Taken together, the membrane does not only attract and retain Ras but also is a key regulator of Ras signaling. This can already be gleaned from the large variability in the sequences of Ras membrane targeting domains, suggesting that localization, environment and orientation are important factors in optimizing the function of Ras isoforms.

  14. Sulfate transport in Penicillium chrysogenum plasma membranes.

    PubMed Central

    Hillenga, D J; Versantvoort, H J; Driessen, A J; Konings, W N

    1996-01-01

    Transport studies with Penicillium chrysogenum plasma membranes fused with cytochrome c oxidase liposomes demonstrate that sulfate uptake is driven by the transmembrane pH gradient and not by the transmembrane electrical potential. Ca2+ and other divalent cations are not required. It is concluded that the sulfate transport system catalyzes the symport of two protons with one sulfate anion. PMID:8682803

  15. Physiology and pathophysiology of renal aquaporins.

    PubMed

    Nielsen, S; Kwon, T H; Christensen, B M; Promeneur, D; Frøkiaer, J; Marples, D

    1999-03-01

    The discovery of aquaporin membrane water channels by Agre and coworkers answered a long-standing biophysical question of how water specifically crosses biologic membranes, and provided insight, at the molecular level, into the fundamental physiology of water balance and the pathophysiology of water balance disorders. Of nine aquaporin isoforms, at least six are known to be present in the kidney at distinct sites along the nephron and collecting duct. Aquaporin-1 (AQP1) is extremely abundant in the proximal tubule and descending thin limb, where it appears to provide the chief route for proximal nephron water reabsorption. AQP2 is abundant in the collecting duct principal cells and is the chief target for vasopressin to regulate collecting duct water reabsorption. Acute regulation involves vasopressin-regulated trafficking of AQP2 between an intracellular reservoir and the apical plasma membrane. In addition, AQP2 is involved in chronic/adaptational regulation of body water balance achieved through regulation of AQP2 expression. Importantly, multiple studies have now identified a critical role of AQP2 in several inherited and acquired water balance disorders. This concerns inherited forms of nephrogenic diabetes insipidus and several, much more common acquired types of nephrogenic diabetes insipidus where AQP2 expression and/or targeting are affected. Conversely, AQP2 expression and targeting appear to be increased in some conditions with water retention such as pregnancy and congestive heart failure. AQP3 and AQP4 are basolateral water channels located in the kidney collecting duct, and AQP6 and AQP7 appear to be expressed at lower abundance at several sites including the proximal tubule. This review focuses mainly on the role of AQP2 in water balance regulation and in the pathophysiology of water balance disorders.

  16. Aquaporin water channels in mammals.

    PubMed

    Ishibashi, Kenichi; Hara, Shigeki; Kondo, Shintaro

    2009-04-01

    Water channels, aquaporins (AQPs), are a family of small integral plasma membrane proteins that primarily transport water across the plasma membrane. There are 13 members (AQP0-12) in humans. This number is final as the human genome project has been completed. They are divided into three subgroups based on the primary sequences: water selective AQPs (AQP0, 1, 2, 4, 5, 6, 8), aquaglyceroporins (AQP3, 7, 9, 10), and superaquaporins (AQP11, 12). Since no specific inhibitors are yet available, functional roles of AQPs are suggested by AQP null mice and humans. Abnormal water metabolism was shown with AQP1, 2, 3, 4, 5 null mice, especially with AQP2 null mice: fatal at neonate due to diabetes insipidus. Abnormal glycerol transport was shown with AQP3, 7, 9 null mice, although they appeared normal. AQP0 null mice suffer from cataracts, although the pathogenesis is not clear. Unexpectedly, AQP11 null mice die from uremia as a result of polycystic kidneys. Interestingly, AQP6, 8, 10, 12 null mice are almost normal. AQP null humans have been reported with AQP0, 1, 2, 3, 7: only AQP2 null humans show an outstanding phenotype, diabetes insipidus. This review summarizes the current knowledge on all mammalian AQPs and hopefully will stimulate future research in both clinical and basic fields.

  17. Cholesterol Asymmetry in Synaptic Plasma Membranes

    PubMed Central

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

    2010-01-01

    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: 1) chronic ethanol consumption; 2) statins; 3) aging; and 4) 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. PMID:21214553

  18. A cautionary note on cosmetics containing ingredients that increase aquaporin-3 expression.

    PubMed

    Verkman, A S

    2008-10-01

    Aquaporin-3 (AQP3) is a membrane transport protein that facilitates water and glycerol transport across cell plasma membranes in the basal layer of keratinocytes in normal skin. Motivated by a relation between AQP3 expression and skin water content, several companies have marketed cosmetics containing ingredients that increase AQP3 expression. However, caution seems warranted in targeting AQP3 to increase skin moisturization based on a recently discovered association in mice between epidermal AQP3 expression and skin tumor formation.

  19. Coordinated Post-translational Responses of Aquaporins to Abiotic and Nutritional Stimuli in Arabidopsis Roots*

    PubMed Central

    di Pietro, Magali; Vialaret, Jérôme; Li, Guo-Wei; Hem, Sonia; Prado, Karine; Rossignol, Michel; Maurel, Christophe; Santoni, Véronique

    2013-01-01

    In plants, aquaporins play a crucial role in regulating root water transport in response to environmental and physiological cues. Controls achieved at the post-translational level are thought to be of critical importance for regulating aquaporin function. To investigate the general molecular mechanisms involved, we performed, using the model species Arabidopsis, a comprehensive proteomic analysis of root aquaporins in a large set of physiological contexts. We identified nine physiological treatments that modulate root hydraulics in time frames of minutes (NO and H2O2 treatments), hours (mannitol and NaCl treatments, exposure to darkness and reversal with sucrose, phosphate supply to phosphate-starved roots), or days (phosphate or nitrogen starvation). All treatments induced inhibition of root water transport except for sucrose supply to dark-grown plants and phosphate resupply to phosphate-starved plants, which had opposing effects. Using a robust label-free quantitative proteomic methodology, we identified 12 of 13 plasma membrane intrinsic protein (PIP) aquaporin isoforms, 4 of the 10 tonoplast intrinsic protein isoforms, and a diversity of post-translational modifications including phosphorylation, methylation, deamidation, and acetylation. A total of 55 aquaporin peptides displayed significant changes after treatments and enabled the identification of specific and as yet unknown patterns of response to stimuli. The data show that the regulation of PIP and tonoplast intrinsic protein abundance was involved in response to a few treatments (i.e. NaCl, NO, and nitrate starvation), whereas changes in the phosphorylation status of PIP aquaporins were positively correlated to changes in root hydraulic conductivity in the whole set of treatments. The identification of in vivo deamidated forms of aquaporins and their stimulus-induced changes in abundance may reflect a new mechanism of aquaporin regulation. The overall work provides deep insights into the in vivo post

  20. Coordinated post-translational responses of aquaporins to abiotic and nutritional stimuli in Arabidopsis roots.

    PubMed

    di Pietro, Magali; Vialaret, Jérôme; Li, Guo-Wei; Hem, Sonia; Prado, Karine; Rossignol, Michel; Maurel, Christophe; Santoni, Véronique

    2013-12-01

    In plants, aquaporins play a crucial role in regulating root water transport in response to environmental and physiological cues. Controls achieved at the post-translational level are thought to be of critical importance for regulating aquaporin function. To investigate the general molecular mechanisms involved, we performed, using the model species Arabidopsis, a comprehensive proteomic analysis of root aquaporins in a large set of physiological contexts. We identified nine physiological treatments that modulate root hydraulics in time frames of minutes (NO and H2O2 treatments), hours (mannitol and NaCl treatments, exposure to darkness and reversal with sucrose, phosphate supply to phosphate-starved roots), or days (phosphate or nitrogen starvation). All treatments induced inhibition of root water transport except for sucrose supply to dark-grown plants and phosphate resupply to phosphate-starved plants, which had opposing effects. Using a robust label-free quantitative proteomic methodology, we identified 12 of 13 plasma membrane intrinsic protein (PIP) aquaporin isoforms, 4 of the 10 tonoplast intrinsic protein isoforms, and a diversity of post-translational modifications including phosphorylation, methylation, deamidation, and acetylation. A total of 55 aquaporin peptides displayed significant changes after treatments and enabled the identification of specific and as yet unknown patterns of response to stimuli. The data show that the regulation of PIP and tonoplast intrinsic protein abundance was involved in response to a few treatments (i.e. NaCl, NO, and nitrate starvation), whereas changes in the phosphorylation status of PIP aquaporins were positively correlated to changes in root hydraulic conductivity in the whole set of treatments. The identification of in vivo deamidated forms of aquaporins and their stimulus-induced changes in abundance may reflect a new mechanism of aquaporin regulation. The overall work provides deep insights into the in vivo post

  1. Rapid gating and anion permeability of an intracellular aquaporin.

    PubMed

    Yasui, M; Hazama, A; Kwon, T H; Nielsen, S; Guggino, W B; Agre, P

    1999-11-11

    Aquaporin (AQP) water-channel proteins are freely permeated by water but not by ions or charged solutes. Although mammalian aquaporins were believed to be located in plasma membranes, rat AQP6 is restricted to intracellular vesicles in renal epithelia. Here we show that AQP6 is functionally distinct from other known aquaporins. When expressed in Xenopus laevis oocytes, AQP6 exhibits low basal water permeability; however, when treated with the known water channel inhibitor, Hg2+, the water permeability of AQP6 oocytes rapidly rises up to tenfold and is accompanied by ion conductance. AQP6 colocalizes with H+-ATPase in intracellular vesicles of acid-secreting alpha-intercalated cells in renal collecting duct. At pH less than 5.5, anion conductance is rapidly and reversibly activated in AQP6 oocytes. Site-directed mutation of lysine to glutamate at position 72 in the cytoplasmic mouth of the pore changes the cation/anion selectivity, but leaves low pH activation intact. Our results demonstrate unusual biophysical properties of an aquaporin, and indicate that anion-channel function may now be explored in a protein with known structure.

  2. Piscine aquaporins: an overview of recent advances.

    PubMed

    Cerdà, Joan; Finn, Roderick Nigel

    2010-12-01

    Aquaporins are a superfamily of integral membrane proteins that facilitate the rapid and yet highly selective flux of water and other small solutes across biological membranes. Since their discovery, they have been documented throughout the living biota, with the majority of research focusing on mammals and plants. Here, we review available data for piscine aquaporins, including Agnatha (jawless fish), Chondrichthyes (chimaeras, sharks, and rays), Dipnoi (lungfishes), and Teleostei (ray-finned bony fishes). Recent evidence suggests that the aquaporin superfamily has specifically expanded in the chordate lineage consequent to serial rounds of whole genome duplication, with teleost genomes harboring the largest number of paralogs. The selective retention and dichotomous clustering of most duplicated paralogs in Teleostei, with differential tissue expression profiles, implies that novel or specialized physiological functions may have evolved in this clade. The recently proposed new nomenclature of the piscine aquaporin superfamily is discussed in relation to the phylogenetic signal and genomic synteny, with the teleost aquaporin-8 paralogs used as a case study to illustrate disparities between the underlying codons, molecular phylogeny, and physical locus. Structural data indicate that piscine aquaporins display similar channel restriction residues found in the tetrapod counterparts, and hence their functional properties seem to be conserved. However, emerging evidence suggests that regulation of aquaporin function in teleosts may have diverged in some cases. Cell localization and experimental studies imply that the physiological roles of piscine aquaporins extend at least to osmoregulation, reproduction, and early development, although in most cases their specific functions remain to be elucidated.

  3. Stomatin interacts with GLUT1/SLC2A1, band 3/SLC4A1, and aquaporin-1 in human erythrocyte membrane domains

    PubMed Central

    Rungaldier, Stefanie; Oberwagner, Walter; Salzer, Ulrich; Csaszar, Edina; Prohaska, Rainer

    2013-01-01

    The widely expressed, homo-oligomeric, lipid raft-associated, monotopic integral membrane protein stomatin and its homologues are known to interact with and modulate various ion channels and transporters. Stomatin is a major protein of the human erythrocyte membrane, where it associates with and modifies the glucose transporter GLUT1; however, previous attempts to purify hetero-oligomeric stomatin complexes for biochemical analysis have failed. Because lateral interactions of membrane proteins may be short-lived and unstable, we have used in situ chemical cross-linking of erythrocyte membranes to fix the stomatin complexes for subsequent purification by immunoaffinity chromatography. To further enrich stomatin, we prepared detergent-resistant membranes either before or after cross-linking. Mass spectrometry of the isolated, high molecular, cross-linked stomatin complexes revealed the major interaction partners as glucose transporter-1 (GLUT1), anion exchanger (band 3), and water channel (aquaporin-1). Moreover, ferroportin-1 (SLC40A1), urea transporter-1 (SLC14A1), nucleoside transporter (SLC29A1), the calcium-pump (Ca-ATPase-4), CD47, and flotillins were identified as stomatin-interacting proteins. These findings are in line with the hypothesis that stomatin plays a role as membrane-bound scaffolding protein modulating transport proteins. PMID:23219802

  4. Stomatin interacts with GLUT1/SLC2A1, band 3/SLC4A1, and aquaporin-1 in human erythrocyte membrane domains.

    PubMed

    Rungaldier, Stefanie; Oberwagner, Walter; Salzer, Ulrich; Csaszar, Edina; Prohaska, Rainer

    2013-03-01

    The widely expressed, homo-oligomeric, lipid raft-associated, monotopic integral membrane protein stomatin and its homologues are known to interact with and modulate various ion channels and transporters. Stomatin is a major protein of the human erythrocyte membrane, where it associates with and modifies the glucose transporter GLUT1; however, previous attempts to purify hetero-oligomeric stomatin complexes for biochemical analysis have failed. Because lateral interactions of membrane proteins may be short-lived and unstable, we have used in situ chemical cross-linking of erythrocyte membranes to fix the stomatin complexes for subsequent purification by immunoaffinity chromatography. To further enrich stomatin, we prepared detergent-resistant membranes either before or after cross-linking. Mass spectrometry of the isolated, high molecular, cross-linked stomatin complexes revealed the major interaction partners as glucose transporter-1 (GLUT1), anion exchanger (band 3), and water channel (aquaporin-1). Moreover, ferroportin-1 (SLC40A1), urea transporter-1 (SLC14A1), nucleoside transporter (SLC29A1), the calcium-pump (Ca-ATPase-4), CD47, and flotillins were identified as stomatin-interacting proteins. These findings are in line with the hypothesis that stomatin plays a role as membrane-bound scaffolding protein modulating transport proteins.

  5. [Expression of aquaporins and its significance in human pulmonary adenocarcinoma cell line SPC-A-1].

    PubMed

    Chen, Jie; Bai, Chunxue; Zhang, Min; Ren, Zhenyi; Hu, Jie

    2004-06-20

    To investigate the expression of aquaporins in human pulmonary adenocarcinoma cell line SPC-A-1. The expressions of aquaporin 1, aquaporin 3, aquaporin 4, and aquaporin 5 in mRNA level and their locations were determined in cell line SPC-A-1 respectively by RT-PCR and immunohistochemistry. The immunohistochemical stain showed aquaporin 3 and aquaporin 5 located on the membrane of SPC-A-1 cell, but no positive stain of aquaporin 1 and aquaporin 4 was observed. Both aquaporin 3 and aquaporin 5 mRNA expressed in SPC-A-1 cell line, and the expression level of aquaporin 5 mRNA was significantly higher than that of aquaporin 3 mRNA ( P < 0.01). Aquaporin 1 and aquaporin 4 mRNA did not express in SPC-A-1 cell line. Aquaporin 3 and aquaporin 5 express in SPC-A-1 cell, and their roles in water transport of SPC-A-1 cell should be further investigated.

  6. Preparation of pure and intact Plasmodium falciparum plasma membrane vesicles and partial characterisation of the plasma membrane ATPase.

    PubMed

    Elandalloussi, Laurence M; Smith, Pete J

    2002-04-26

    In host erythrocytes, the malaria parasite must contend with ion and drug transport across three membranes; its own plasma membrane, the parasitophorous membrane and the host plasma membrane. Isolation of pure and intact Plasmodium falciparum plasma membrane would provide a suitable model to elucidate the possible role played by the parasite plasma membrane in ion balance and drug transport. This study describes a procedure for isolating parasite plasma membrane from P. falciparum-infected erythrocytes. With this method, the trophozoites released by saponin treatment were cleansed of erythrocyte membranes using anti-erythrocyte antibodies fixed to polystyrene beads. These trophozoites were then biotinylated and the parasite plasma membrane was disrupted by nitrogen cavitation. This process allows the membranes to reform into vesicles. The magnetic streptavidin beads bind specifically to the biotinylated parasite plasma membrane vesicles facilitating their recovery with a magnet. These vesicles can then be easily released from the magnetic beads by treatment with dithiotreithol. The parasite plasma membrane showed optimal ATPase activity at 2 mM ATP and 2 mM Mg2+. It was also found that Ca2+ could not substitute for Mg2+ ATPase activity in parasite plasma membranes whereas activity was completely preserved when Mn2+ was used instead of Mg2+. Other nucleoside triphosphates tested were hydrolysed as efficiently as ATP, while the nucleoside monophosphate AMP was not. We have described the successful isolation of intact P. falciparum plasma membrane vesicles free of contaminating organelles and determined the experimental conditions for optimum ATPase activity.

  7. Aquaporins: important but elusive drug targets

    PubMed Central

    Verkman, Alan S.; Anderson, Marc O.; Papadopoulos, Marios C.

    2014-01-01

    The aquaporins (AQPs) are a family of small, integral membrane proteins that facilitate water transport across the plasma membranes of cells in response to osmotic gradients. Data from knockout mice support the involvement of AQPs in epithelial fluid secretion, cell migration, brain oedema and adipocyte metabolism, which suggests that modulation of AQP function or expression could have therapeutic potential in oedema, cancer, obesity, brain injury, glaucoma and several other conditions. Moreover, loss-of-function mutations in human AQPs cause congenital cataracts (AQP0) and nephrogenic diabetes insipidus (AQP2), and autoantibodies against AQP4 cause the autoimmune demyelinating disease neuromyelitis optica. Although some potential AQP modulators have been identified, challenges associated with the development of better modulators include the druggability of the target and the suitability of the assay methods used to identify modulators. PMID:24625825

  8. Proteomic approaches to identify cold-regulated plasma membrane proteins.

    PubMed

    Takahashi, Daisuke; Nakayama, Takato; Miki, Yushi; Kawamura, Yukio; Uemura, Matsuo

    2014-01-01

    Plasma membrane is the primary determinant of freezing tolerance in plants because of its central role in freeze-thaw cycle. Changes in the plasma membrane proteins have been one of the major research areas in plant cold acclimation. To obtain comprehensive profiles of the plasma membrane proteomes and their changes during the cold acclimation process, a plasma membrane purification method using a dextran-polyethylene glycol two polymer system and a mass spectrometry-based shotgun proteomics method using nano-LC-MS/MS for the plasma membrane proteins are described. The proteomic results obtained are further applied to label-free protein semiquantification.

  9. Protein quality control at the plasma membrane

    PubMed Central

    Okiyoneda, Tsukasa; Apaja, Pirjo M.; Lukacs, Gergely L.

    2011-01-01

    Cellular proteostasis (or protein homeostasis) depends on the timely folding and disposal of conformationally damaged polypeptides during their life span at all subcellular locations. This process is particularly important for membrane proteins confined to the cell surface with critical regulatory role in cellular homoeostasis and intercellular communication. Accumulating evidences indicate that membrane proteins exported from the endoplasmic reticulum (ER) are subjected to peripheral quality control (QC) along the late secretory and endocytic pathways, as well as at the plasma membrane (PM). Recently identified components of the PM QC recognition and effector mechanisms responsible for ubiquitination and lysosomal degradation of conformationally damaged PM proteins uncovered striking similarities to and differences from that of the ER QC machinery. Possible implications of the peripheral protein QC activity in phenotypic modulation of conformational diseases are also outlined. PMID:21571517

  10. Protein quality control at the plasma membrane.

    PubMed

    Okiyoneda, Tsukasa; Apaja, Pirjo M; Lukacs, Gergely L

    2011-08-01

    Cellular proteostasis (or protein homeostasis) depends on the timely folding and disposal of conformationally damaged polypeptides during their life span at all subcellular locations. This process is particularly important for membrane proteins confined to the cell surface with crucial regulatory role in cellular homoeostasis and intercellular communication. Accumulating evidences indicate that membrane proteins exported from the endoplasmic reticulum (ER) are subjected to peripheral quality control (QC) along the late secretory and endocytic pathways, as well as at the plasma membrane (PM). Recently identified components of the PM QC recognition and effector mechanisms responsible for ubiquitination and lysosomal degradation of conformationally damaged PM proteins uncovered striking similarities to and differences from that of the ER QC machinery. Possible implications of the peripheral protein QC activity in phenotypic modulation of conformational diseases are also outlined. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

  11. 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.

  12. Osmotic water permeability of rat intestinal brush border membrane vesicles: involvement of aquaporin-7 and aquaporin-8 and effect of metal ions.

    PubMed

    Tritto, Simona; Gastaldi, Giulia; Zelenin, Sergey; Grazioli, Monica; Orsenigo, Maria Novella; Ventura, Ulderico; Laforenza, Umberto; Zelenina, Marina

    2007-12-01

    Water channels AQP7 and AQP8 may be involved in transcellular water movement in the small intestine. We show that both AQP7 and AQP8 mRNA are expressed in rat small intestine. Immunoblot and immunohistochemistry experiments demonstrate that AQP7 and AQP8 proteins are present in the apical brush border membrane of intestinal epithelial cells. We investigated the effect of several metals and pH on the osmotic water permeability (Pf) of brush border membrane vesicles (BBMVs) and of AQP7 and AQP8 expressed in a cell line. Hg2+, Cu2+, and Zn2+ caused a significant decrease in the BBMV Pf, whereas Ni2+ and Li+ had no effect. AQP8-transfected cells showed a reduction in Pf in the presence of Hg2+ and Cu2+, whereas AQP7-transfected cells were insensitive to all tested metals. The Pf of both BBMVs and cells transfected with AQP7 and AQP8 was not affected by pH changes within the physiological range, and the Pf of BBMVs alone was not affected by phlorizin or amiloride. Our results indicate that AQP7 and AQP8 may play a role in water movement via the apical domain of small intestine epithelial cells. AQP8 may contribute to the water-imbalance-related clinical symptoms apparent after ingestion of high doses of Hg2+ and Cu2+.

  13. Labeling the plasma membrane with TMA-DPH.

    PubMed

    Chazotte, Brad

    2011-05-01

    INTRODUCTION TMA-DPH (trimethylamine-diphenylhexatriene) is a fluorescent membrane probe that has classically been used to label the outer leaflet of a membrane bilayer, to label the outer leaflet of the plasma membrane in cells, and to report on membrane dynamics using the techniques of fluorescence polarization and/or fluorescence lifetime. This probe has also been used to follow exocytosis and endocytosis of labeled plasma membranes. The interaction of the aqueous environment with mitochondrial inner membrane dynamics has also been studied following the fluorescence polarization and the lifetime of TMA-DPH. This protocol describes the use of TMA-DPH to label the plasma membrane.

  14. Phsyiological relevance of aquaporins: luxury or necessity?

    PubMed

    van Os, C H; Kamsteeg, E J; Marr, N; Deen, P M

    2000-08-01

    Aquaporins are members of a large family of pore-forming intrinsic membrane proteins, the MIP family. Based on their permeability properties they are now further subdivided into aquaporins, with real water-selective pores, and aquaglyceroporins with slightly less selective pores. Aquaporins are expressed in a large variety of tissues throughout the body but in most situations it is not clear whether their presence is necessary for the proper physiological function of these tissues. This review focuses on recent insight into the physiological relevance of aquaporins gained from studying aquaporin knockout mouse models and from diseases, on new surprising findings related to gating and selectivity, and on the consequences of tetramerization for routing and the genetics of nephrogenic diabetes insipidus. The active fluid transport in proximal tubules and in salivary glands is seriously compromised by aquaporin deletion. This is in contrast to lung, airways and stomach, where active fluid transport proceeds unhindered in the face of greatly reduced water permeabilities due to aquaporin deletion. Therefore, aquaporins seem to be a necessity at extreme high rates of active fluid transport but appear to be more of a luxury at medium or low fluid transport rates.

  15. Aquaporins in the digestive system.

    PubMed

    Matsuzaki, Toshiyuki; Tajika, Yuki; Ablimit, Abdushukur; Aoki, Takeo; Hagiwara, Haruo; Takata, Kuniaki

    2004-06-01

    Fluid transfer such as secretion and absorption is one of the major functions of the digestive system. Aquaporins are water channel proteins providing water transfer across the cellular membrane. At least six aquaporin isoforms are expressed in the digestive system. Aquaporin-1 (AQP1) is widely distributed in endothelial cells of capillaries and small vessels as well as in the central lacteals in the small intestine. AQP1 is also present in the duct system in the pancreas, liver, and bile duct. AQP3 is mainly expressed in the epithelia of the upper digestive tract from the oral cavity to the stomach and of the lower digestive tract from the distal colon to the anus. AQP4 is present in the parietal cells of the stomach and in the intestinal epithelia. AQP5 is expressed in acinar cells of the salivary, pyloric, and duodenal glands. AQP8 is expressed in the intestinal epithelia, salivary glands, pancreas, and liver. AQP9 is present in the liver and intestinal goblet cells. Aquaporins have important roles in the digestive system, such as AQP5 in saliva secretion, as shown by the studies on AQP5-null mice. In addition, water transfer across the digestive epithelia seems to occur not only via aquaporins but also via other transporter or channel systems. Copyright 2004 The Clinical Electron Microscopy Society of Japan

  16. Aquaporin-1 and HCO3−–Cl− transporter-mediated transport of CO2 across the human erythrocyte membrane

    PubMed Central

    Blank, Michael E; Ehmke, Heimo

    2003-01-01

    Recent studies have suggested that aquaporin-1 (AQP1) as well as the HCO3−–Cl− transporter may be involved in CO2 transport across biological membranes, but the physiological importance of this route of gas transport remained unknown. We studied CO2 transport in human red blood cell ghosts at physiological temperatures (37 °C). Replacement of inert with CO2-containing gas above a stirred cell suspension caused an outside-to-inside directed CO2 gradient and generated a rapid biphasic intracellular acidification. The gradient of the acidifying gas was kept small to favour high affinity entry of CO2 passing the membrane. All rates of acidification except that of the approach to physicochemical equilibrium of the uncatalysed reaction were restricted to the intracellular environment. Inhibition of carbonic anhydrase (CA) demonstrated that CO2-induced acidification required the catalytic activity of CA. Blockade of the function of either AQP1 (by HgCl2 at 65 μM) or the HCO3−–Cl− transporter (by DIDS at 15 μM) completely prevented fast acidification. These data indicate that, at low chemical gradients for CO2, nearly the entire CO2 transport across the red cell membrane is mediated by AQP1 and the HCO3−–Cl− transporter. Therefore, these proteins may function as high affinity sites for CO2 transport across the erythrocyte membrane. PMID:12754312

  17. Cellular membrane collapse by atmospheric-pressure plasma jet

    SciTech Connect

    Kim, Kangil; Sik Yang, Sang E-mail: ssyang@ajou.ac.kr; Jun Ahn, Hak; Lee, Jong-Soo E-mail: ssyang@ajou.ac.kr; Lee, Jae-Hyeok; Kim, Jae-Ho

    2014-01-06

    Cellular membrane dysfunction caused by air plasma in cancer cells has been studied to exploit atmospheric-pressure plasma jets for cancer therapy. Here, we report that plasma jet treatment of cervical cancer HeLa cells increased electrical conductivity across the cellular lipid membrane and caused simultaneous lipid oxidation and cellular membrane collapse. We made this finding by employing a self-manufactured microelectrode chip. Furthermore, increased roughness of the cellular lipid membrane and sequential collapse of the membrane were observed by atomic force microscopy following plasma jet treatment. These results suggest that the cellular membrane catastrophe occurs via coincident altered electrical conductivity, lipid oxidation, and membrane roughening caused by an atmospheric-pressure plasma jet, possibly resulting in cellular vulnerability to reactive species generated from the plasma as well as cytotoxicity to cancer cells.

  18. Cellular membrane collapse by atmospheric-pressure plasma jet

    NASA Astrophysics Data System (ADS)

    Kim, Kangil; Jun Ahn, Hak; Lee, Jae-Hyeok; Kim, Jae-Ho; Sik Yang, Sang; Lee, Jong-Soo

    2014-01-01

    Cellular membrane dysfunction caused by air plasma in cancer cells has been studied to exploit atmospheric-pressure plasma jets for cancer therapy. Here, we report that plasma jet treatment of cervical cancer HeLa cells increased electrical conductivity across the cellular lipid membrane and caused simultaneous lipid oxidation and cellular membrane collapse. We made this finding by employing a self-manufactured microelectrode chip. Furthermore, increased roughness of the cellular lipid membrane and sequential collapse of the membrane were observed by atomic force microscopy following plasma jet treatment. These results suggest that the cellular membrane catastrophe occurs via coincident altered electrical conductivity, lipid oxidation, and membrane roughening caused by an atmospheric-pressure plasma jet, possibly resulting in cellular vulnerability to reactive species generated from the plasma as well as cytotoxicity to cancer cells.

  19. Aquaporins in skeletal muscle: reassessment of the functional role of aquaporin-4.

    PubMed

    Frigeri, Antonio; Nicchia, Grazia Paola; Balena, Rosalba; Nico, Beatrice; Svelto, Maria

    2004-05-01

    Aquaporin-4 (AQP4) is the major water channel of the neuromuscular system, but its physiological function in both perivascular astrocytes and skeletal muscle sarcolemma is unclear. The purpose of this study was to assess the following in skeletal muscle: a) the expression of all cloned water cannels; b) the functional role of AQP4 using sarcolemma vesicles purified by means of several fractionation methods, and c) the functional effect of AQP4 reduction in mdx mice, the animal model of Duchenne muscular dystrophy (DMD). Immunofluorescence and immunoblot experiments performed with affinity purified antibodies revealed that only AQP1 and AQP4 are expressed in mouse skeletal muscle: AQP1 in endothelial cells of continuous capillaries and AQP4 on the plasma membrane of muscle fiber. Plasma membrane vesicle purification was performed with a procedure extensively used to purify and characterize dystrophin-associated proteins (DAPs) from rabbit skeletal muscle. Western blot analysis showed strong co-enrichment of the analyzed DAPs and AQP4, indicating that the membrane vesicle preparation was highly enriched in sarcolemma. Stopped-flow light-scattering measurements showed high osmotic water permeability of sarcolemma vesicles (approximately 150 microm/s) compatible with the AQP-mediated pathway for water movement. Sarcolemma vesicles prepared from mdx mice revealed, in parallel with AQP4 disappearance from the plasma membrane, a strong reduction in water permeability compared with wild-type mice. Altogether, these results demonstrate high AQP4-mediated water permeability of the skeletal muscle sarcolemma. Expression of sarcolemmal AQP4 together with that of vascular AQP1 may be responsible for the fast water transfer from the blood into the muscle during intense activity. These data imply an important role for aquaporins in skeletal muscle physiology as well as an involvement of AQP4 in the molecular alterations that occur in the muscle of DMD patients.

  20. Aquaporin Expression Correlates with Freeze Tolerance in Baker's Yeast, and Overexpression Improves Freeze Tolerance in Industrial Strains

    PubMed Central

    Tanghe, An; Van Dijck, Patrick; Dumortier, Françoise; Teunissen, Aloys; Hohmann, Stefan; Thevelein, Johan M.

    2002-01-01

    Little information is available about the precise mechanisms and determinants of freeze resistance in baker's yeast, Saccharomyces cerevisiae. Genomewide gene expression analysis and Northern analysis of different freeze-resistant and freeze-sensitive strains have now revealed a correlation between freeze resistance and the aquaporin genes AQY1 and AQY2. Deletion of these genes in a laboratory strain rendered yeast cells more sensitive to freezing, while overexpression of the respective genes, as well as heterologous expression of the human aquaporin gene hAQP1, improved freeze tolerance. These findings support a role for plasma membrane water transport activity in determination of freeze tolerance in yeast. This appears to be the first clear physiological function identified for microbial aquaporins. We suggest that a rapid, osmotically driven efflux of water during the freezing process reduces intracellular ice crystal formation and resulting cell damage. Aquaporin overexpression also improved maintenance of the viability of industrial yeast strains, both in cell suspensions and in small doughs stored frozen or submitted to freeze-thaw cycles. Furthermore, an aquaporin overexpression transformant could be selected based on its improved freeze-thaw resistance without the need for a selectable marker gene. Since aquaporin overexpression does not seem to affect the growth and fermentation characteristics of yeast, these results open new perspectives for the successful development of freeze-resistant baker's yeast strains for use in frozen dough applications. PMID:12450819

  1. Aquaporin expression correlates with freeze tolerance in baker's yeast, and overexpression improves freeze tolerance in industrial strains.

    PubMed

    Tanghe, An; Van Dijck, Patrick; Dumortier, Françoise; Teunissen, Aloys; Hohmann, Stefan; Thevelein, Johan M

    2002-12-01

    Little information is available about the precise mechanisms and determinants of freeze resistance in baker's yeast, Saccharomyces cerevisiae. Genomewide gene expression analysis and Northern analysis of different freeze-resistant and freeze-sensitive strains have now revealed a correlation between freeze resistance and the aquaporin genes AQY1 and AQY2. Deletion of these genes in a laboratory strain rendered yeast cells more sensitive to freezing, while overexpression of the respective genes, as well as heterologous expression of the human aquaporin gene hAQP1, improved freeze tolerance. These findings support a role for plasma membrane water transport activity in determination of freeze tolerance in yeast. This appears to be the first clear physiological function identified for microbial aquaporins. We suggest that a rapid, osmotically driven efflux of water during the freezing process reduces intracellular ice crystal formation and resulting cell damage. Aquaporin overexpression also improved maintenance of the viability of industrial yeast strains, both in cell suspensions and in small doughs stored frozen or submitted to freeze-thaw cycles. Furthermore, an aquaporin overexpression transformant could be selected based on its improved freeze-thaw resistance without the need for a selectable marker gene. Since aquaporin overexpression does not seem to affect the growth and fermentation characteristics of yeast, these results open new perspectives for the successful development of freeze-resistant baker's yeast strains for use in frozen dough applications.

  2. Putative Role of Aquaporins in Variable Hydraulic Conductance of Leaves in Response to Light1

    PubMed Central

    Cochard, Hervé; Venisse, Jean-Stéphane; Barigah, Têtè Sévérien; Brunel, Nicole; Herbette, Stéphane; Guilliot, Agnès; Tyree, Melvin T.; Sakr, Soulaiman

    2007-01-01

    Molecular and physiological studies in walnut (Juglans regia) are combined to establish the putative role of leaf plasma membrane aquaporins in the response of leaf hydraulic conductance (Kleaf) to irradiance. The effects of light and temperature on Kleaf are described. Under dark conditions, Kleaf was low, but increased by 400% upon exposure to light. In contrast to dark conditions, Kleaf values of light-exposed leaves responded to temperature and 0.1 mm cycloheximide treatments. Furthermore, Kleaf was not related to stomatal aperture. Data of real-time reverse transcription-polymerase chain reaction showed that Kleaf dynamics were tightly correlated with the transcript abundance of two walnut aquaporins (JrPIP2,1 and JrPIP2,2). Low Kleaf in the dark was associated with down-regulation, whereas high Kleaf in the light was associated with up-regulation of JrPIP2. Light responses of Kleaf and aquaporin transcripts were reversible and inhibited by cycloheximide, indicating the importance of de novo protein biosynthesis in this process. Our results indicate that walnut leaves can rapidly change their hydraulic conductance and suggest that these changes can be explained by regulation of plasma membrane aquaporins. Model simulation suggests that variable leaf hydraulic conductance in walnut might enhance leaf gas exchanges while buffering leaf water status in response to ambient light fluctuations. PMID:17114274

  3. Putative role of aquaporins in variable hydraulic conductance of leaves in response to light.

    PubMed

    Cochard, Hervé; Venisse, Jean-Stéphane; Barigah, Têtè Sévérien; Brunel, Nicole; Herbette, Stéphane; Guilliot, Agnès; Tyree, Melvin T; Sakr, Soulaiman

    2007-01-01

    Molecular and physiological studies in walnut (Juglans regia) are combined to establish the putative role of leaf plasma membrane aquaporins in the response of leaf hydraulic conductance (K(leaf)) to irradiance. The effects of light and temperature on K(leaf) are described. Under dark conditions, K(leaf) was low, but increased by 400% upon exposure to light. In contrast to dark conditions, K(leaf) values of light-exposed leaves responded to temperature and 0.1 mm cycloheximide treatments. Furthermore, K(leaf) was not related to stomatal aperture. Data of real-time reverse transcription-polymerase chain reaction showed that K(leaf) dynamics were tightly correlated with the transcript abundance of two walnut aquaporins (JrPIP2,1 and JrPIP2,2). Low K(leaf) in the dark was associated with down-regulation, whereas high K(leaf) in the light was associated with up-regulation of JrPIP2. Light responses of K(leaf) and aquaporin transcripts were reversible and inhibited by cycloheximide, indicating the importance of de novo protein biosynthesis in this process. Our results indicate that walnut leaves can rapidly change their hydraulic conductance and suggest that these changes can be explained by regulation of plasma membrane aquaporins. Model simulation suggests that variable leaf hydraulic conductance in walnut might enhance leaf gas exchanges while buffering leaf water status in response to ambient light fluctuations.

  4. The mitochondria-plasma membrane contact site.

    PubMed

    Westermann, Benedikt

    2015-08-01

    Mitochondria are dynamic organelles that are highly motile and frequently fuse and divide. It has recently become clear that their complex behavior is governed to a large extent by interactions with other cellular structures. This review will focus on a mitochondria-plasma membrane tethering complex that was recently discovered and molecularly analyzed in budding yeast, the Num1/Mdm36 complex. This complex attaches mitochondria to the cell cortex and ensures that a portion of the organelles is retained in mother cells during cell division. At the same time, it supports mitochondrial division and integrates mitochondrial dynamics into cellular architecture. Recent evidence suggests that similar mechanisms might exist also in mammalian cells.

  5. Membrane potential modulates plasma membrane phospholipid dynamics and K-Ras signaling

    PubMed Central

    Zhou, Yong; Wong, Ching-On; Cho, Kwang-jin; van der Hoeven, Dharini; Liang, Hong; Thakur, Dhananiay P.; Luo, Jialie; Babic, Milos; Zinsmaier, Konrad E.; Zhu, Michael X.; Hu, Hongzhen; Venkatachalam, Kartik; Hancock, John F.

    2015-01-01

    Plasma membrane depolarization can trigger cell proliferation, but how membrane potential influences mitogenic signaling is uncertain. Here, we show that plasma membrane depolarization induces nanoscale reorganization of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate but not other anionic phospholipids. K-Ras, which is targeted to the plasma membrane by electrostatic interactions with phosphatidylserine, in turn undergoes enhanced nanoclustering. Depolarization-induced changes in phosphatidylserine and K-Ras plasma membrane organization occur in fibroblasts, excitable neuroblastoma cells, and Drosophila neurons in vivo and robustly amplify K-Ras–dependent mitogen-activated protein kinase (MAPK) signaling. Conversely, plasma membrane repolarization disrupts K-Ras nanoclustering and inhibits MAPK signaling. By responding to voltage-induced changes in phosphatidylserine spatiotemporal dynamics, K-Ras nanoclusters set up the plasma membrane as a biological field-effect transistor, allowing membrane potential to control the gain in mitogenic signaling circuits. PMID:26293964

  6. SIGNAL TRANSDUCTION. Membrane potential modulates plasma membrane phospholipid dynamics and K-Ras signaling.

    PubMed

    Zhou, Yong; Wong, Ching-On; Cho, Kwang-jin; van der Hoeven, Dharini; Liang, Hong; Thakur, Dhananiay P; Luo, Jialie; Babic, Milos; Zinsmaier, Konrad E; Zhu, Michael X; Hu, Hongzhen; Venkatachalam, Kartik; Hancock, John F

    2015-08-21

    Plasma membrane depolarization can trigger cell proliferation, but how membrane potential influences mitogenic signaling is uncertain. Here, we show that plasma membrane depolarization induces nanoscale reorganization of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate but not other anionic phospholipids. K-Ras, which is targeted to the plasma membrane by electrostatic interactions with phosphatidylserine, in turn undergoes enhanced nanoclustering. Depolarization-induced changes in phosphatidylserine and K-Ras plasma membrane organization occur in fibroblasts, excitable neuroblastoma cells, and Drosophila neurons in vivo and robustly amplify K-Ras-dependent mitogen-activated protein kinase (MAPK) signaling. Conversely, plasma membrane repolarization disrupts K-Ras nanoclustering and inhibits MAPK signaling. By responding to voltage-induced changes in phosphatidylserine spatiotemporal dynamics, K-Ras nanoclusters set up the plasma membrane as a biological field-effect transistor, allowing membrane potential to control the gain in mitogenic signaling circuits.

  7. 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.

  8. Characterization of plant plasma membrane antigens. Progress report

    SciTech Connect

    Galbraith, D.W.

    1986-01-01

    The library of monoclonal antibodies, which are directed against membrane bound antigens of protoplast plasma membrane, are being characterized by immunoprecipitation, immunoaffinity chromatography, and by Western blotting of SDS gels. Progress on these studies is reported here. (DT)

  9. A plasma membrane template for macropinocytic cups

    PubMed Central

    Veltman, Douwe M; Williams, Thomas D; Bloomfield, Gareth; Chen, Bi-Chang; Betzig, Eric; Insall, Robert H; Kay, Robert R

    2016-01-01

    Macropinocytosis is a fundamental mechanism that allows cells to take up extracellular liquid into large vesicles. It critically depends on the formation of a ring of protrusive actin beneath the plasma membrane, which develops into the macropinocytic cup. We show that macropinocytic cups in Dictyostelium are organised around coincident intense patches of PIP3, active Ras and active Rac. These signalling patches are invariably associated with a ring of active SCAR/WAVE at their periphery, as are all examined structures based on PIP3 patches, including phagocytic cups and basal waves. Patch formation does not depend on the enclosing F-actin ring, and patches become enlarged when the RasGAP NF1 is mutated, showing that Ras plays an instructive role. New macropinocytic cups predominantly form by splitting from existing ones. We propose that cup-shaped plasma membrane structures form from self-organizing patches of active Ras/PIP3, which recruit a ring of actin nucleators to their periphery. DOI: http://dx.doi.org/10.7554/eLife.20085.001 PMID:27960076

  10. Proteomic analysis of plasma membrane and tonoplast from the leaves of mangrove plant Avicennia officinalis.

    PubMed

    Krishnamurthy, Pannaga; Tan, Xing Fei; Lim, Teck Kwang; Lim, Tit-Meng; Kumar, Prakash P; Loh, Chiang-Shiong; Lin, Qingsong

    2014-11-01

    In order to understand the salt tolerance and secretion in mangrove plant species, gel electrophoresis coupled with LC-MS-based proteomics was used to identify key transport proteins in the plasma membrane (PM) and tonoplast fractions of Avicennia officinalis leaves. PM and tonoplast proteins were purified using two-aqueous-phase partitioning and density gradient centrifugation, respectively. Forty of the 254 PM proteins and 31 of the 165 tonoplast proteins identified were predicted to have transmembrane domains. About 95% of the identified proteins could be classified based on their functions. The major classes of proteins were predicted to be involved in transport, metabolic processes, defense/stress response, and signal transduction, while a few of the proteins were predicted to be involved in other functions such as membrane trafficking. The main classes of transporter proteins identified included H(+) -ATPases, ATP-binding cassette transporters, and aquaporins, all of which could play a role in salt secretion. These data will serve as the baseline membrane proteomic dataset for Avicennia species. Further, this information can contribute to future studies on understanding the mechanism of salt tolerance in halophytes in addition to salt secretion in mangroves. All MS data have been deposited in the ProteomeXchange with identifier PXD000837 (http://proteomecentral.proteomexchange.org/dataset/PXD000837).

  11. 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.

  12. Pollen Aquaporins: The Solute Factor

    PubMed Central

    Pérez Di Giorgio, Juliana A.; Soto, Gabriela C.; Muschietti, Jorge P.; Amodeo, Gabriela

    2016-01-01

    In the recent years, the biophysical properties and presumed physiological role of aquaporins (AQPs) have been expanded to specialized cells where water and solute exchange are crucial traits. Complex but unique processes such as stomatal movement or pollen hydration and germination have been addressed not only by identifying the specific AQP involved but also by studying how these proteins integrate and coordinate cellular activities and functions. In this review, we referred specifically to pollen-specific AQPs and analyzed what has been assumed in terms of transport properties and what has been found in terms of their physiological role. Unlike that in many other cells, the AQP machinery in mature pollen lacks plasma membrane intrinsic proteins, which are extensively studied for their high water capacity exchange. Instead, a variety of TIPs and NIPs are expressed in pollen. These findings have altered the initial understanding of AQPs and water exchange to consider specific and diverse solutes that might be critical to sustaining pollen’s success. The spatial and temporal distribution of the pollen AQPs also reflects a regulatory mechanism that allowing a properly adjusting water and solute exchange. PMID:27881985

  13. Pollen Aquaporins: The Solute Factor.

    PubMed

    Pérez Di Giorgio, Juliana A; Soto, Gabriela C; Muschietti, Jorge P; Amodeo, Gabriela

    2016-01-01

    In the recent years, the biophysical properties and presumed physiological role of aquaporins (AQPs) have been expanded to specialized cells where water and solute exchange are crucial traits. Complex but unique processes such as stomatal movement or pollen hydration and germination have been addressed not only by identifying the specific AQP involved but also by studying how these proteins integrate and coordinate cellular activities and functions. In this review, we referred specifically to pollen-specific AQPs and analyzed what has been assumed in terms of transport properties and what has been found in terms of their physiological role. Unlike that in many other cells, the AQP machinery in mature pollen lacks plasma membrane intrinsic proteins, which are extensively studied for their high water capacity exchange. Instead, a variety of TIPs and NIPs are expressed in pollen. These findings have altered the initial understanding of AQPs and water exchange to consider specific and diverse solutes that might be critical to sustaining pollen's success. The spatial and temporal distribution of the pollen AQPs also reflects a regulatory mechanism that allowing a properly adjusting water and solute exchange.

  14. Activation of muscarinic receptors in rat parotid acinar cells induces AQP5 trafficking to nuclei and apical plasma membrane.

    PubMed

    Cho, Gota; Bragiel, Aneta M; Wang, Di; Pieczonka, Tomasz D; Skowronski, Mariusz T; Shono, Masayuki; Nielsen, Søren; Ishikawa, Yasuko

    2015-04-01

    The subcellular distribution of aquaporin-5 (AQP5) in rat parotid acinar cells in response to muscarinic acetylcholine receptor (mAChR) activation remains unclear. Immunoconfocal and immunoelectron microscopy were used to visualize the distribution of AQP5 in parotid acinar cells. Western blotting was used to analyze AQP5 levels in membranes. To clarify the characteristics of membrane domains associated with AQP5, detergent solubility and sucrose-density flotation experiments were performed. Under control conditions, AQP5 was diffusely distributed on the apical plasma membrane (APM) and apical plasmalemmal region and throughout the cytoplasm. Upon mAChR activation, AQP5 was predominantly located in the nucleus, APM and lateral plasma membrane (LPM). Subsequently, localization of AQP5 in the nucleus, APM and LPM was decreased. Prolonged atropine treatment inhibited mAChR agonist-induced translocation of AQP5 to the nucleus, APM and LPM. AQP5 levels were enhanced in isolated nuclei and nuclear membranes prepared from parotid tissues incubated with mAChR agonist. mAChR agonist induced AQP5 levels in both soluble and insoluble nuclear fractions solubilized with Triton X-100 or Lubrol WX. Small amounts of AQP5 in nuclei were detected using low-density sucrose gradient. When AQP5 was present in the nuclear membrane, nuclear size decreased. The activation of mAChR induced AQP5 translocation to the nucleus, APM and LPM, and AQP5 may trigger water transport across the nuclear membrane and plasma membrane in rat parotid acinar cells. AQP5 translocates to the nuclear membrane and may trigger the movement of water, inducing shrinkage of the nucleus and the start of nuclear functions. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Aquaporins in the kidney: from molecules to medicine.

    PubMed

    Nielsen, Søren; Frøkiaer, Jørgen; Marples, David; Kwon, Tae-Hwan; Agre, Peter; Knepper, Mark A

    2002-01-01

    The discovery of aquaporin-1 (AQP1) answered the long-standing biophysical question of how water specifically crosses biological membranes. In the kidney, at least seven aquaporins are expressed at distinct sites. AQP1 is extremely abundant in the proximal tubule and descending thin limb and is essential for urinary concentration. AQP2 is exclusively expressed in the principal cells of the connecting tubule and collecting duct and is the predominant vasopressin-regulated water channel. AQP3 and AQP4 are both present in the basolateral plasma membrane of collecting duct principal cells and represent exit pathways for water reabsorbed apically via AQP2. Studies in patients and transgenic mice have demonstrated that both AQP2 and AQP3 are essential for urinary concentration. Three additional aquaporins are present in the kidney. AQP6 is present in intracellular vesicles in collecting duct intercalated cells, and AQP8 is present intracellularly at low abundance in proximal tubules and collecting duct principal cells, but the physiological function of these two channels remains undefined. AQP7 is abundant in the brush border of proximal tubule cells and is likely to be involved in proximal tubule water reabsorption. Body water balance is tightly regulated by vasopressin, and multiple studies now have underscored the essential roles of AQP2 in this. Vasopressin regulates acutely the water permeability of the kidney collecting duct by trafficking of AQP2 from intracellular vesicles to the apical plasma membrane. The long-term adaptational changes in body water balance are controlled in part by regulated changes in AQP2 and AQP3 expression levels. Lack of functional AQP2 is seen in primary forms of diabetes insipidus, and reduced expression and targeting are seen in several diseases associated with urinary concentrating defects such as acquired nephrogenic diabetes insipidus, postobstructive polyuria, as well as acute and chronic renal failure. In contrast, in conditions

  16. Characterization and differential expression analysis of Toxocara canis aquaporin-1 gene.

    PubMed

    Luo, Yong-Fang; Hu, Ling; Ma, Guang-Xu; Luo, Yong-Li; Yin, Sha-Sha; Xiong, Yi; Zhu, Xing-Quan; Zhou, Rong-Qiong

    2016-09-01

    Toxocara canis is an intestinal nematode of canids with a worldwide distribution, causing an important but neglected parasitic zoonosis in humans. Aquaporins (AQP) are a family of water channel proteins, which function as membrane channels to regulate water homeostasis. In this study, the coding sequence of aquaporin-1 gene of T. canis (Tc-aqp-1) was cloned and characterized. The obtained Tc-aqp-1 coding sequence was 933 bp in length, which predicted to encode 311 amino acids. Two conserved asparagine-proline-alanine (NPA) motifs were identified in the multiple sequence alignments. Phylogenetic analysis revealed the closest relationship between T. canis and Opisthorchis viverrini based on aquaporin-1 amino acid sequence. A structure was predicted with ligand binding sites predicted at H93, N95, N226, L94, I79, and I210 and with active sites predicted at I256 and G207. Gene Ontology (GO) annotations predicted its cellular component term of integral component of plasma membrane (GO: 0005887), molecular function term of channel activity (GO: 0015250), and biological process term of water transport (GO: 0006833). Tissue expression analysis revealed that the Tc-aqp-1 was highly expressed in the intestine of adult male. The findings of the present study provide the basis for further functional studies of T. canis aquaporin-1.

  17. Lectin-Magnetic Beads for Plasma Membrane Isolation.

    PubMed

    Lee, Yu-Chen; Liu, Hsuan-Chen; Chuang, Carol; Lin, Sue-Hwa

    2015-07-01

    Plasma membrane proteins mainly function to transmit external signals into the cell. Many plasma membrane receptor tyrosine kinases (e.g., HER2 and EGFR) are known to mediate oncogenic progression, making them prime targets for cancer therapy. Recently, it has become important to identify plasma membrane proteins that are differentially expressed in normal versus cancer cells, in drug-sensitive versus drug-resistant cells, or among tumor cells that metastasize to different organ sites because these differentially expressed membrane proteins may lead to the identification of therapeutic targets or diagnostic markers. In addition, there is an increased interest in identifying cell-surface proteins that could serve as markers for stem cells, progenitor cells, or cells of different lineages. Traditionally, membrane isolation requires multiple centrifugation steps to isolate different organelles based on their density. With the advent of affinity matrix technology, it is possible to separate organelles based on their molecular differences. A defining characteristic of the plasma membrane is that plasma membrane proteins are more extensively glycosylated than are intracellular membrane proteins. As a result, affinity chromatography employing lectin, a carbohydrate-binding protein, is commonly used to isolate plasma membrane proteins. We have extended this concept for plasma membrane isolation by using concanavalin A (ConA), a lectin with mannose specificity. Here we describe a protocol that uses immobilized ConA bound to magnetic beads to isolate plasma membranes from homogenized cell lysates. The captured plasma membrane proteins are then solubilized from the ConA-magnetic beads by detergents in the presence of a competing sugar, methyl α-mannopyranoside.

  18. 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.

  19. Plasma membrane of Beta vulgaris storage root shows high water channel activity regulated by cytoplasmic pH and a dual range of calcium concentrations.

    PubMed

    Alleva, Karina; Niemietz, Christa M; Sutka, Moira; Maurel, Christophe; Parisi, Mario; Tyerman, Stephen D; Amodeo, Gabriela

    2006-01-01

    Plasma membrane vesicles isolated by two-phase partitioning from the storage root of Beta vulgaris show atypically high water permeability that is equivalent only to those reported for active aquaporins in tonoplast or animal red cells (Pf=542 microm s(-1)). The values were determined from the shrinking kinetics measured by stopped-flow light scattering. This high Pf was only partially inhibited by mercury (HgCl2) but showed low activation energy (Ea) consistent with water permeation through water channels. To study short-term regulation of water transport that could be the result of channel gating, the effects of pH, divalent cations, and protection against dephosphorylation were tested. The high Pf observed at pH 8.3 was dramatically reduced by medium acidification. Moreover, intra-vesicular acidification (corresponding to the cytoplasmic face of the membrane) shut down the aquaporins. De-phosphorylation was discounted as a regulatory mechanism in this preparation. On the other hand, among divalent cations, only calcium showed a clear effect on aquaporin activity, with two distinct ranges of sensitivity to free Ca2+ concentration (pCa 8 and pCa 4). Since the normal cytoplasmic free Ca2+ sits between these ranges it allows for the possibility of changes in Ca2+ to finely up- or down-regulate water channel activity. The calcium effect is predominantly on the cytoplasmic face, and inhibition corresponds to an increase in the activation energy for water transport. In conclusion, these findings establish both cytoplasmic pH and Ca2+ as important regulatory factors involved in aquaporin gating.

  20. Actin cytoskeleton remodeling governs aquaporin-4 localization in astrocytes.

    PubMed

    Nicchia, Grazia Paola; Rossi, Andrea; Mola, Maria Grazia; Procino, Giuseppe; Frigeri, Antonio; Svelto, Maria

    2008-12-01

    Aquaporin-4 (AQP4) is constitutively concentrated in the plasma membrane of the perivascular glial processes, and its expression is altered in certain pathological conditions associated with brain edema or altered glial migration. When astrocytes are grown in culture, they lose their characteristic star-like shape and AQP4 continuous plasma membrane localization observed in vivo. In this study, we differentiated primary astrocyte cultures with cAMP and lovastatin, both able to induce glial stellation through a reorganization of F-actin cytoskeleton, and obtained AQP4 selectively localized on the cell plasma membrane associated with an increase in the plasma membrane water transport level, but only cAMP induced an increase in AQP4 total protein expression. Phosphorylation experiments indicated that AQP4 in astrocytes is neither phosphorylated nor a substrate of PKA. Depolymerization of F-actin cytoskeleton performed by cytochalasin-D suggested that F-actin cytoskeleton plays a primary role for AQP4 plasma membrane localization and during cell adhesion. Finally, AQP4 knockdown does not compromise the ability of astrocytes to stellate in the presence of cAMP, indicating that astrocyte stellation is independent of AQP4.

  1. [Updated detection of the function of sperm plasma membrane].

    PubMed

    Zhou, Xin; Xia, Xin-Yi; Huang, Yu-Feng

    2010-08-01

    The sperm plasma membrane is rich in polyunsaturated fatty acids and a variety of proteins, and its function is associated with sperm capacitation, acrosome reaction and sperm-egg fusion. Sperm fertilizability can be predicted by detecting the function of the sperm plasma membrane, which is performed mainly with the following five techniques: sperm hypoosmotic swelling test, Eosin gamma water test, sperm membrane lipid peroxidation determination, seminal plasma superoxide dismutase determination, and flow cytometry. The evaluation of the function of sperm plasma membrane can be applied in detecting semen quality, selecting semen centrifugation, assessing the quality and fertilizability of sex-sorted sperm, improving cryopreservation, and guiding the optimization of intracytoplasmic sperm injection. This review presents an update on the principles, methods and steps of the detection of sperm plasma membrane function, as well as an overview of its status quo and application.

  2. Biogenesis of plasma membrane glycoproteins. Purification and properties of two rat liver plasma membrane glycoproteins.

    PubMed

    Elovson, J

    1980-06-25

    As a preliminary to a study of the biogenesis of individual plasma membrane glycoproteins, the marker enzyme nucleotide pyrophosphatase (NPPase) and a major rat liver plasma membrane sialoprotein, subsequently found to be identical with the enzyme dipeptidyl peptidase IV (DPP IV), were purified 10,000- and 2,000-fold, respectively, from rat liver. Both were amphipathic proteins which formed defined micellar complexes with detergents and aggregated in their absence. Gel filtration, sucrose density gradient centrifugation, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed the Triton X-100 complex of NPPase to contain a single 150,000-dalton peptide, while that of DPP IV was composed of two 120,000-dalton subunits; each complex also contained about 150,000-dalton Triton X-100. Trypsin cleaved the detergent complexes with release of major hydrophilic fragments which no longer bound detergent micelles; the accompanying change in peptide size was small for NPPase and undetectable for DPP IV, which also retained the dimer structure of its native form. DPP IV was the only major glycoprotein in rat liver plasma membrane which bound strongly to wheat germ agglutinin. Monospecific rabbit antibodies against NPPase and DPP IV precipitated the antigens without affecting their enzymatic activities.

  3. Induced expression and functional effects of aquaporin-1 in human leukocytes in sepsis

    PubMed Central

    2013-01-01

    in β-mercaptoethanol, which abrogated the action of mercury inhibition. Conclusions Aquaporin-1 is induced in leukocytes of patients with ICU-acquired sepsis and exhibits higher expression in septic shock. This phenomenon may be due to LPS-triggered NF-κB activation that can also lead to alterations in plasma membrane permeability. PMID:24028651

  4. Phosphorylation-dependent trafficking of plasma membrane proteins in animal and plant cells.

    PubMed

    Offringa, Remko; Huang, Fang

    2013-09-01

    In both unicellular and multicellular organisms, transmembrane (TM) proteins are sorted to and retained at specific membrane domains by endomembrane trafficking mechanisms that recognize sorting signals in the these proteins. The trafficking and distribution of plasma membrane (PM)-localized TM proteins (PM proteins), especially of those PM proteins that show an asymmetric distribution over the PM, has received much attention, as their proper PM localization is crucial for elementary signaling and transport processes, and defects in their localization often lead to severe disease symptoms or developmental defects. The subcellular localization of PM proteins is dynamically regulated by post-translational modifications, such as phosphorylation and ubiquitination. These modificaitons mostly occur on sorting signals that are located in the larger cytosolic domains of the cargo proteins. Here we review the effects of phosphorylation of PM proteins on their trafficking, and present the key examples from the animal field that have been subject to studies for already several decades, such as that of aquaporin 2 and the epidermal growth factor receptor. Our knowledge on cargo trafficking in plants is largely based on studies of the family of PIN FORMED (PIN) carriers that mediate the efflux of the plant hormone auxin. We will review what is known on the subcellular distribution and trafficking of PIN proteins, with a focus on how this is modulated by phosphorylation, and identify and discuss analogies and differences in trafficking with the well-studied animal examples.

  5. Aquaporins in Obesity.

    PubMed

    da Silva, Inês Vieira; Soveral, Graça

    2017-01-01

    Obesity is one of the most important metabolic disorders of this century and is associated with a cluster of the most dangerous cardiovascular disease risk factors, such as insulin resistance and diabetes , dyslipidemia and hypertension , collectively named Metabolic Syndrome. The role of aquaporins in glycerol metabolism facilitating glycerol release from the adipose tissue and distribution to various tissues and organs, unveils these membrane channels as important players in lipid balance and energy homeostasis and points to their involvement in a variety of pathophysiological mechanisms including insulin resistance, obesity and diabetes.This review summarizes the physiologic role of aquaglyceroporins in glycerol metabolism and lipid homeostasis, describing their specific tissue distribution, their involvement in glycerol balance and their implication in obesity and fat-related metabolic complications. The development of specify pharmacologic modulators able to regulate aquaglyceroporins expression and function , in particular AQP7 in adipose tissue, might constitute a novel approach for controlling obesity and other metabolic disorders.

  6. LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum.

    PubMed

    Larsson, Karin E; Kjellberg, J Magnus; Tjellström, Henrik; Sandelius, Anna Stina

    2007-11-28

    The phospholipids of the plant plasma membrane are synthesized in the endoplasmic reticulum (ER). The majority of these lipids reach the plasma membrane independently of the secretory vesicular pathway. Phospholipid delivery to the mitochondria and chloroplasts of plant cells also bypasses the secretory pathway and here it has been proposed that lysophospholipids are transported at contact sites between specific regions of the ER and the respective organelle, followed by lysophospholipid acylation in the target organelle. To test the hypothesis that a corresponding mechanism operates to transport phospholipids to the plasma membrane outside the secretory pathway, we investigated whether lysolipid acylation occurs also in the plant plasma membrane and whether this membrane, like the chloroplasts and mitochondria, is in close contact with the ER. The plant plasma membrane readily incorporated the acyl chain of acyl-CoA into phospholipids. Oleic acid was preferred over palmitic acid as substrate and acyl incorporation occurred predominantly into phosphatidylcholine (PC). Phospholipase A2 stimulated the reaction, as did exogenous lysoPC when administered in above critical micellar concentrations. AgNO3 was inhibitory. The lysophospholipid acylation reaction was higher in a membrane fraction that could be washed off the isolated plasma membranes after repeated freezing and thawing cycles in a medium with lowered pH. This fraction exhibited several ER-like characteristics. When plasma membranes isolated from transgenic Arabidopsis expressing green fluorescent protein in the ER lumen were observed by confocal microscopy, membranes of ER origin were associated with the isolated plasma membranes. We conclude that a lysoPC acylation activity is associated with plant plasma membranes and cannot exclude a PC transacylase activity. It is highly plausible that the enzyme(s) resides in a fraction of the ER, closely associated with the plasma membrane, or in both. We suggest that

  7. LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum

    PubMed Central

    Larsson, Karin E; Kjellberg, J Magnus; Tjellström, Henrik; Sandelius, Anna Stina

    2007-01-01

    Background The phospholipids of the plant plasma membrane are synthesized in the endoplasmic reticulum (ER). The majority of these lipids reach the plasma membrane independently of the secretory vesicular pathway. Phospholipid delivery to the mitochondria and chloroplasts of plant cells also bypasses the secretory pathway and here it has been proposed that lysophospholipids are transported at contact sites between specific regions of the ER and the respective organelle, followed by lysophospholipid acylation in the target organelle. To test the hypothesis that a corresponding mechanism operates to transport phospholipids to the plasma membrane outside the secretory pathway, we investigated whether lysolipid acylation occurs also in the plant plasma membrane and whether this membrane, like the chloroplasts and mitochondria, is in close contact with the ER. Results The plant plasma membrane readily incorporated the acyl chain of acyl-CoA into phospholipids. Oleic acid was preferred over palmitic acid as substrate and acyl incorporation occurred predominantly into phosphatidylcholine (PC). Phospholipase A2 stimulated the reaction, as did exogenous lysoPC when administered in above critical micellar concentrations. AgNO3 was inhibitory. The lysophospholipid acylation reaction was higher in a membrane fraction that could be washed off the isolated plasma membranes after repeated freezing and thawing cycles in a medium with lowered pH. This fraction exhibited several ER-like characteristics. When plasma membranes isolated from transgenic Arabidopsis expressing green fluorescent protein in the ER lumen were observed by confocal microscopy, membranes of ER origin were associated with the isolated plasma membranes. Conclusion We conclude that a lysoPC acylation activity is associated with plant plasma membranes and cannot exclude a PC transacylase activity. It is highly plausible that the enzyme(s) resides in a fraction of the ER, closely associated with the plasma membrane

  8. Aquaporins in poplar: what a difference a symbiont makes!

    PubMed

    Marjanović, Zaklina; Uehlein, Norbert; Kaldenhoff, Ralf; Zwiazek, Janusz J; Weiss, Michael; Hampp, Rüdiger; Nehls, Uwe

    2005-10-01

    The formation of ectomycorrhizas, a tight association between fine roots of trees and certain soil fungi, improves plant nutrition in a nutrient-limited environment and may increase plant survival under water stress conditions. To investigate the impact of mycorrhiza formation on plant water uptake, seven genes coding for putative water channel proteins (aquaporins) were isolated from a poplar ectomycorrhizal cDNA library. Four out of the seven genes were preferentially expressed in roots. Mycorrhiza formation resulted in an increased transcript level for three of these genes, two of which are the most prominently expressed aquaporins in roots. When expressed in Xenopus laevis oocytes, the corresponding proteins of both genes were able to transport water. Together, these data indicate, that the water transport capacity of the plasma membrane of root cells is strongly increased in mycorrhized plants. Measurements of the hydraulic conductance of intact root systems revealed an increased water transport capacity of mycorrhized poplar roots. These data, however, also indicate that changes in the properties of the plasma membrane as well as those of the apoplast are responsible for the increased root hydraulic conductance in ectomycorrhizal symbiosis.

  9. 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.

  10. 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

  11. The plasma membrane calcium ATPase and disease.

    PubMed

    Tempel, B L; Shilling, D J

    2007-01-01

    The plasma membrane calcium ATPase (PMCA) uses energy to pump calcium (Ca2+) ions out of the cytosol into the extracellular milieu, usually against a strong chemical gradient. This energy expenditure is necessary to maintain a relatively low intracellular net Ca2+ load. Mammals have four genes (ATP2B1-ATP2B4), encoding the proteins PMCA1 through PMCA4. Transcripts from each of these genes are alternatively spliced to generate several variant proteins that are in turn post-translationally modified in a variety of ways. Expressed ubiquitously and with some level of functional redundancy in most vital tissues, only one of the four genes--Atp2b2--has been causally linked through naturally occuring mutations to disease in mammals: specifically to deafness and ataxia in spontaneous mouse mutants. In humans, a missense amino acid substitution in PMCA2 modifies the severity of hearing loss. Targeted null mutations of the Atp2b1 and Atp2b4 genes in mouse are embryonic lethal and cause a sperm motility defect, respectively. These phenotypes point to complex human diseases like hearing loss, cardiac function and infertility. Changes in PMCA expression are associated with other diseases including cataract formation, carciniogenesis, diabetes, and cardiac hypertension and hypertrophy. Severity of these diseases may be affected by subtle changes in expression of the PMCA isoforms expressed in those tissues.

  12. Aquaporin Expression in Normal and Pathological Skeletal Muscles: A Brief Review with Focus on AQP4

    PubMed Central

    Wakayama, Yoshihiro

    2010-01-01

    Freeze-fracture electron microscopy enabled us to observe the molecular architecture of the biological membranes. We were studying the myofiber plasma membranes of health and disease by using this technique and were interested in the special assembly called orthogonal arrays (OAs). OAs were present in normal myofiber plasma membranes and were especially numerous in fast twitch type 2 myofibers; while OAs were lost from sarcolemmal plasma membranes of severely affected muscles with dystrophinopathy and dysferlinopathy but not with caveolinopathy. In the mid nineties of the last century, the OAs turned out to be a water channel named aquaporin 4 (AQP4). Since this discovery, several groups of investigators have been studying AQP4 expression in diseased muscles. This review summarizes the papers which describe the expression of OAs, AQP4, and other AQPs at the sarcolemma of healthy and diseased muscle and discusses the possible role of AQPs, especially that of AQP4, in normal and pathological skeletal muscles. PMID:20339523

  13. Identification of lipopolysaccharide-interacting plasma membrane-type proteins in Arabidopsis thaliana.

    PubMed

    Vilakazi, Cornelius S; Dubery, Ian A; Piater, Lizelle A

    2017-02-01

    Lipopolysaccharide (LPS) is an amphiphatic bacterial glycoconjugate found on the external membrane of Gram-negative bacteria. This endotoxin is considered as a microbe-associated molecular pattern (MAMP) molecule and has been shown to elicit defense responses in plants. Here, LPS-interacting proteins from Arabidopsis thaliana plasma membrane (PM)-type fractions were captured and identified in order to investigate those involved in LPS perception and linked to triggering of innate immune responses. A novel proteomics-based affinity-capture strategy coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed for the enrichment and identification of LPS-interacting proteins. As such, LPS isolated from Burkholderia cepacia (LPSB.cep.) was immobilized on three independent and distinct affinity-based matrices to serve as bait for interacting proteins from A. thaliana leaf and callus tissue. These were resolved by 1D electrophoresis and identified by mass spectrometry. Proteins specifically bound to LPSB.cep. have been implicated in membrane structure (e.g. COBRA-like and tubulin proteins), membrane trafficking and/or transport (e.g. soluble NSF attachment protein receptor (SNARE) proteins, patellin, aquaporin, PM instrinsic proteins (PIP) and H(+)-ATPase), signal transduction (receptor-like kinases and calcium-dependent protein kinases) as well as defense/stress responses (e.g. hypersensitive-induced response (HIR) proteins, jacalin-like lectin domain-containing protein and myrosinase-binding proteins). The novel affinity-capture strategy for the enrichment of LPS-interacting proteins proved to be effective, especially in the binding of proteins involved in plant defense responses, and can thus be used to elucidate LPS-mediated molecular recognition and disease mechanism(s).

  14. Plasma membrane reorganization induced by chemical transformation in cultura

    SciTech Connect

    Packard, B.S.

    1984-04-01

    Induction of increased rigidity in the plasma membrane paralleling properties associated with a transformed state was suggested by two experiments. Fluorescence recovery after photobleaching (FRAP) indicated the induction of an environment in the plasma membrane where the synthetic fluorescent phospholipid collarein was immobile on the FRAP timescale. The other technique revealed the binding of epidermal growth factor (EGF) to a cryptic class of receptors which become accessible upon chemical transformation. These two lines of evidence are consistent with a reorganization of the plasma membrane induced by tumor promoters. 110 references, 38 figures, 4 tables.

  15. Proton Pumping of the Yeast Plasma Membrane H+-ATPase

    DTIC Science & Technology

    1993-08-16

    function of the yeast plasma membrane H+- ATPase. This ATPase is a P-type cation transporter composed of a single protein of 100,000 Da molecular...August 16, 1993 ] Final 25 Sep 89 - 14 May 94 / 4. TITLE AND SUBTITLE S UDN UBR Proton Pumping of the Yeast Plasma Membrane HW-AT~ase G. AUTOR(S)DAALO3...Maximum 200 words) This proposal was to study the structure and function of the yeast plasma membrane H+-ATPase. We I proposed to study I )the

  16. Significance of oxygen transport through aquaporins.

    PubMed

    Zwiazek, Janusz J; Xu, Hao; Tan, Xiangfeng; Navarro-Ródenas, Alfonso; Morte, Asunción

    2017-01-12

    Aquaporins are membrane integral proteins responsible for the transmembrane transport of water and other small neutral molecules. Despite their well-acknowledged importance in water transport, their significance in gas transport processes remains unclear. Growing evidence points to the involvement of plant aquaporins in CO2 delivery for photosynthesis. The role of these channel proteins in the transport of O2 and other gases may also be more important than previously envisioned. In this study, we examined O2 permeability of various human, plant, and fungal aquaporins by co-expressing heterologous aquaporin and myoglobin in yeast. Two of the most promising O2-transporters (Homo sapiens AQP1 and Nicotiana tabacum PIP1;3) were confirmed to facilitate O2 transport in the spectrophotometric assay using yeast protoplasts. The over-expression of NtPIP1;3 in yeasts significantly increased their O2 uptake rates in suspension culture. In N. tabacum roots subjected to hypoxic hydroponic conditions, the transcript levels of the O2-transporting aquaporin NtPIP1;3 significantly increased after the seven-day hypoxia treatment, which was accompanied by the increase of ATP levels in the apical root segments. Our results suggest that the functional significance of aquaporin-mediated O2 transport and the possibility of controlling the rate of transmembrane O2 transport should be further explored.

  17. Significance of oxygen transport through aquaporins

    PubMed Central

    Zwiazek, Janusz J.; Xu, Hao; Tan, Xiangfeng; Navarro-Ródenas, Alfonso; Morte, Asunción

    2017-01-01

    Aquaporins are membrane integral proteins responsible for the transmembrane transport of water and other small neutral molecules. Despite their well-acknowledged importance in water transport, their significance in gas transport processes remains unclear. Growing evidence points to the involvement of plant aquaporins in CO2 delivery for photosynthesis. The role of these channel proteins in the transport of O2 and other gases may also be more important than previously envisioned. In this study, we examined O2 permeability of various human, plant, and fungal aquaporins by co-expressing heterologous aquaporin and myoglobin in yeast. Two of the most promising O2-transporters (Homo sapiens AQP1 and Nicotiana tabacum PIP1;3) were confirmed to facilitate O2 transport in the spectrophotometric assay using yeast protoplasts. The over-expression of NtPIP1;3 in yeasts significantly increased their O2 uptake rates in suspension culture. In N. tabacum roots subjected to hypoxic hydroponic conditions, the transcript levels of the O2-transporting aquaporin NtPIP1;3 significantly increased after the seven-day hypoxia treatment, which was accompanied by the increase of ATP levels in the apical root segments. Our results suggest that the functional significance of aquaporin-mediated O2 transport and the possibility of controlling the rate of transmembrane O2 transport should be further explored. PMID:28079178

  18. 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.

  19. 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.

  20. Constitutive overexpression of soybean plasma membrane intrinsic protein GmPIP1;6 confers salt tolerance

    PubMed Central

    2014-01-01

    Background Under saline conditions, plant growth is depressed via osmotic stress and salt can accumulate in leaves leading to further depression of growth due to reduced photosynthesis and gas exchange. Aquaporins are proposed to have a major role in growth of plants via their impact on root water uptake and leaf gas exchange. In this study, soybean plasma membrane intrinsic protein 1;6 (GmPIP1;6) was constitutively overexpressed to evaluate the function of GmPIP1;6 in growth regulation and salt tolerance in soybean. Results GmPIP1;6 is highly expressed in roots as well as reproductive tissues and the protein targeted to the plasma membrane in onion epidermis. Treatment with 100 mM NaCl resulted in reduced expression initially, then after 3 days the expression was increased in root and leaves. The effects of constitutive overexpression of GmPIP1;6 in soybean was examined under normal and salt stress conditions. Overexpression in 2 independent lines resulted in enhanced leaf gas exchange, but not growth under normal conditions compared to wild type (WT). With 100 mM NaCl, net assimilation was much higher in the GmPIP1;6-Oe and growth was enhanced relative to WT. GmPIP1;6-Oe plants did not have higher root hydraulic conductance (Lo) under normal conditions, but were able to maintain Lo under saline conditions compared to WT which decreased Lo. GmPIP1;6-Oe lines grown in the field had increased yield resulting mainly from increased seed size. Conclusions The general impact of overexpression of GmPIP1;6 suggests that it may be a multifunctional aquaporin involved in root water transport, photosynthesis and seed loading. GmPIP1;6 is a valuable gene for genetic engineering to improve soybean yield and salt tolerance. PMID:24998596

  1. Aquaporins: Highly Regulated Channels Controlling Plant Water Relations1

    PubMed Central

    Chaumont, François; Tyerman, Stephen D.

    2014-01-01

    Plant growth and development are dependent on tight regulation of water movement. Water diffusion across cell membranes is facilitated by aquaporins that provide plants with the means to rapidly and reversibly modify water permeability. This is done by changing aquaporin density and activity in the membrane, including posttranslational modifications and protein interaction that act on their trafficking and gating. At the whole organ level aquaporins modify water conductance and gradients at key “gatekeeper” cell layers that impact on whole plant water flow and plant water potential. In this way they may act in concert with stomatal regulation to determine the degree of isohydry/anisohydry. Molecular, physiological, and biophysical approaches have demonstrated that variations in root and leaf hydraulic conductivity can be accounted for by aquaporins but this must be integrated with anatomical considerations. This Update integrates these data and emphasizes the central role played by aquaporins in regulating plant water relations. PMID:24449709

  2. HeLa cell plasma membranes. I. 5'-Nucleotidase and ouabain-sensitive ATPase as markers for plasma membranes.

    PubMed

    Johnsen, S; Stokke, T; Prydz, H

    1974-11-01

    A method for the preparation of HeLa cell plasma membrane ghosts is described. The purity of the plasma membrane fraction was examined by phase contrast and electron microscopy, by chemical analysis, and by assay of marker enzymes. Data on the composition of the plasma membrane fraction are given. It was observed that the distribution pattern of 5'-nucleotidase activity among the subcellular fractions differed from that of ouabain-sensitive ATPase. In addition, the specific activity of 5'-nucleotidase did not follow the distribution of the membrane ghosts. Thus, this enzyme would seem unsuitable as a plasma membrane marker. A complete balance sheet for marker enzyme activities during the fractionation is necessary for the calculation of increase in specific activity because the activities of both 5'-nucleotidase and ouabain-sensitive ATPase might change during the fractionation procedures.

  3. CHANGING ELECTRICAL CONSTANTS OF THE FUNDULUS EGG PLASMA MEMBRANE

    PubMed Central

    Kao, C. Y.

    1956-01-01

    Electrical constants of the plasma membrane of the Fundulus egg have been measured with microelectrodes by the transient method. No consistent and significant membrane potential was measured. Membrane capacity averages 0.63 µF/cm.2 for both unactivated and activated eggs. Membrane resistance averages 3450 ohm-cm.2 in the unactivated eggs, but increases 2 to 7 times to an average of 13,290 ohm-cm.2 in the fully activated state. In a hypertonic sucrose solution, the swelling of the egg proper is accompanied by a rapid fall of membrane resistance towards that in the unactivated state. The changes of the membrane resistance are interpreted as probably caused by alterations in the effective pore size in the plasma membrane. PMID:13357740

  4. Plasma Membrane Intrinsic Proteins SlPIP2;1, SlPIP2;7 and SlPIP2;5 Conferring Enhanced Drought Stress Tolerance in Tomato

    PubMed Central

    Li, Ren; Wang, Jinfang; Li, Shuangtao; Zhang, Lei; Qi, Chuandong; Weeda, Sarah; Zhao, Bing; Ren, Shuxin; Guo, Yang-Dong

    2016-01-01

    The function of aquaporin (AQP) protein in transporting water is crucial for plants to survive in drought stress. With 47 homologues in tomato (Solanum lycopersicum) were reported, but the individual and integrated functions of aquaporins involved in drought response remains unclear. Here, three plasma membrane intrinsic protein genes, SlPIP2;1, SlPIP2;7 and SlPIP2;5, were identified as candidate aquaporins genes because of highly expressed in tomato roots. Assay on expression in Xenopus oocytes demonstrated that SlPIP2s protein displayed water channel activity and facilitated water transport into the cells. With real-time PCR and in situ hybridization analysis, SlPIP2s were considered to be involved in response to drought treatment. To test its function, transgenic Arabidopsis and tomato lines overexpressing SlPIP2;1, SlPIP2;7 or SlPIP2;5 were generated. Compared with wild type, the over-expression of SlPIP2;1, SlPIP2;7 or SlPIP2;5 transgenic Arabidopsis and tomato plants all showed significantly higher hydraulic conductivity levels and survival rates under both normal and drought conditions. Taken together, this study concludes that aquaporins (SlPIP2;1, SlPIP2;7 and SlPIP2;5) contribute substantially to root water uptake in tomato plants through improving plant water content and maintaining osmotic balance. PMID:27545827

  5. Electrostatic anchoring precedes stable membrane attachment of SNAP25/SNAP23 to the plasma membrane

    PubMed Central

    Weber, Pascal; Batoulis, Helena; Rink, Kerstin M; Dahlhoff, Stefan; Pinkwart, Kerstin; Söllner, Thomas H; Lang, Thorsten

    2017-01-01

    The SNAREs SNAP25 and SNAP23 are proteins that are initially cytosolic after translation, but then become stably attached to the cell membrane through palmitoylation of cysteine residues. For palmitoylation to occur, membrane association is a prerequisite, but it is unclear which motif may increase the affinities of the proteins for the target membrane. In experiments with rat neuroendocrine cells, we find that a few basic amino acids in the cysteine-rich region of SNAP25 and SNAP23 are essential for plasma membrane targeting. Reconstitution of membrane-protein binding in a liposome assay shows that the mechanism involves protein electrostatics between basic amino acid residues and acidic lipids such as phosphoinositides that play a primary role in these interactions. Hence, we identify an electrostatic anchoring mechanism underlying initial plasma membrane contact by SNARE proteins, which subsequently become palmitoylated at the plasma membrane. DOI: http://dx.doi.org/10.7554/eLife.19394.001 PMID:28240595

  6. A phylogenetic framework for the aquaporin family in eukaryotes.

    PubMed

    Zardoya, R; Villalba, S

    2001-05-01

    A comprehensive evolutionary analysis of aquaporins, a family of intrinsic membrane proteins that function as water channels, was conducted to establish groups of homology (i.e., to identify orthologues and paralogues) within the family and to gain insights into the functional constraints acting on the structure of the aquaporin molecule structure. Aquaporins are present in all living organisms, and therefore, they provide an excellent opportunity to further our understanding of the broader biological significance of molecular evolution by gene duplication followed by functional and structural specialization. Based on the resulting phylogeny, the 153 channel proteins analyzed were classified into six major paralogous groups: (1) GLPs, or glycerol-transporting channel proteins, which include mammalian AQP3, AQP7, and AQP9, several nematode paralogues, a yeast paralogue, and Escherichia coli GLP; (2) AQPs, or aquaporins, which include metazoan AQP0, AQP1, AQP2, AQP4, AQP5, and AQP6; (3) PIPs, or plasma membrane intrinsic proteins of plants, which include PIP1 and PIP2; (4) TIPs, or tonoplast intrinsic proteins of plants, which include alphaTIP, gammaTIP, and deltaTIP; (5) NODs, or nodulins of plants; and (6) AQP8s, or metazoan aquaporin 8 proteins. Of these groups, AQPs, PIPs, and TIPs cluster together. According to the results, the capacity to transport glycerol shown by several members of the family was acquired only early in the history of the family. The new phylogeny reveals that several water channel proteins are misclassified and require reassignment, whereas several previously undetermined ones can now be classified with confidence. The deduced phylogenetic framework was used to characterize the molecular features of water channel proteins. Three motifs are common to all family members: AEF (Ala-Glu-Phe), which is located in the N-terminal domain; and two NPA (Asp-Pro-Ala) boxes, which are located in the center and C-terminal domains, respectively. Other

  7. 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.

  8. ISOLATION OF PLASMA MEMBRANE FRAGMENTS FROM HELA CELLS

    PubMed Central

    Boone, Charles W.; Ford, Lincoln E.; Bond, Howard E.; Stuart, Donald C.; Lorenz, Dianne

    1969-01-01

    A method for isolating plasma membrane fragments from HeLa cells is described. The procedure starts with the preparation of cell membrane "ghosts," obtained by gentle rupture of hypotonically swollen cells, evacuation of most of the cell contents by repeated washing, and isolation of the ghosts on a discontinuous sucrose density gradient. The ghosts are then treated by minimal sonication (5 sec) at pH 8.6, which causes the ghost membranes to pinch off into small vesicles but leaves any remaining larger intracellular particulates intact and separable by differential centrifugation. The ghost membrane vesicles are then subjected to isopycnic centrifugation on a 20–50% w/w continuous sucrose gradient in tris-magnesium buffer, pH 8.6. A band of morphologically homogeneous smooth vesicles, derived principally from plasma membrane, is recovered at 30–33% (peak density = 1.137). The plasma membrane fraction contained a Na-K-activated ATPase activity of 1.5 µmole Pi/hr per mg, 3% RNA, and 13.8% of the NADH-cytochrome c reductase activity of a heavier fraction from the same gradient which contained mitochondria and rough endoplasmic vesicles. The plasma membranes of viable HeLa cells were marked with 125I-labeled horse antibody and followed through the isolation procedure. The specific antibody binding of the plasma membrane vesicle fraction was increased 49-fold over that of the original whole cells. PMID:4239370

  9. Dynamic regulation of aquaporin-4 water channels in neurological disorders

    PubMed Central

    Hsu, Ying; Tran, Minh; Linninger, Andreas A.

    2015-01-01

    Aquaporin-4 water channels play a central role in brain water regulation in neurological disorders. Aquaporin-4 is abundantly expressed at the astroglial endfeet facing the cerebral vasculature and the pial membrane, and both its expression level and subcellular localization significantly influence brain water transport. However, measurements of aquaporin-4 levels in animal models of brain injury often report opposite trends of change at the injury core and the penumbra. Furthermore, aquaporin-4 channels play a beneficial role in brain water clearance in vasogenic edema, but a detrimental role in cytotoxic edema and exacerbate cell swelling. In light of current evidence, we still do not have a complete understanding of the role of aquaporin-4 in brain water transport. In this review, we propose that the regulatory mechanisms of aquaporin-4 at the transcriptional, translational, and post-translational levels jointly regulate water permeability in the short and long time scale after injury. Furthermore, in order to understand why aquaporin-4 channels play opposing roles in cytotoxic and vasogenic edema, we discuss experimental evidence on the dynamically changing osmotic gradients between blood, extracellular space, and the cytosol during the formation of cytotoxic and vasogenic edema. We conclude with an emerging picture of the distinct osmotic environments in cytotoxic and vasogenic edema, and propose that the directions of aquaporin-4-mediated water clearance in these two types of edema are distinct. The difference in water clearance pathways may provide an explanation for the conflicting observations of the roles of aquaporin-4 in edema resolution. PMID:26526878

  10. Paracrine signaling through plasma membrane hemichannels☆

    PubMed Central

    Wang, Nan; De Bock, Marijke; Decrock, Elke; Bol, Mélissa; Gadicherla, Ashish; Vinken, Mathieu; Rogiers, Vera; Bukauskas, Feliksas F.; Bultynck, Geert; Leybaert, Luc

    2013-01-01

    Plasma membrane hemichannels composed of connexin (Cx) proteins are essential components of gap junction channels but accumulating evidence suggests functions of hemichannels beyond the communication provided by junctional channels. Hemichannels not incorporated into gap junctions, called unapposed hemichannels, can open in response to a variety of signals, electrical and chemical, thereby forming a conduit between the cell’s interior and the extracellular milieu. Open hemichannels allow the bidirectional passage of ions and small metabolic or signaling molecules of below 1–2 kDa molecular weight. In addition to connexins, hemichannels can also be formed by pannexin (Panx) proteins and current evidence suggests that Cx26, Cx32, Cx36, Cx43 and Panx1, form hemichannels that allow the diffusive release of paracrine messengers. In particular, the case is strong for ATP but substantial evidence is also available for other messengers like glutamate and prostaglandins or metabolic substances like NAD+ or glutathione. While this field is clearly in expansion, evidence is still lacking at essential points of the paracrine signaling cascade that includes not only messenger release, but also downstream receptor signaling and consequent functional effects. The data available at this moment largely derives from in vitro experiments and still suffers from the difficulty of separating the functions of connexin-based hemichannels from gap junctions and from pannexin hemichannels. However, messengers like ATP or glutamate have universal roles in the body and further defining the contribution of hemichannels as a possible release pathway is expected to open novel avenues for better understanding their contribution to a variety of physiological and pathological processes. This article is part of a Special Issue entitled: The Communicating junctions, roles and dysfunctions. PMID:22796188

  11. Composite plasma polymerized sulfonated polystyrene membrane for PEMFC

    SciTech Connect

    Nath, Bhabesh Kumar; Khan, Aziz; Chutia, Joyanti

    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 chemical 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.

  12. An effective plasma membrane proteomics approach for small tissue samples

    PubMed Central

    Smolders, Katrien; Lombaert, Nathalie; Valkenborg, Dirk; Baggerman, Geert; Arckens, Lutgarde

    2015-01-01

    Advancing the quest for new drug targets demands the development of innovative plasma membrane proteome research strategies applicable to small, functionally defined tissue samples. Biotinylation of acute tissue slices and streptavidin pull-down followed by shotgun proteomics allowed the selective extraction and identification of >1,600 proteins of which >60% are associated with the plasma membrane, including (G-protein coupled) receptors, ion channels and transporters, and this from mm3-scale tissue. PMID:26047021

  13. 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.

  14. Antibodies raised against tobacco aquaporins of the PIP2 class label viscin tissue of the explosive dwarf mistletoe fruit.

    PubMed

    Ross Friedman, C M; Ross, B N; Martens, G D

    2010-01-01

    Dwarf mistletoes, genus Arceuthobium, are parasitic flowering plants and forest pests. In western North America, Arceuthobium americanum (lodgepole pine dwarf mistletoe) is principally found on Pinus contorta var. latifolia (lodgepole pine). Dwarf mistletoes disperse their seeds by an explosive process that involves the buildup of hydrostatic pressure within a mucilaginous fruit tissue called the 'viscin'. Living viscin tissue envelops the discharged seeds. This study examined the possibility that aquaporins, critical in plant water relations, might be found in the dwarf mistletoe fruit, specifically the viscin cells. An antibody raised against a tobacco plasma membrane intrinsic 2 (PIP2) aquaporin was used with a gold-labeled secondary antibody to probe dwarf mistletoe fruit at various developmental stages. Viscin cell plasma membranes were successfully labeled with the anti-tobacco probe, and the validity of the immunolabeling was supported by Western blot analysis, showing a strong signal at about 30 kDa, which is at the expected size of a PIP2. A definitive immunolabeling pattern, supported by quantification of gold signal per membrane length, was observed: viscin cells sampled early in development had abundant gold label at their plasma membranes (1.93 +/- 0.13 to 2.13 +/- 0.33 gold particles per microm membrane), while other areas of the cells had no discernible label. Viscin cells sampled near the time of explosive discharge had significantly less label at the plasma membrane (0.21 gold particles +/- 0.11 per microm membrane, P < 0.05), and label was seen at vesicular membranes. Aquaporins likely have a role in directing water to the viscin mucilage early in development, but are retrieved via endocytosis to prevent excess water loss from viscin cells when discharge is imminent.

  15. Insights into structural mechanisms of gating induced regulation of aquaporins.

    PubMed

    Sachdeva, Ruchi; Singh, Balvinder

    2014-04-01

    Aquaporin family comprises of transmembrane channels that are specialized in conducting water and certain small, uncharged molecules across cell membranes. Essential roles of aquaporins in various physiological and pathophysiological conditions have attracted great scientific interest. Pioneering structural studies on aquaporins have almost solved the basic question of mechanism of selective water transport through these channels. Another important structural aspect of aquaporins which seeks attention is that how the flow of water through the channel is regulated by the mechanism of gating. Aquaporins are also regulated at the protein level, i.e. by trafficking which includes changes in their expression levels in the membrane. Availability of high resolution structures along with numerous molecular dynamics simulation studies have helped to gain an understanding of the structural mechanisms by which water flux through aquaporins is controlled. This review will summarize the highlights regarding structural features of aquaporins, mechanisms governing water permeation, proton exclusion and substrate specificity, and describe the structural insights into the mechanisms of aquaporin gating whereby water conduction is regulated by post translational modifications, such as phosphorylation.

  16. Plasma Membrane Ca-ATPase of Radish Seedlings 1

    PubMed Central

    Rasi-Caldogno, Franca; Carnelli, Antonella; De Michelis, Maria I.

    1992-01-01

    The effect of calmodulin on the activity of the plasma membrane Ca-ATPase was investigated on plasma membranes purified from radish (Raphanus sativus L.) seedlings. Calmodulin stimulated the hydrolytic activity and the transport activity of the plasma membrane Ca-ATPase to comparable extents in a manner dependent on the free Ca2+ concentration. Stimulation was marked at low, nonsaturating Ca2+ concentrations and decreased increasing Ca2+, so that the effect of calmodulin resulted in an increase of the apparent affinity of the enzyme for free Ca2+. The pattern of calmodulin stimulation of the plasma membrane Ca-ATPase activity was substantially the same at pH 6.9 and 7.5, in the presence of ATP or ITP, and when calmodulin from radish seeds was used rather than that from bovine brain. At pH 6.9 in the presence of 5 micromolar free Ca2+, stimulation of the plasma membrane Ca-ATPase was saturated by 30 to 50 micrograms per milliliter bovine brain calmodulin. The calmodulin antagonist calmidazolium inhibited both basal and calmodulin-stimulated plasma membrane Ca-ATPase activity to comparable extents. PMID:16668747

  17. Facilitative plasma membrane transporters function during ER transit

    PubMed Central

    Takanaga, Hitomi; Frommer, Wolf B.

    2010-01-01

    Although biochemical studies suggested a high permeability of the endoplasmic reticulum (ER) membrane for small molecules, proteomics identified few specialized ER transporters. To test functionality of transporters during ER passage, we tested whether glucose transporters (GLUTs, SGLTs) destined for the plasma membrane are active during ER transit. HepG2 cells were characterized by low-affinity ER transport activity, suggesting that ER uptake is protein mediated. The much-reduced capacity of HEK293T cells to take up glucose across the plasma membrane correlated with low ER transport. Ectopic expression of GLUT1, -2, -4, or -9 induced GLUT isoform-specific ER transport activity in HEK293T cells. In contrast, the Na+-glucose cotransporter SGLT1 mediated efficient plasma membrane glucose transport but no detectable ER uptake, probably because of lack of a sufficient sodium gradient across the ER membrane. In conclusion, we demonstrate that GLUTs are sufficient for mediating ER glucose transport en route to the plasma membrane. Because of the low volume of the ER, trace amounts of these uniporters contribute to ER solute import during ER transit, while uniporters and cation-coupled transporters carry out export from the ER, together potentially explaining the low selectivity of ER transport. Expression levels and residence time of transporters in the ER, as well as their coupling mechanisms, could be key determinants of ER permeability.—Takanaga, H., Frommer, W. B. Facilitative plasma membrane transporters function during ER transit. PMID:20354141

  18. Enhancement of polycarbonate membrane permeability due to plasma polymerization precursors

    NASA Astrophysics Data System (ADS)

    Çökeliler, Dilek

    2013-03-01

    The diffusivity of different species through a membrane depends on several factors to illustrate the structure of the matrix, molecular size and concentration of the species and temperature. This study concerns the use of the low-pressure plasma process with different monomers to confer surface chemical character to polycarbonate membranes without altering their bulk properties for change membrane permeability. Track-etched polycarbonate membranes with 0.03 μm pore sizes were modified by plasma polymerization technique with two precursors; acrylic acid and allylamine in radio frequency discharge at certain plasma process conditions (discharge power: 20 W, exposure time: 10 min, frequency: 13.56 MHz). The transport properties of model organic acid (citric acid) was studied through unmodified and modified polycarbonate membranes by using diffusion cell system. Such plasma treated membranes were characterized by scanning electron microscopy, X-ray photoelectron spectroscopy and surface energy changes were studied by static contact angle measurements. These results showed that the change of surface properties could be used to improve the transport properties of the target substrates. The diffusion of citric acid through plasma treated polycarbonate membrane was increased about 54.1 ± 3.5% with precursor: allylamine while it was decreased 48.7 ± 2.5% with precursor acrylic acid. It was observed that the presences of proper functional group (like amino) in surfaces of pores can raise the affinity to citric acid and improve its transport rate.

  19. Huntingtin associates with acidic phospholipids at the plasma membrane.

    PubMed

    Kegel, Kimberly B; Sapp, Ellen; Yoder, Jennifer; Cuiffo, Benjamin; Sobin, Lindsay; Kim, Yun J; Qin, Zheng-Hong; Hayden, Michael R; Aronin, Neil; Scott, David L; Isenberg, Gerhard; Goldmann, Wolfgang H; DiFiglia, Marian

    2005-10-28

    We have identified a domain in the N terminus of huntingtin that binds to membranes. A three-dimensional homology model of the structure of the binding domain predicts helical HEAT repeats, which emanate a positive electrostatic potential, consistent with a charge-based mechanism for membrane association. An amphipathic helix capable of inserting into pure lipid bilayers may serve to anchor huntingtin to the membrane. In cells, N-terminal huntingtin fragments targeted to regions of plasma membrane enriched in phosphatidylinositol 4,5-bisphosphate, receptor bound-transferrin, and endogenous huntingtin. N-terminal huntingtin fragments with an expanded polyglutamine tract aberrantly localized to intracellular regions instead of plasma membrane. Our data support a new model in which huntingtin directly binds membranes through electrostatic interactions with acidic phospholipids.

  20. Preparation of Artificial Plasma Membrane Mimicking Vesicles with Lipid Asymmetry

    PubMed Central

    Lin, Qingqing; London, Erwin

    2014-01-01

    Lipid asymmetry, the difference in lipid distribution across the lipid bilayer, is one of the most important features of eukaryotic cellular membranes. However, commonly used model membrane vesicles cannot provide control of lipid distribution between inner and outer leaflets. We recently developed methods to prepare asymmetric model membrane vesicles, but facile incorporation of a highly controlled level of cholesterol was not possible. In this study, using hydroxypropyl-α-cyclodextrin based lipid exchange, a simple method was devised to prepare large unilamellar model membrane vesicles that closely resemble mammalian plasma membranes in terms of their lipid composition and asymmetry (sphingomyelin (SM) and/or phosphatidylcholine (PC) outside/phosphatidylethanolamine (PE) and phosphatidylserine (PS) inside), and in which cholesterol content can be readily varied between 0 and 50 mol%. We call these model membranes “artificial plasma membrane mimicking” (“PMm”) vesicles. Asymmetry was confirmed by both chemical labeling and measurement of the amount of externally-exposed anionic lipid. These vesicles should be superior and more realistic model membranes for studies of lipid-lipid and lipid-protein interaction in a lipid environment that resembles that of mammalian plasma membranes. PMID:24489974

  1. Preparation of artificial plasma membrane mimicking vesicles with lipid asymmetry.

    PubMed

    Lin, Qingqing; London, Erwin

    2014-01-01

    Lipid asymmetry, the difference in lipid distribution across the lipid bilayer, is one of the most important features of eukaryotic cellular membranes. However, commonly used model membrane vesicles cannot provide control of lipid distribution between inner and outer leaflets. We recently developed methods to prepare asymmetric model membrane vesicles, but facile incorporation of a highly controlled level of cholesterol was not possible. In this study, using hydroxypropyl-α-cyclodextrin based lipid exchange, a simple method was devised to prepare large unilamellar model membrane vesicles that closely resemble mammalian plasma membranes in terms of their lipid composition and asymmetry (sphingomyelin (SM) and/or phosphatidylcholine (PC) outside/phosphatidylethanolamine (PE) and phosphatidylserine (PS) inside), and in which cholesterol content can be readily varied between 0 and 50 mol%. We call these model membranes "artificial plasma membrane mimicking" ("PMm") vesicles. Asymmetry was confirmed by both chemical labeling and measurement of the amount of externally-exposed anionic lipid. These vesicles should be superior and more realistic model membranes for studies of lipid-lipid and lipid-protein interaction in a lipid environment that resembles that of mammalian plasma membranes.

  2. Structural Determinants of Oligomerization of the Aquaporin-4 Channel.

    PubMed

    Kitchen, Philip; Conner, Matthew T; Bill, Roslyn M; Conner, Alex C

    2016-03-25

    The aquaporin (AQP) family of integral membrane protein channels mediate cellular water and solute flow. Although qualitative and quantitative differences in channel permeability, selectivity, subcellular localization, and trafficking responses have been observed for different members of the AQP family, the signature homotetrameric quaternary structure is conserved. Using a variety of biophysical techniques, we show that mutations to an intracellular loop (loop D) of human AQP4 reduce oligomerization. Non-tetrameric AQP4 mutants are unable to relocalize to the plasma membrane in response to changes in extracellular tonicity, despite equivalent constitutive surface expression levels and water permeability to wild-type AQP4. A network of AQP4 loop D hydrogen bonding interactions, identified using molecular dynamics simulations and based on a comparative mutagenic analysis of AQPs 1, 3, and 4, suggest that loop D interactions may provide a general structural framework for tetrameric assembly within the AQP family.

  3. Structural Determinants of Oligomerization of the Aquaporin-4 Channel*

    PubMed Central

    Kitchen, Philip; Conner, Matthew T.; Bill, Roslyn M.; Conner, Alex C.

    2016-01-01

    The aquaporin (AQP) family of integral membrane protein channels mediate cellular water and solute flow. Although qualitative and quantitative differences in channel permeability, selectivity, subcellular localization, and trafficking responses have been observed for different members of the AQP family, the signature homotetrameric quaternary structure is conserved. Using a variety of biophysical techniques, we show that mutations to an intracellular loop (loop D) of human AQP4 reduce oligomerization. Non-tetrameric AQP4 mutants are unable to relocalize to the plasma membrane in response to changes in extracellular tonicity, despite equivalent constitutive surface expression levels and water permeability to wild-type AQP4. A network of AQP4 loop D hydrogen bonding interactions, identified using molecular dynamics simulations and based on a comparative mutagenic analysis of AQPs 1, 3, and 4, suggest that loop D interactions may provide a general structural framework for tetrameric assembly within the AQP family. PMID:26786101

  4. 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.

  5. Plasma Membrane Repair in Health and Disease

    PubMed Central

    Demonbreun, Alexis R.; McNally, Elizabeth M.

    2016-01-01

    Since an intact membrane is required for normal cellular homeostasis, membrane repair is essential for cell survival. Human genetic studies, combined with the development of novel animal models and refinement of techniques to study cellular injury, have now uncovered series of repair proteins highly relevant for human health. Many of the deficient repair pathways manifest in skeletal muscle, where defective repair processes result in myopathies or other forms of muscle disease. Dysferlin is a membrane-associated protein implicated in sarcolemmal repair and also linked to other membrane functions including the maintenance of transverse tubules in muscle. MG53, annexins, and Eps15-homology domain (EHD)-containing proteins interact with dysferlin to form a membrane repair complex and similarly have roles in membrane trafficking in muscle. These molecular features of membrane repair are not unique to skeletal muscle, but rather skeletal muscle, due to its high demands, is more dependent on an efficient repair process. Phosphatidylserine and phosphatidylinositol 4, 5 bisphosphate, as well as Ca2+, are central regulators of membrane organization during repair. Given the importance of muscle health in disease and in aging, these pathways are targets to enhance muscle function and recovery from injury. PMID:26781830

  6. 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

  7. Discovery of the aquaporins and development of the field.

    PubMed

    Carbrey, Jennifer M; Agre, Peter

    2009-01-01

    The study of water transport began long before the molecular identification of water channels with studies of water-permeable tissues. The discovery of the first aquaporin, AQP1, occurred during experiments focused on the identity of the Rh blood group antigens. Since then the field has expanded dramatically to study aquaporins in all types of organisms. In mammals, some of the aquaporins transport only water. However, there are some family members that collectively transport a diverse set of solutes. The aquaporins can be regulated by factors that affect channel permeability or subcellular localization. An extensive set of studies examines the physiological role of many of the mammalian aquaporins. However, much is still to be discovered about the physiological role of this membrane protein family.

  8. 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

  9. The human plasma membrane peripherome: visualization and analysis of interactions.

    PubMed

    Nastou, Katerina C; Tsaousis, Georgios N; Kremizas, Kimon E; Litou, Zoi I; Hamodrakas, Stavros J

    2014-01-01

    A major part of membrane function is conducted by proteins, both integral and peripheral. Peripheral membrane proteins temporarily adhere to biological membranes, either to the lipid bilayer or to integral membrane proteins with noncovalent interactions. The aim of this study was to construct and analyze the interactions of the human plasma membrane peripheral proteins (peripherome hereinafter). For this purpose, we collected a dataset of peripheral proteins of the human plasma membrane. We also collected a dataset of experimentally verified interactions for these proteins. The interaction network created from this dataset has been visualized using Cytoscape. We grouped the proteins based on their subcellular location and clustered them using the MCL algorithm in order to detect functional modules. Moreover, functional and graph theory based analyses have been performed to assess biological features of the network. Interaction data with drug molecules show that ~10% of peripheral membrane proteins are targets for approved drugs, suggesting their potential implications in disease. In conclusion, we reveal novel features and properties regarding the protein-protein interaction network created by peripheral proteins of the human plasma membrane.

  10. Molecular dynamics study of the archaeal aquaporin AqpM

    PubMed Central

    2011-01-01

    Background Aquaporins are a large family of transmembrane channel proteins that are present throughout all domains of life and are implicated in human disorders. These channels, allow the passive but selective movement of water and other small neutral solutes across cell membranes. Aquaporins have been classified into two sub-families: i) strict aquaporins that only allow the passage of water and ii) the less selective aquaglyceroporins that transport water and other neutral solutes, such as glycerol, CO2 or urea. Recently, the identification and characterization of a number of archaeal and bacterial aquaporins suggested the existence of a third sub-family; one that is neither a strict aquaporin nor an aquaglyceroporin. The function and phylogeny of this third family is still a matter of debate. Results Twenty nanosecond molecular dynamics (MD) simulation of a fully hydrated tetramer of AqpM embedded in a lipid bilayer permitted predictions to be made of key biophysical parameters including: single channel osmotic permeability constant (pf), single channel diffusive permeability constant (pd), channel radius, potential water occupancy of the channel and water orientation inside the pore. These properties were compared with those of well characterized representatives of the two main aquaporin sub-families. Results show that changes in the amino acid composition of the aromatic/arginine region affect the size and polarity of the selectivity filter (SF) and could help explain the difference in water permeability between aquaporins. In addition, MD simulation results suggest that AqpM combines characteristics of strict aquaporins, such as the narrow SF and channel radius, with those of aquaglyceroporins, such as a more hydrophobic and less polar SF. Conclusions MD simulations of AqpM extend previous evidence that this archaeal aquaporin exhibits hybrid features intermediate between the two known aquaporin sub-families, supporting the idea that it may constitute a

  11. Aquaporins and plant transpiration.

    PubMed

    Maurel, Christophe; Verdoucq, Lionel; Rodrigues, Olivier

    2016-11-01

    Although transpiration and aquaporins have long been identified as two key components influencing plant water status, it is only recently that their relations have been investigated in detail. The present review first examines the various facets of aquaporin function in stomatal guard cells and shows that it involves transport of water but also of other molecules such as carbon dioxide and hydrogen peroxide. At the whole plant level, changes in tissue hydraulics mediated by root and shoot aquaporins can indirectly impact plant transpiration. Recent studies also point to a feedback effect of transpiration on aquaporin function. These mechanisms may contribute to the difference between isohydric and anisohydric stomatal regulation of leaf water status. The contribution of aquaporins to transpiration control goes far beyond the issue of water transport during stomatal movements and involves emerging cellular and long-distance signalling mechanisms which ultimately act on plant growth.

  12. Ex vivo preclinical evaluation of membrane plasma separators.

    PubMed

    Matsubara, S; Wojcicki, J M; Sueoka, A; Horiuchi, T; Matsugane, T; Starre, J J; Smith, J W; Malchesky, P S; Nosé, Y

    1984-05-01

    Four different types of hollow-fiber membrane plasma separators, constructed of cellulose acetate, polyvinyl alcohol, polyethylene, and polymethylmethacrylate membranes, were evaluated in ex vivo dog perfusions under conditions simulating their clinical use. An arteriovenous (A-V) fistula constructed in the dogs for blood access enabled repeated access to be achieved without surgical intervention. All modules produced transient leukopenia and a reduction of platelet counts. The polymethylmethacrylate module showed minimum reductions of white blood cell counts and CH50. The early leukocyte count reduction in membrane plasmapheresis is most likely related to the magnitude of complement activation by the membrane, as is seen with hemodialysis.

  13. Influence of training habits on exercise-induced changes in plasma atrial and brain natriuretic peptide and urinary excretion of aquaporin-2 in healthy man.

    PubMed

    Bentzen, H; Pedersen, R S; Nyvad, O; Pedersen, E B

    2002-01-01

    The purpose of this study was to quantify the influence of training habits on the changes in plasma atrial natriuretic peptide (ANP), plasma brain natriuretic peptide (BNP) and urine aquaporin-2 (u-AQP2) during exercise by studying trained and untrained healthy subjects. Eleven trained subjects (7 males, 4 females) and 10 untrained subjects (8 males, 2 females) performed a maximal aerobic exercise test. ANP and BNP were determined every 3 min and at maximum exercise by radioimmunoassay (RIA), and u-AQP2 was determined before and after the exercise test by RIA. The absolute increase in ANP during exercise was higher in the trained subjects (trained subjects: 5.6 pmol/L; untrained subjects: 2.4 pmol/L, p < 0.05) and was positively correlated to ANP at rest (p < 0.03). The maximum absolute increase in BNP during exercise was the same in the two groups (trained subjects: 0.5 pmol/L; untrained subjects: 0.6 pmol/L, NS) and tended to correlate positively with resting BNP in the trained subjects (p = 0.07). Exercise did not change u-AQP2 excretion in either trained subjects (rest: 372 ng/mmol creatinine; exercise: 314 ng/mmol creatinine, NS) or untrained subjects (rest: 263 ng/mmol creatinine; exercise: 338 ng/mmol creatinine, NS). The absolute increase in ANP during exercise was higher in trained subjects than in untrained subjects and was positively correlated to ANP at rest. This might reflect the normal cardiovascular adaptation to exercise. The increase in BNP during exercise was unrelated to training habits. Training habits did not affect the u-AQP2 excretion during exercise.

  14. 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.

  15. Ca2+ induces clustering of membrane proteins in the plasma membrane via electrostatic interactions

    PubMed Central

    Zilly, Felipe E; Halemani, Nagaraj D; Walrafen, David; Spitta, Luis; Schreiber, Arne; Jahn, Reinhard; Lang, Thorsten

    2011-01-01

    Membrane proteins and membrane lipids are frequently organized in submicron-sized domains within cellular membranes. Factors thought to be responsible for domain formation include lipid–lipid interactions, lipid–protein interactions and protein–protein interactions. However, it is unclear whether the domain structure is regulated by other factors such as divalent cations. Here, we have examined in native plasma membranes and intact cells the role of the second messenger Ca2+ in membrane protein organization. We find that Ca2+ at low micromolar concentrations directly redistributes a structurally diverse array of membrane proteins via electrostatic effects. Redistribution results in a more clustered pattern, can be rapid and triggered by Ca2+ influx through voltage-gated calcium channels and is reversible. In summary, the data demonstrate that the second messenger Ca2+ strongly influences the organization of membrane proteins, thus adding a novel and unexpected factor that may control the domain structure of biological membranes. PMID:21364530

  16. Plasma Membrane Ca-ATPase of Radish Seedlings 1

    PubMed Central

    Carnelli, Antonella; De Michelis, Maria I.; Rasi-Caldogno, Franca

    1992-01-01

    In this work, we exploited the capability of the plasma membrane Ca-ATPase to utilize ITP as a substrate to study its characteristics in plasma membrane vesicles purified from radish (Raphanus sativus L.) seedlings. The majority of the ITPase activity of plasma membrane was Ca2+-dependent. The Ca2+-dependent ITPase activity was Mg2+-dependent and was stimulated by the calcium ionophore A23187. It was inhibited by erythrosin B (concentration giving 50% inhibition, 50 nanomolar) and by vanadate (concentration giving 50% inhibition, 3 micromolar) and displayed a broad pH optimum around pH 7.2 to 7.5. Both the hydrolytic and the transport activity of the plasma membrane Ca-ATPase were half-saturated by Ca2+ in the micromolar concentration range. No major effect of EGTA on the saturation kinetics of the enzyme was observed. The affinity of the plasma membrane Ca-ATPase for Ca2+ was about fourfold higher at pH 7.5 than at pH 6.9. The Ca2+-dependent ITPase activity was stimulated about twofold by polyoxyethylene 20 cetyl ether, although it was inhibited by Triton X-100 and by lysolecithin. PMID:16668746

  17. Plant plasma membrane proteomics for improving cold tolerance.

    PubMed

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

    2013-01-01

    Plants are always exposed to various stresses. We have focused on freezing stress, which causes serious problems for agricultural management. When plants suffer freeze-induced damage, the plasma membrane is thought to be the primary site of injury because of its central role in regulation of various cellular processes. Cold tolerant species, however, adapt to such freezing conditions by modifying cellular components and functions (cold acclimation). One of the most important adaptation mechanisms to freezing is alteration of plasma membrane compositions and functions. Advanced proteomic technologies have succeeded in identification of many candidates that may play roles in adaptation of the plasma membrane to freezing stress. Proteomics results suggest that adaptations of plasma membrane functions to low temperature are associated with alterations of protein compositions during cold acclimation. Some of proteins identified by proteomic approaches have been verified their functional roles in freezing tolerance mechanisms further. Thus, accumulation of proteomic results in the plasma membrane is of importance for application to molecular breeding efforts to increase cold tolerance in crops.

  18. Facilitative plasma membrane transporters function during ER transit.

    PubMed

    Takanaga, Hitomi; Frommer, Wolf B

    2010-08-01

    Although biochemical studies suggested a high permeability of the endoplasmic reticulum (ER) membrane for small molecules, proteomics identified few specialized ER transporters. To test functionality of transporters during ER passage, we tested whether glucose transporters (GLUTs, SGLTs) destined for the plasma membrane are active during ER transit. HepG2 cells were characterized by low-affinity ER transport activity, suggesting that ER uptake is protein mediated. The much-reduced capacity of HEK293T cells to take up glucose across the plasma membrane correlated with low ER transport. Ectopic expression of GLUT1, -2, -4, or -9 induced GLUT isoform-specific ER transport activity in HEK293T cells. In contrast, the Na(+)-glucose cotransporter SGLT1 mediated efficient plasma membrane glucose transport but no detectable ER uptake, probably because of lack of a sufficient sodium gradient across the ER membrane. In conclusion, we demonstrate that GLUTs are sufficient for mediating ER glucose transport en route to the plasma membrane. Because of the low volume of the ER, trace amounts of these uniporters contribute to ER solute import during ER transit, while uniporters and cation-coupled transporters carry out export from the ER, together potentially explaining the low selectivity of ER transport. Expression levels and residence time of transporters in the ER, as well as their coupling mechanisms, could be key determinants of ER permeability.

  19. Protein Targeting to Exosomes/Microvesicles by Plasma Membrane Anchors*

    PubMed Central

    Shen, Beiyi; Wu, Ning; Yang, Jr-Ming; Gould, Stephen J.

    2011-01-01

    Animal cells secrete small vesicles, otherwise known as exosomes and microvesicles (EMVs). A short, N-terminal acylation tag can target a highly oligomeric cytoplasmic protein, TyA, into secreted vesicles (Fang, Y., Wu, N., Gan, X., Yan, W., Morell, J. C., and Gould, S. J. (2007) PLoS Biol. 5, 1267–1283). However, it is not clear whether this is true for other membrane anchors or other highly oligomeric, cytoplasmic proteins. We show here that a variety of plasma membrane anchors can target TyA-GFP to sites of vesicle budding and into EMVs, including: (i) a myristoylation tag; (ii) a phosphatidylinositol-(4,5)-bisphosphate (PIP2)-binding domain; (iii), a phosphatidylinositol-(3,4,5)-trisphosphate-binding domain; (iv) a prenylation/palmitoylation tag, and (v) a type-1 plasma membrane protein, CD43. However, the relative budding efficiency induced by these plasma membrane anchors varied over a 10-fold range, from 100% of control (AcylTyA-GFP) for the myristoylation tag and PIP2-binding domain, to one-third or less for the others, respectively. Targeting TyA-GFP to endosome membranes by fusion to a phosphatidylinositol 3-phosphate-binding domain induced only a slight budding of TyA-GFP, ∼2% of control, and no budding was observed when TyA-GFP was targeted to Golgi membranes via a phosphatidylinositol 4-phosphate-binding domain. We also found that a plasma membrane anchor can target two other highly oligomeric, cytoplasmic proteins to EMVs. These observations support the hypothesis that plasma membrane anchors can target highly oligomeric, cytoplasmic proteins to EMVs. Our data also provide additional parallels between EMV biogenesis and retrovirus budding, as the anchors that induced the greatest budding of TyA-GFP are the same as those that mediate retrovirus budding. PMID:21300796

  20. Plasma membrane appearance of phosphatidylethanolamine in stimulated macrophages

    SciTech Connect

    Sandra, A.; Cai, J. )

    1991-07-01

    Mouse peritoneal macrophages were labeled with (1-3H)ethanolamine, and the presence of radioactive (3H)phosphatidylethanolamine (PE) at the plasma membrane was monitored by reacting the cells with trinitrobenzene sulfonic acid (TNBS) under nonpenetrating conditions. Macrophages stimulated with either the calcium ionophore A23187 or zymosan demonstrated a larger proportion of radiolabeled PE in the plasma membrane than control, nonstimulated cells. In experiments in which macrophages were labeled with ethanolamine for increasing times, appearance of membrane 3(H)PE was stimulated as early as after 2 hr of labeling. Macrophages labeled for 24 hr, then stimulated and returned to fresh medium still reflected a higher amount of membrane 3(H)PE at 2 hr after the stimulation, suggesting stimulation results in long-term alterations in plasma membrane lipids. Protease-peptone-elicited macrophages, which are not stimulated by zymosan or ionophore, did not exhibit an increase in membrane 3(H)PE upon stimulation. The size of the TNBS-accessible radiolabeled PE pool increased proportionately with a second stimulation; however, a subsequent labeling of the cells with TNBS after brief warming increased the TNBS-accessible pool in control cells only. As shown in previous studies, macrophage stimulation resulted in an increased incorporation of lipid precursors into phospholipid. The mass of plasma membrane Tnp-PE relative to mass of PE was not increased in ionophore-treated macrophages in contrast to a small (approximately 22%) increase in zymosan-treated cells. These results are suggestive of alterations in lipid synthesis in stimulated macrophages and possible long-term changes in the structure and function of the plasma membrane of macrophages following stimulation.

  1. Effect of BCD Plasma on a Bacteria Cell Membrane

    NASA Astrophysics Data System (ADS)

    Nasrin, Navabsafa; Hamid, Ghomi; Maryam, Nikkhah; Soheila, Mohades; Hossein, Dabiri; Saeed, Ghasemi

    2013-07-01

    Abstract Cell membrane rupture is considered to be one of the probable mechanisms for bacterial inactivation using barrier corona discharge (BCD) plasma. In this paper, the effect of the BCD plasma on the Escherichia coli (E. coli) bacteria cell wall was investigated through two analytical methods; Adenosine-5'-triphosphate (ATP) assay and Atomic Force Microscopy (AFM). The ATP assay results indicate an increase in the ATP content of samples which were exposed to the BCD plasma. This implies the bacteria cell rupture. Moreover, AFM images confirm a serious damage of the bacteria cell wall under the influence of the bactericidal agents of the plasma.

  2. Detergent-resistant membrane subfractions containing proteins of plasma membrane, mitochondrial, and internal membrane origins.

    PubMed

    Mellgren, Ronald L

    2008-04-24

    HEK293 cell detergent-resistant membranes (DRMs) isolated by the standard homogenization protocol employing a Teflon pestle homogenizer yielded a prominent opaque band at approximately 16% sucrose upon density gradient ultracentrifugation. In contrast, cell disruption using a ground glass tissue homogenizer generated three distinct DRM populations migrating at approximately 10%, 14%, and 20% sucrose, named DRM subfractions A, B, and C, respectively. Separation of the DRM subfractions by mechanical disruption suggested that they are physically associated within the cellular environment, but can be dissociated by shear forces generated during vigorous homogenization. All three DRM subfractions possessed cholesterol and ganglioside GM1, but differed in protein composition. Subfraction A was enriched in flotillin-1 and contained little caveolin-1. In contrast, subfractions B and C were enriched in caveolin-1. Subfraction C contained several mitochondrial membrane proteins, including mitofilin and porins. Only subfraction B appeared to contain significant amounts of plasma membrane-associated proteins, as revealed by cell surface labeling studies. A similar distribution of DRM subfractions, as assessed by separation of flotillin-1 and caveolin-1 immunoreactivities, was observed in CHO cells, in 3T3-L1 adipocytes, and in HEK293 cells lysed in detergent-free carbonate. Teflon pestle homogenization of HEK293 cells in the presence of the actin-disrupting agent latrunculin B generated DRM subfractions A-C. The microtubule-disrupting agent vinblastine did not facilitate DRM subfraction separation, and DRMs prepared from fibroblasts of vimentin-null mice were present as a single major band on sucrose gradients, unless pre-treated with latrunculin B. These results suggest that the DRM subfractions are interconnected by the actin cytoskeleton, and not by microtubes or vimentin intermediate filaments. The subfractions described may prove useful in studying discrete protein

  3. 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

  4. Ras diffusion is sensitive to plasma membrane viscosity.

    PubMed

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

    2005-08-01

    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 DiIC(16) and DiIC(18). 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-beta-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.

  5. 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.

  6. Plasma membrane organization and function: moving past lipid rafts.

    PubMed

    Kraft, Mary L

    2013-09-01

    "Lipid raft" is the name given to the tiny, dynamic, and ordered domains of cholesterol and sphingolipids that are hypothesized to exist in the plasma membranes of eukaryotic cells. According to the lipid raft hypothesis, these cholesterol- and sphingolipid-enriched domains modulate the protein-protein interactions that are essential for cellular function. Indeed, many studies have shown that cellular levels of cholesterol and sphingolipids influence plasma membrane organization, cell signaling, and other important biological processes. Despite 15 years of research and the application of highly advanced imaging techniques, data that unambiguously demonstrate the existence of lipid rafts in mammalian cells are still lacking. This Perspective summarizes the results that challenge the lipid raft hypothesis and discusses alternative hypothetical models of plasma membrane organization and lipid-mediated cellular function.

  7. Detection of glycoproteins in the Acanthamoeba plasma membrane

    SciTech Connect

    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 presence 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.

  8. Spatial distributions of phosphorylated membrane proteins aquaporin 0 and MP20 across young and aged human lenses.

    PubMed

    Gutierrez, Danielle B; Garland, Donita L; Schwacke, John H; Hachey, David L; Schey, Kevin L

    2016-08-01

    In the human ocular lens it is now realized that post-translational modifications can alter protein function and/or localization in fiber cells that no longer synthesize proteins. The specific sites of post-translational modification to the abundant ocular lens membrane proteins AQP0 and MP20 have been previously identified and their functional effects are emerging. To further understand how changes in protein function and/or localization induced by these modifications alter lens homeostasis, it is necessary to determine the spatial distributions of these modifications across the lens. In this study, a quantitative LC-MS approach was used to determine the spatial distributions of phosphorylated AQP0 and MP20 peptides from manually dissected, concentric layers of fiber cells from young and aged human lenses. The absolute amounts of phosphorylation were determined for AQP0 Ser235 and Ser229 and for MP20 Ser170 in fiber cells from the lens periphery to the lens center. Phosphorylation of AQP0 Ser229 represented a minor portion of the total phosphorylated AQP0. Changes in spatial distributions of phosphorylated APQ0 Ser235 and MP20 Ser170 correlated with regions of physiological interest in aged lenses, specifically, where barriers to water transport and extracellular diffusion form.

  9. Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase

    PubMed Central

    De Michelis, Maria Ida; Pugliarello, Maria Chiara; Rasi-Caldogno, Franca

    1989-01-01

    The characteristics of fusicoccin binding were investigated in microsomes from 24-h-old radish (Raphanus sativus L.) seedlings. The time course of fusicoccin binding depended on fusicoccin concentration: equilibrium was reached much faster at 10 nanomolar fusicoccin than at 0.3 nanomolar fusicoccin. Scatchard analysis of equilibrium binding as a function of fusicoccin concentration indicated a single class of receptor sites with a Kd of 1.8 nanomolar and a site density of 6.3 picomoles per milligram protein. Similar values (Kd 1.7 nanomolar and site density 7 picomoles per milligram protein) were obtained from the analysis of the dependence of equilibrium binding on membrane concentration at fixed fusicoccin concentrations. Fusicoccin binding comigrated with the plasma membrane H+-ATPase in an equilibrium sucrose density gradient: both activities formed a sharp peak (1.18 grams per milliliter) clearly distinct from that of markers of other membranes which all peaked at lower densities. The saturation profiles of fusicoccin binding and of fusicoccin-induced activation of the plasma membrane H+-ATPase, measured under identical conditions, were similar, supporting the view that fusicoccin-induced activation of the plasma membrane H+-ATPase is mediated by fusicoccin binding to its plasma membrane receptor. PMID:16666723

  10. Endosomal recycling controls plasma membrane area during mitosis.

    PubMed

    Boucrot, Emmanuel; Kirchhausen, Tomas

    2007-05-08

    The shape and total surface of a cell and its daughters change during mitosis. Many cells round up during prophase and metaphase and reacquire their extended and flattened shape during cytokinesis. How does the total area of plasma membrane change to accommodate these morphological changes and by what mechanism is control of total membrane area achieved? Using single-cell imaging methods, we have found that the amount of plasma membrane in attached cells in culture decreases at the beginning of mitosis and recovers rapidly by the end. Clathrin-based endocytosis is normal throughout all phases of cell division, whereas recycling of internalized membranes back to the cell surface slows considerably during the rounding up period and resumes at the time at which recovery of cell membrane begins. Interference with either one of these processes by genetic or chemical means impairs cell division. The total cell-membrane area recovers even in the absence of a functional Golgi apparatus, which would be needed for export of newly synthesized membrane lipids and proteins. We propose a mechanism by which modulation of endosomal recycling controls cell area and surface expression of membrane-bound proteins during cell division.

  11. Palmitoylation of POTE family proteins for plasma membrane targeting

    SciTech Connect

    Das, Sudipto; Ise, Tomoko; Nagata, Satoshi; Maeda, Hiroshi; Bera, Tapan K.; Pastan, Ira

    2007-11-23

    The POTE gene family is composed of 13 paralogs and likely evolved by duplications and remodeling of the human genome. One common property of POTE proteins is their localization on the inner aspect of the plasma membrane. To determine the structural elements required for membrane localization, we expressed mutants of different POTEs in 293T cells as EGFP fusion proteins. We also tested their palmitoylation by a biotin-switch assay. Our data indicate that the membrane localizations of different POTEs are mediated by similar 3-4 short cysteine rich repeats (CRRs) near the amino-terminuses and that palmitoylation on paired cysteine residues in each CRR motif is responsible for the localization. Multiple palmitoylation in the small CRRs can result in the strong association of whole POTEs with plasma membrane.

  12. Contribution of aquaporin 9 and multidrug resistance-associated protein 2 to differential sensitivity to arsenite between primary cultured chorion and amnion cells prepared from human fetal membranes

    SciTech Connect

    Yoshino, Yuta; Yuan, Bo; Kaise, Toshikazu; Takeichi, Makoto; Tanaka, Sachiko; Hirano, Toshihiko; Kroetz, Deanna L.; Toyoda, Hiroo

    2011-12-15

    Arsenic trioxide (arsenite, As{sup III}) has shown a remarkable clinical efficacy, whereas its side effects are still a serious concern. Therefore, it is critical to understand the effects of As{sup III} on human-derived normal cells for revealing the mechanisms underlying these side effects. We examined the effects of As{sup III} on primary cultured chorion (C) and amnion (A) cells prepared from human fetal membranes. A significant dose-dependent As{sup III}-mediated cytotoxicity was observed in the C-cells accompanied with an increase of lactate dehydrogenase (LDH) release. Higher concentrations of As{sup III} were required for the A-cells to show cytotoxicity and LDH release, suggesting that the C-cells were more sensitive to As{sup III} than the A-cells. The expression levels of aquaporin 9 (AQP9) were approximately 2 times higher in the C-cells than those in the A-cells. Both intracellular arsenic accumulation and its cytotoxicity in the C-cells were significantly abrogated by sorbitol, a competitive AQP9 inhibitor, in a dose-dependent manner. The protein expression levels of multidrug resistance-associated protein (MRP) 2 were downregulated by As{sup III} in the C-cells, but not in the A-cells. No significant differences in the expression levels of MRP1 were observed between C- and A-cells. The protein expression of P-glycoprotein (P-gp) was hardly detected in both cells, although a detectable amount of its mRNA was observed. Cyclosporine A, a broad-spectrum inhibitor for ABC transporters, and MK571, a MRP inhibitor, but not PGP-4008, a P-gp specific inhibitor, potently sensitized both cells to As{sup III}-mediated cytotoxicity. These results suggest that AQP9 and MRP2 are involved in controlling arsenic accumulation in these normal cells, which then contribute to differential sensitivity to As{sup III} cytotoxicity between these cells. -- Highlights: Black-Right-Pointing-Pointer Examination of effect of As{sup III} on primary cultured chorion (C) and amnion

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

    PubMed

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

    2017-03-13

    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(CH3)-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.

  14. Transient disruptions of aortic endothelial cell plasma membranes.

    PubMed

    Yu, Q C; McNeil, P L

    1992-12-01

    Cells of gut, skin, and muscle frequently suffer transient survivable plasma membrane disruptions ("wounds") under physiological conditions, but it is not known whether endothelial cells of the aorta, which are constantly exposed to hemodynamically generated mechanical forces, similarly are injured in vivo. We have used serum albumin as a molecular probe for identifying endothelial cells of the rat aorta that incurred and survived transient plasma membrane wounds in vivo. Such wounded endothelial cells were in fact observed in the aortas of all rats examined. However, the percentage of wounded cells in the total aortic endothelial population varied remarkably between individuals ranging from 1.4% to 17.9% with a mean of 6.5% (+/- 4.6% SD). Wounded endothelial cells were heterogeneously distributed, being found in distinct clusters often in the shape of streaks aligned with the long axis of the vessel, or in the shape of partial or complete rims surrounding bifurcation openings, such as the ostia of the intercostal arteries. Physical exercise (running) did not increase the frequency of aortic endothelial cell membrane wounding, nor did spontaneous hypertension. Surprisingly, 80% of mitotic endothelial cell figures were identified as wounded. This article identified a previously unrecognized form of endothelial cell injury, survivable disruptions of the plasma membrane, and shows that injury to the endothelial cells of the normal aorta is far more commonplace than previously suspected. Plasma membrane wounding of endothelial cells could be linked to the initiation of atherosclerosis.

  15. 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.

  16. Cloning, functional characterization, and co-expression studies of a novel aquaporin (FaPIP2;1) of strawberry fruit

    PubMed Central

    Alleva, Karina; Marquez, Mercedes; Villarreal, Natalia; Mut, Paula; Bustamante, Claudia; Bellati, Jorge; Martínez, Gustavo; Civello, Marcos; Amodeo, Gabriela

    2010-01-01

    In strawberry, the putative participation of aquaporins should be considered during fruit ripening. Furthermore, the availability of different firmness cultivars in this non-climacteric fruit is a very useful tool to determine their involvement in softening. In a previous work, the cloning of a strawberry fruit-specific aquaporin, FaPIP1;1, which showed an expression profile associated with fruit ripening was reported. Here, FaPIP2;1, an aquaporin subtype of PIP2 was cloned and its functional characterization in Xenopus oocytes determined. The FaPIP2;1 gene encodes a water channel with high water permeability (Pf) that is regulated by cytosolic pH. Interestingly, the co-expression of both FaPIP subtypes resulted in an enhancement of water permeability, showing Pf values that exceeds their individual contribution. The expression pattern of both aquaporin subtypes in two cultivars with contrasting fruit firmness showed that the firmer cultivar (Camarosa) has a higher accumulation of FaPIP1 and FaPIP2 mRNAs during fruit ripening when compared with the softer cultivar (Toyonoka). In conclusion, not only FaPIP aquaporins showed an expression pattern associated with fruit firmness but it was also shown that the enhancement of water transfer through the plasma membrane is coupled to the presence/absence of the co-expression of both subtypes. PMID:20663858

  17. Therapeutic plasmapheresis using membrane plasma separation.

    PubMed

    Sinha, Aditi; Tiwari, Anand Narain; Chanchlani, Rahul; Seetharamanjaneyulu, V; Hari, Pankaj; Bagga, Arvind

    2012-08-01

    The authors present their experience with therapeutic plasmapheresis (TPE) using membrane filters at the pediatric dialysis unit of a referral center. Between January 2006 and December 2010, 486 sessions of TPE were performed in 39 patients (range 6-17 y), chiefly for atypical hemolytic uremic syndrome (HUS, n = 22), crescentic glomerulonephritis (n = 8) and focal segmental glomerulosclerosis (n = 5). Satisfactory response was noted in 32 patients, particularly with HUS (n = 22) or crescentic glomerulonephritis (n = 6). Adverse effects included chills or urticaria (n = 8 sessions), hypocalcemia (n = 6) and hypotension (n = 5). The present findings highlight the safety, efficacy and feasibility of TPE using membrane filtration.

  18. An endosome-to-plasma membrane pathway involved in trafficking of a mutant plasma membrane ATPase in yeast.

    PubMed

    Luo, W j; Chang, A

    2000-02-01

    The plasma membrane ATPase, encoded by PMA1, is delivered to the cell surface via the secretory pathway. Previously, we characterized a temperature-sensitive pma1 mutant in which newly synthesized Pma1-7 is not delivered to the plasma membrane but is mislocalized instead to the vacuole at 37 degrees C. Several vps mutants, which are defective in vacuolar protein sorting, suppress targeting-defective pma1 by allowing mutant Pma1 to move once again to the plasma membrane. In this study, we have analyzed trafficking in the endosomal system by monitoring the movement of Pma1-7 in vps36, vps1, and vps8 mutants. Upon induction of expression, mutant Pma1 accumulates in the prevacuolar compartment in vps36 cells. After chase, a fraction of newly synthesized Pma1-7 is delivered to the plasma membrane. In both vps1 and vps8 cells, newly synthesized mutant Pma1 appears in small punctate structures before arrival at the cell surface. Nevertheless, biosynthetic membrane traffic appears to follow different routes in vps8 and vps1: the vacuolar protein-sorting receptor Vps10p is stable in vps8 but not in vps1. Furthermore, a defect in endocytic delivery to the vacuole was revealed in vps8 (and vps36) but not vps1 by endocytosis of the bulk membrane marker FM 4-64. Moreover, in vps8 cells, there is defective down-regulation from the cell surface of the mating receptor Ste3, consistent with persistent receptor recycling from an endosomal compartment to the plasma membrane. These data support a model in which mutant Pma1 is diverted from the Golgi to the surface in vps1 cells. We hypothesize that in vps8 and vps36, in contrast to vps1, mutant Pma1 moves to the surface via endosomal intermediates, implicating an endosome-to-surface traffic pathway.

  19. Maize black Mexican sweet suspension cultured cells are a convenient tool for studying aquaporin activity and regulation.

    PubMed

    Cavez, Damien; Hachez, Charles; Chaumont, François

    2009-09-01

    Aquaporins (AQPs) are channel proteins that facilitate and regulate the permeation of water across biological membranes. Black Mexican sweet suspension cultured cells are a convenient model for studying the regulation of maize AQP expression and activity. Among other advantages, a single cell system allows the contribution of plasma membrane AQPs (PIPs, plasma membrane intrinsic proteins) to the membrane water permeability coefficient (P(f)) to be determined using biophysical measurement methods, such as the cell pressure probe or protoplast swelling assay. We generated a transgenic cell culture line expressing a tagged version of ZmPIP2;6 and used this material to demonstrate that the ZmPIP2;6 and ZmPIP2;1 isoforms physically interact. This kind of interaction could be an additional mechanism for regulating membrane water permeability by acting on the activity and/or trafficking of PIP hetero-oligomers.

  20. Remodeling of the postsynaptic plasma membrane during neural development

    PubMed Central

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

    2016-01-01

    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. PMID:27535429

  1. Mechanical properties of the plasma membrane of isolated plant protoplasts

    SciTech Connect

    Wolfe, J.; Steponkus, P.L.

    1983-01-01

    The volume of isolated protoplasts of rye (Secale cereale L. cv Puma) in a suspending solution at constant concentration is shown to be negligibly changed by tensions in the plasma membrane which approach that tension necessary to lyse them. This allows a detailed investigation of the plasma membrane stress-strain relation by micropipette aspiration. Over periods less than a second, the membrane behaves as an elastic two-dimensional fluid with an area modulus of elasticity of 230 millinewtons per meter. Over longer periods, the stress-strain relation approaches a surface energy law--the resting tension is independent of area and has a value of the order 100 micronewtons per meter. Over longer periods the untensioned area, which is defined as the area that would be occupied by the molecules in the membrane at any given time if the tension were zero, increases with time under large imposed tensions and decreases under sufficiently small tension. It is proposed that these long term responses are the result of exchange of material between the plane of the membrane and a reservoir of membrane material. The irreversibility of large contractions in area is demonstrated directly, and the behavior of protoplasts during osmotically induced cycles of contraction and expansion is explained in terms of the membrane stress-strain relation.

  2. 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.

  3. Cortical actin and the plasma membrane: inextricably intertwined.

    PubMed

    Köster, Darius V; Mayor, Satyajit

    2016-02-01

    The plasma membrane serves as a barrier, separating the cell from its external environment. Simultaneously it acts as a site for information transduction, entry of nutrients, receptor signaling, and adapts to the shape of the cell. This requires local control of organization at multiple scales in this heterogeneous fluid lipid bilayer with a plethora of proteins and a closely juxtaposed dynamic cortical cytoskeleton. New membrane models highlight the influence of the underlying cortical actin on the diffusion of membrane components. Myosin motors as well as proteins that remodel actin filaments have additionally been implicated in defining the organization of many membrane constituents. Here we provide a perspective of the intimate relationship of the membrane lipid matrix and the underlying cytoskeleton. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. 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.

  5. 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.

  6. Molecular mechanisms and drug development in aquaporin water channel diseases: water channel aquaporin-2 of kidney collecting duct cells.

    PubMed

    Takata, Kuniaki; Tajika, Yuki; Matsuzaki, Toshiyuki; Aoki, Takeo; Suzuki, Takeshi; Abduxukur, Ablimit; Hagiwara, Haruo

    2004-11-01

    Aquaporin-2 (AQP2) is one of the membrane water channel proteins expressed in principal cells of the kidney collecting ducts. In the basal state, AQP2 resides in the storage vesicles localized in the subapical cytoplasm. Upon stimulation with vasopressin, AQP2 is translocated to the apical plasma membrane by the exocytic fusion of the storage vesicles with the apical membrane. This translocation enables the transepithelial reabsorption of water from the lumen to the interstitium via AQP2 at the apical membrane and AQP3/AQP4 at the basolateral membrane. AQP2-storage vesicles are distinct from the endoplasmic reticulum, Golgi apparatus, trans-Golgi network, and lysosomes. The early endosomal marker EEA1 is colocalized with some of AQP2 vesicles. Further analyses in Madin-Darby canine kidney (MDCK) cells transfected with AQP2 revealed that subapical Rab11-positive/EEA1-negative smaller vesicles constitute part of the AQP2 storage vesicles for the translocation to the apical membrane. Termination of stimulation results in the retrieval of AQP2 to the larger EEA1-positive early endosomal compartment. AQP2 is then transferred to the subapical storage compartment in a PI3-kinase-dependent manner. GLUT4 is an isoform of glucose transporters whose localization is also regulated by vesicular trafficking induced by insulin stimulation. Comparison of the intracellular localization of AQP2 with GLUT4 suggests distinct regulation of AQP2 trafficking.

  7. PSD-95 mediates membrane clustering of the human plasma membrane Ca2+ pump isoform 4b.

    PubMed

    Padányi, Rita; Pászty, Katalin; Strehler, Emanuel E; Enyedi, Agnes

    2009-06-01

    Besides the control of global calcium changes, specific plasma membrane calcium ATPase (PMCA) isoforms are involved in the regulation of local calcium signals. Although local calcium signaling requires the confinement of signaling molecules into microdomains, little is known about the specific organization of PMCA molecules within the plasma membrane. Here we show that co-expression with the postsynaptic density-95 (PSD-95) scaffolding protein increased the plasma membrane expression of PMCA4b and redistributed the pump into clusters. The clustering of PMCA4b was fully dependent on the presence of its PDZ-binding sequence. Using the fluorescence recovery after photobleaching (FRAP) technique, we show that the lateral membrane mobility of the clustered PMCA4b is significantly lower than that of the non-clustered molecules. Disruption of the actin-based cytoskeleton by cytochalasin D resulted in increased cluster size. Our results suggest that PSD-95 promotes the formation of high-density PMCA4b microdomains in the plasma membrane and that the membrane cytoskeleton plays an important role in the regulation of this process.

  8. Aquaporin-facilitated transmembrane diffusion of hydrogen peroxide.

    PubMed

    Bienert, Gerd P; Chaumont, François

    2014-05-01

    Hydrogen peroxide (H2O2) is an important signaling compound that has recently been identified as a new substrate for several members of the aquaporin superfamily in various organisms. Evidence is emerging about the physiological significance of aquaporin-facilitated H2O2 diffusion. This review summarizes current knowledge about aquaporin-facilitated H2O2 diffusion across cellular membranes. It focuses on physicochemical and experimental evidence demonstrating the involvement of aquaporins in the transport of this redox signaling compound and discusses the regulation and structural prerequisites of these channels to transmit this signal. It also provides perspectives about the potential importance of aquaporin-facilitated H2O2 diffusion processes and places this knowledge in the context of the current understanding of transmembrane redox signaling processes. Specific aquaporin isoforms facilitate the passive diffusion of H2O2 across biological membranes and control H2O2 membrane permeability and signaling in living organisms. Redox signaling is a very important process regulating the physiology of cells and organisms in a similar way to the well-characterized hormonal and calcium signaling pathways. Efficient transmembrane diffusion of H2O2, a key molecule in the redox signaling network, requires aquaporins and makes these channels important players in this signaling process. Channel-mediated membrane transport allows the fine adjustment of H2O2 levels in the cytoplasm, intracellular organelles, the apoplast, and the extracellular space, which are essential for it to function as a signal molecule. This article is part of a Special Issue entitled Aquaporins. © 2013.

  9. Aquaporins and leaf hydraulics: poplar sheds new light.

    PubMed

    Lopez, David; Venisse, Jean-Stéphane; Fumanal, Boris; Chaumont, François; Guillot, Esther; Daniels, Mark J; Cochard, Hervé; Julien, Jean-Louis; Gousset-Dupont, Aurélie

    2013-12-01

    To help understand leaf hydraulic conductance (Kleaf) modulation under high irradiance, well-watered poplars (Populus trichocarpa Torr. & Gray ex Hook and Populus nigra L.) were studied diurnally at molecular and ecophysiological scales. Transcriptional and translational modulations of plasma membrane intrinsic protein (PIP) aquaporins were evaluated in leaf samples during diurnal time courses. Among the 15 poplar PIP genes, a subset of two PIP1s and seven PIP2s are precociously induced within the first hour of the photoperiod concomitantly with a Kleaf increase. Since expression patterns were cyclic and reproducible over several days, we hypothesized that endogenous signals could be involved in PIP transcriptional regulation. To address this question, plants were submitted to forced darkness during their subjective photoperiod and compared with their control counterparts, which showed that some PIP1s and PIP2s have circadian regulation while others did not. Promoter analysis revealed that a large number of hormone, light, stress response and circadian elements are present. Finally, involvement of aquaporins is supported by the reduction of Kleaf by HgCl2 treatment.

  10. Conversion of aquaporin 6 from an anion channel to a water-selective channel by a single amino acid substitution

    PubMed Central

    Liu, Kun; Kozono, David; Kato, Yasuhiro; Agre, Peter; Hazama, Akihiro; Yasui, Masato

    2005-01-01

    Aquaporin (AQP) 6 belongs to the aquaporin water channel family. Unlike other aquaporins, AQP6 functions not as a water channel but as an anion-selective channel. Single-channel analyses have shown AQP6 to flicker rapidly between closed and open status. The atomic structure of AQP1 and amino acid sequence alignments of the mammalian aquaporins reveal two well conserved glycine residues: Gly-57 in transmembrane helix (TM) 2 and Gly-173 in TM5 reside at the contact point where the two helices cross in human AQP1. Uniquely, all known mammalian orthologs of AQP6 have an asparagine residue (Asn-60) at the position corresponding to Gly-57. Here we show that a single residue substitution (N60G in rat AQP6) totally eliminates the anion permeability of AQP6 when expressed in Xenopus oocytes, but the N60G oocytes exhibit significantly higher osmotic water permeability under basal conditions. Replacement of the glycine at this site in AQP0, AQP1, and AQP2 blocked expression of the mutants at the oocyte plasma membrane. We propose that the asparagine residue at the contact point between TM2 and TM5 in AQP6 may function as a teeter board needed for rapid structural oscillations during anion permeation. PMID:15671159

  11. Conversion of aquaporin 6 from an anion channel to a water-selective channel by a single amino acid substitution.

    PubMed

    Liu, Kun; Kozono, David; Kato, Yasuhiro; Agre, Peter; Hazama, Akihiro; Yasui, Masato

    2005-02-08

    Aquaporin (AQP) 6 belongs to the aquaporin water channel family. Unlike other aquaporins, AQP6 functions not as a water channel but as an anion-selective channel. Single-channel analyses have shown AQP6 to flicker rapidly between closed and open status. The atomic structure of AQP1 and amino acid sequence alignments of the mammalian aquaporins reveal two well conserved glycine residues: Gly-57 in transmembrane helix (TM) 2 and Gly-173 in TM5 reside at the contact point where the two helices cross in human AQP1. Uniquely, all known mammalian orthologs of AQP6 have an asparagine residue (Asn-60) at the position corresponding to Gly-57. Here we show that a single residue substitution (N60G in rat AQP6) totally eliminates the anion permeability of AQP6 when expressed in Xenopus oocytes, but the N60G oocytes exhibit significantly higher osmotic water permeability under basal conditions. Replacement of the glycine at this site in AQP0, AQP1, and AQP2 blocked expression of the mutants at the oocyte plasma membrane. We propose that the asparagine residue at the contact point between TM2 and TM5 in AQP6 may function as a teeter board needed for rapid structural oscillations during anion permeation.

  12. Overexpression of Laccaria bicolor aquaporin JQ585595 alters root water transport properties in ectomycorrhizal white spruce (Picea glauca) seedlings.

    PubMed

    Xu, Hao; Kemppainen, Minna; El Kayal, Walid; Lee, Seong Hee; Pardo, Alejandro G; Cooke, Janice E K; Zwiazek, Janusz J

    2015-01-01

    The contribution of hyphae to water transport in ectomycorrhizal (ECM) white spruce (Picea glauca) seedlings was examined by altering expression of a major water-transporting aquaporin in Laccaria bicolor. Picea glauca was inoculated with wild-type (WT), mock transgenic or L. bicolor aquaporin JQ585595-overexpressing (OE) strains and exposed to root temperatures ranging from 5 to 20°C to examine the root water transport properties, physiological responses and plasma membrane intrinsic protein (PIP) expression in colonized plants. Mycorrhization increased shoot water potential, transpiration, net photosynthetic rates, root hydraulic conductivity and root cortical cell hydraulic conductivity in seedlings. At 20°C, OE plants had higher root hydraulic conductivity compared with WT plants and the increases were accompanied by higher expression of P. glauca PIP GQ03401_M18.1 in roots. In contrast to WT L. bicolor, the effects of OE fungi on root and root cortical cell hydraulic conductivities were abolished at 10 and 5°C in the absence of major changes in the examined transcript levels of P. glauca root PIPs. The results provide evidence for the importance of fungal aquaporins in root water transport of mycorrhizal plants. They also demonstrate links between hyphal water transport, root aquaporin expression and root water transport in ECM plants.

  13. Aquaporin functionality in relation to H+-ATPase activity in root cells of Capsicum annuum grown under salinity.

    PubMed

    Martínez-Ballesta, M. Carmen; Martínez, Vicente; Carvajal, Micaela

    2003-03-01

    As water and nutrient uptake should be related in the response of plants to salinity, the aim of this paper is to establish whether or not aquaporin functionality is related to H+-ATPase activity in root cells of pepper (Capsicum annuum L.) plants. Thus, H+-ATPase activity was measured in plasma membrane vesicles isolated from roots and aquaporin functionality was measured using a cell pressure probe in intact roots. Salinity was applied as 60 mM NaCl or 60 mM KCl, to determine which ion (Na+, K+ or Cl-) is producing the effects. We also investigated whether the effects of both salts were ameliorated by Ca2+. Similar results were obtained for cell hydraulic conductivity, Lpc, and H+-ATPase activity, large reductions in the presence at NaCl or KCl and an ameliorative effect of Ca2+. However, fusicoccin (an activator of H+-ATPase) did not alter osmotic water permeability of protoplasts isolated from roots. Addition of Hg2+ inhibited both ATPase and aquaporins, but ATPase also contains Hg-binding sites. Therefore, the results indicate that H+-ATPase and aquaporin activities may not be related in pepper plants.

  14. Inhibition of microbial growth on chitosan membranes by plasma treatment.

    PubMed

    de Oliveira Cardoso Macêdo, Marina; de Macêdo, Haroldo Reis Alves; Gomes, Dayanne Lopes; de Freitas Daudt, Natália; Rocha, Hugo Alexandre Oliveira; Alves, Clodomiro

    2013-11-01

    The use of polymeric medical devices has stimulated the development of new sterilization methods. The traditional techniques rely on ethylene oxide, but there are many questions concerning the carcinogenic properties of the ethylene oxide residues adsorbed on the materials after processing. Another common technique is the gamma irradiation process, but it is costly, its safe operation requires an isolated site, and it also affects the bulk properties of the polymers. The use of gas plasma is an elegant alternative sterilization technique. The plasma promotes efficient inactivation of the microorganisms, minimizes damage to the materials, and presents very little danger for personnel and the environment. In this study we used plasma for microbial inhibition of chitosan membranes. The membranes were treated with oxygen, methane, or argon plasma for different time periods (15, 30, 45, or 60 min). For inhibition of microbial growth with oxygen plasma, the time needed was 60 min. For the methane plasma, samples were successfully treated after 30, 45, and 60 min. For argon plasma, all treatment periods were effective. © 2013 Wiley Periodicals, Inc. and International Center for Artificial Organs and Transplantation.

  15. Mammalian gamete plasma membranes re-assessments and reproductive implications

    USDA-ARS?s Scientific Manuscript database

    Establishment of the diploid status occurs with the fusion of female and male gametes. Both the mammalian oocyte and spermatozoa are haploid cells surrounded with plasma membranes that are rich in various proteins playing a crucial role during fertilization. Fertilization is a complex and ordered st...

  16. 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.

  17. Exclusive photorelease of signalling lipids at the plasma membrane

    PubMed Central

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

    2015-01-01

    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. PMID:26686736

  18. Selective association of lipoprotein cholesteryl esters with liver plasma membranes.

    PubMed

    Rinninger, F; Jaeckle, S; Greten, H; Windler, E

    1993-02-24

    High-density lipoprotein (HDL) cholesteryl esters are taken up by hepatocytes without parallel uptake of HDL apolipoproteins. This selective uptake of HDL cholesteryl esters is mediated by a non-endocytotic mechanism. Recently, selective uptake of cholesteryl esters also from low-density lipoprotein (LDL) was demonstrated. In this study, the role of the plasma membrane in selective uptake by the liver was investigated. Plasma membranes were prepared from rat liver or from human Hep G2 hepatoma cells. Human HDL3 (d = 1.125-1.21 g/ml) was either radioiodinated or labeled with [3H]cholesteryl oleate. Human low-density lipoprotein (d = 1.019-1.05 g/ml) was labeled in its protein and in its lipid moiety as well. Labeled lipoproteins, unlabeled lipoproteins and membranes were incubated. After separation by ultracentrifugation, apparent lipoprotein particle association with membranes was determined. Plasma membranes from rat liver and Hep G2 cells bound 125I-HDL3, indicating specific HDL3 particle binding. With both types of membrane, apparent HDL3 particle association according to [3H]cholesteryl oleate-labeled HDL3 was in significant excess on that due to 125I-HDL3. This indicates selective, i.e., particle binding independent, association of cholesteryl esters with the membrane. Excess unlabeled HDL3 competed for selective association, indicating a specific process. Selective association of HDL3 cholesteryl esters was concentration-, time-, temperature-dependent; however, parameters differed from HDL3 particle binding. HDL3 was modified by nitration; this modification inhibited HDL3 particle binding in contrast to unchanged selective association. These results suggested distinct membrane sites for HDL3 particle binding and selective cholesteryl ester association. Regulation of selective association was investigated. Hep G2 cells were cholesterol-loaded or cholesterol-depleted. Cellular cholesterol-loading down-regulated selective association of HDL3 cholesteryl esters

  19. Aquaporin-6: An intracellular vesicle water channel protein in renal epithelia.

    PubMed

    Yasui, M; Kwon, T H; Knepper, M A; Nielsen, S; Agre, P

    1999-05-11

    All characterized mammalian aquaporins (AQPs) are localized to plasma membranes where they function chiefly to mediate water transport across cells. Here we show that AQP6 is localized exclusively in intracellular membranes in renal epithelia. By using a polyclonal antibody to the C terminus of AQP6, immunoblots revealed a major 30-kDa band in membranes from rat renal cortex and medulla. Endoglycosidase treatment demonstrated presence of an intracellular high mannose glycan on each subunit. Sequential ultracentrifugation of rat kidney homogenates confirmed that AQP6 resides predominantly in vesicular fractions, and immunohistochemical and immunoelectron microscopic studies confirmed that >98% of AQP6 is located in intracellular membrane vesicles. In glomeruli, AQP6 is present in membrane vesicles within podocyte cell bodies and foot processes. In proximal tubules, AQP6 is also abundant in membrane vesicles within the subapical compartment of segment 2 and segment 3 cells, but was not detected in the brush border or basolateral membranes. In collecting duct, AQP6 resides in intracellular membrane vesicles in apical, mid, and basolateral cytoplasm of type A intercalated cells, but was not observed in the plasma membrane. Unlike other members of the AQP family, the unique distribution in intracellular membrane vesicles in multiple types of renal epithelia indicates that AQP6 is not simply involved in transcellular fluid absorption. Moreover, our studies predict that AQP6 participates in distinct physiological functions such as glomerular filtration, tubular endocytosis, and acid-base metabolism.

  20. Aquaporin-6: An intracellular vesicle water channel protein in renal epithelia

    PubMed Central

    Yasui, Masato; Kwon, Tae-Hwan; Knepper, Mark A.; Nielsen, Søren; Agre, Peter

    1999-01-01

    All characterized mammalian aquaporins (AQPs) are localized to plasma membranes where they function chiefly to mediate water transport across cells. Here we show that AQP6 is localized exclusively in intracellular membranes in renal epithelia. By using a polyclonal antibody to the C terminus of AQP6, immunoblots revealed a major 30-kDa band in membranes from rat renal cortex and medulla. Endoglycosidase treatment demonstrated presence of an intracellular high mannose glycan on each subunit. Sequential ultracentrifugation of rat kidney homogenates confirmed that AQP6 resides predominantly in vesicular fractions, and immunohistochemical and immunoelectron microscopic studies confirmed that >98% of AQP6 is located in intracellular membrane vesicles. In glomeruli, AQP6 is present in membrane vesicles within podocyte cell bodies and foot processes. In proximal tubules, AQP6 is also abundant in membrane vesicles within the subapical compartment of segment 2 and segment 3 cells, but was not detected in the brush border or basolateral membranes. In collecting duct, AQP6 resides in intracellular membrane vesicles in apical, mid, and basolateral cytoplasm of type A intercalated cells, but was not observed in the plasma membrane. Unlike other members of the AQP family, the unique distribution in intracellular membrane vesicles in multiple types of renal epithelia indicates that AQP6 is not simply involved in transcellular fluid absorption. Moreover, our studies predict that AQP6 participates in distinct physiological functions such as glomerular filtration, tubular endocytosis, and acid-base metabolism. PMID:10318966

  1. 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.

  2. 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.

  3. Biochemical properties of platelet microparticle membranes formed during exocytosis resemble organelles more than plasma membrane.

    PubMed

    Olas, Beata; Lundell, Kerstin; Holmsen, Holm; Fukami, Miriam H

    2002-08-14

    Studies of [3H]glycerol turnover in phosphatidylcholine (PC) in platelets revealed two metabolic pools, a 'low turnover PC' in collagen-induced microparticles with specific radioactivity only 10% of that found in the 'high turnover PC' of bulk platelet PC. Isolated organelle fractions of [3H]glycerol-labelled platelets contained [3H]PC with specific radioactivities about 20% of that in membrane fractions. These results together with studies on distribution of concanavalin A-FITC and GPlb, a plasma membrane receptor, indicate that microparticles formed during exocytosis are not simple vesiculations of plasma membrane, but they seem rather to originate from a relatively metabolically static membrane pool not accessible to extracellular reagents.

  4. The aquaporin TcAQP1 of the desert truffle Terfezia claveryi is a membrane pore for water and CO(2) transport.

    PubMed

    Navarro-Ródenas, Alfonso; Ruíz-Lozano, Juan Manuel; Kaldenhoff, Ralf; Morte, Asunción

    2012-02-01

    Terfezia claveryi is a hypogeous mycorrhizal fungus belonging to the so-called "desert truffles," with a good record as an edible fungus and of considerable economic importance. T. claveryi improves the tolerance to water stress of the host plant Helianthemum almeriense, for which, in field conditions, symbiosis with T. claveryi is valuable for its survival. We have characterized cDNAs from T. claveryi and identified a sequence related to the aquaporin gene family. The full-length sequence was obtained by rapid amplification of cDNA ends and was named TcAQP1. This aquaporin gene encoded a functional water-channel protein, as demonstrated by heterologous expression assays in Saccharomyces cerevisiae. The mycorrhizal fungal aquaporin increased both water and CO(2) conductivity in the heterologous expression system. The expression patterns of the TcAQP1 gene in mycelium, under different water potentials, and in mycorrhizal plants are discussed. The high levels of water conductivity of TcAQP1 could be related to the adaptation of this mycorrhizal fungus to semiarid areas. The CO(2) permeability of TcAQP1 could be involved in the regulation of T. claveryi growth during presymbiotic phases, making it a good candidate to be considered a novel molecular signaling channel in mycorrhizal fungi.

  5. Hierarchical organization of the plasma membrane: investigations by single-molecule tracking vs. fluorescence correlation spectroscopy.

    PubMed

    Kusumi, Akihiro; Shirai, Yuki M; Koyama-Honda, Ikuko; Suzuki, Kenichi G N; Fujiwara, Takahiro K

    2010-05-03

    Single-molecule tracking and fluorescence correlation spectroscopy (FCS) applied to the plasma membrane in living cells have allowed a number of unprecedented observations, thus fostering a new basic understanding of molecular diffusion, interaction, and signal transduction in the plasma membrane. It is becoming clear that the plasma membrane is a heterogeneous entity, containing diverse structures on nano-meso-scales (2-200 nm) with a variety of lifetimes, where certain membrane molecules stay together for limited durations. Molecular interactions occur in the time-dependent inhomogeneous two-dimensional liquid of the plasma membrane, which might be a key for plasma membrane functions.

  6. High Expression of the Tonoplast Aquaporin ZmTIP1 in Epidermal and Conducting Tissues of Maize1

    PubMed Central

    Barrieu, François; Chaumont, François; Chrispeels, Maarten J.

    1998-01-01

    Aquaporins are integral membrane proteins of the tonoplast and the plasma membrane that facilitate the passage of water through these membranes. Because of their potentially important role in regulating water flow in plants, studies documenting aquaporin gene expression in specialized tissues involved in water and solute transport are important. We used in situ hybridization to examine the expression pattern of the tonoplast aquaporin ZmTIP1 in different organs of maize (Zea mays L.). This tonoplast water channel is highly expressed in the root epidermis, the root endodermis, the small parenchyma cells surrounding mature xylem vessels in the root and the stem, phloem companion cells and a ring of cells around the phloem strand in the stem and the leaf sheath, and the basal endosperm transfer cells in developing kernels. We postulate that the high level of expression of ZmTIP1 in these tissues facilitates rapid flow of water through the tonoplast to permit osmotic equilibration between the cytosol and the vacuolar content, and to permit rapid transcellular water flow through living cells when required. PMID:9701571

  7. Drought, salt and wounding stress induce the expression of the plasma membrane intrinsic protein 1 gene in poplar (Populus alba×P. tremula var. glandulosa).

    PubMed

    Bae, Eun-Kyung; Lee, Hyoshin; Lee, Jae-Soon; Noh, Eun-Woon

    2011-09-01

    Water uptake across cell membranes is a principal requirement for plant growth at both the cellular and whole-plant levels; water movement through plant membranes is regulated by aquaporins (AQPs) or major intrinsic proteins (MIPs). We examined the expression characteristics of the poplar plasma membrane intrinsic protein 1 gene (PatPIP1), a type of MIP, which was isolated from a suspension cell cDNA library of Populus alba×P. tremula var. glandulosa. Examination of protoplasts expressing the p35S-PatPIP1::sGFP fusion protein revealed that the protein was localized in the plasma membrane. Northern blot analysis revealed that the gene was strongly expressed in poplar roots and leaves. Gene expression was inducible by abiotic factors including drought, salinity, cold temperatures and wounding, and also by plant hormones including gibberellic acid, jasmonic acid and salicylic acid. Since we found that the PatPIP1 gene was strongly expressed in response to mannitol, NaCl, jasmonic acid and wounding, we propose that PatPIP1 plays an essential role in the defense of plants against water stress.

  8. Expression of multiple AQP4 pools in the plasma membrane and their association with the dystrophin complex.

    PubMed

    Nicchia, Grazia Paola; Cogotzi, Laura; Rossi, Andrea; Basco, Davide; Brancaccio, Andrea; Svelto, Maria; Frigeri, Antonio

    2008-06-01

    Altered aquaporin-4 (AQP4) expression has been reported in brain edema, tumors, muscular dystrophy, and neuromyelitis optica. However, the plasma membrane organization of AQP4 and its interaction with proteins such as the dystrophin-associated protein complex are not well understood. In this study, we used sucrose density gradient ultracentrifugation and 2D blue native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed the expression of several AQP4 multi-subunit complexes (pools) of different sizes, ranging from > 1 MDa to approximately 500 kDa and containing different ratios of the 30/32 kDa AQP4 isoforms, indicative of orthogonal arrays of particles of various sizes. A high molecular weight pool co-purified with dystrophin and beta-dystroglycan and was drastically reduced in the skeletal muscle of mdx3cv mice, which have no dystrophin. The number and size of the AQP4 pools were the same in the kidney where dystrophin is not expressed, suggesting the presence of dystrophin-like proteins for their expression. We found that AQP2 is expressed only in one major pool of approximately 500 kDa, indicating that the presence of different pools is a peculiarity of AQP4 rather than a widespread feature in the AQP family. Finally, in skeletal muscle caveolin-3 did not co-purify with any AQP4 pool, indicating the absence of interaction of the two proteins and confirming that caveolae and orthogonal arrays of particles are two independent plasma membrane microdomains. These results contribute to a better understanding of AQP4 membrane organization and raise the possibility that abnormal expression of specific AQP4 pools may be found in pathological states.

  9. Lipid signalling dynamics at the β-cell plasma membrane.

    PubMed

    Wuttke, Anne

    2015-04-01

    Pancreatic β-cells are clustered in islets of Langerhans and secrete insulin in response to increased concentrations of circulating glucose. Insulin in turn acts on liver, muscle and fat tissue to store energy and normalize the blood glucose level. Inappropriate insulin release may lead to impaired glucose tolerance and diabetes. In addition to glucose, other nutrients, neural stimuli and hormonal stimuli control insulin secretion. Many of these signals are perceived at the plasma membrane, which is also the site where insulin granules undergo exocytosis. Therefore, it is not surprising that membrane lipids play an important role in the regulation of insulin secretion. β-cells release insulin in a pulsatile fashion. Signalling lipids integrate the nutrient and neurohormonal inputs to fine-tune, shape and co-ordinate the pulsatility. An important group of signalling lipids are phosphoinositides and their downstream messengers. This MiniReview will discuss new insights into lipid signalling dynamics in β-cells obtained from live-cell imaging experiments with fluorescent translocation biosensors. The plasma membrane concentration of several phosphoinositides and of their downstream messengers changes rapidly upon nutrient or neurohormonal stimulation. Glucose induces the most complex spatio-temporal patterns, typically involving oscillations of messenger concentrations, which sometimes are locally restricted. The tightly controlled levels of lipid messengers can mediate specific binding of downstream effectors to the plasma membrane, contributing to the appropriate regulation of insulin secretion. © 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

  10. Active transport of calcium in Neurospora plasma membrane vesicles.

    PubMed Central

    Stroobant, P; Scarborough, G A

    1979-01-01

    Functionally inverted plasma membrane vesicles isolated from the eukaryotic microorganism Neurospora crassa catalyze Mg2+/ATP-dependent Ca2+ uptake. Inhibitors induced efflux studies and isotope-exchange experiments indicate that the Ca2+ is accumulated inside the vesicles against a concentration gradient of about 40-fold, and that the majority of the transported Ca2+ is present essentially in free solution. Comparisons of Mg2+/ATP-driven 45Ca2+ uptake and [14C]SCN-uptake with respect to the Mg2+/ATP concentration dependence, the effects of inhibitors, and the nucleotide and divalent cation specificities indicate that the energy for Ca2+ accumulation is derived from ATP hydrolysis catalyzed by the electrogenic plasma membrane ATPase. Energized Ca2+ uptake is stimulated by the permeant anion SCN- to a degree that varies reciprocally with the ability of this anion to dissipate the membrane potential, and is inhibited by K+ in the presence of nigericin. All of these data point to the conclusion that the active transport of Ca2+ across the Neurospora plasma membrane takes place via a Ca2+/H+ antiporter, which functions to pump Ca2+ out of the intact cell. PMID:40223

  11. A membrane reservoir at the cell surface: unfolding the plasma membrane to fuel cell shape change.

    PubMed

    Figard, Lauren; Sokac, Anna Marie

    2014-01-01

    Cell surface expansion is a necessary part of cell shape change. One long-standing hypothesis proposes that membrane for this expansion comes from the flattening out of cell surface projections such as microvilli and membrane folds. Correlative EM data of cells undergoing phagocytosis, cytokinesis, and morphogenesis has hinted at the existence of such an unfolding mechanism for decades; but unfolding has only recently been confirmed using live-cell imaging and biophysical approaches. Considering the wide range of cells in which plasma membrane unfolding has now been reported, it likely represents a fundamental mechanism of cell shape change.

  12. 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.

  13. Effects of freezing and cold acclimation on the plasma membrane of isolated protoplasts

    SciTech Connect

    Steponkus, P.L.

    1990-01-01

    The goals of this project are to provide a mechanistic understanding of freeze/dehydration induced mesomorphic phase transitions if the plasma membrane of winter cereals. Topics discussed include freezing tolerance, hydration characteristics of plasma membrane lipids force-distance relationships of lipid bilayers, and phase behavior of plasma membrane lipids. (KD)

  14. Spatiotemporal Analysis of Differential Akt Regulation in Plasma Membrane Microdomains

    PubMed Central

    Gao, Xinxin

    2008-01-01

    As a central kinase in the phosphatidylinositol 3-kinase pathway, Akt has been the subject of extensive research; yet, spatiotemporal regulation of Akt in different membrane microdomains remains largely unknown. To examine dynamic Akt activity in membrane microdomains in living cells, we developed a specific and sensitive fluorescence resonance energy transfer-based Akt activity reporter, AktAR, through systematic testing of different substrates and fluorescent proteins. Targeted AktAR reported higher Akt activity with faster activation kinetics within lipid rafts compared with nonraft regions of plasma membrane. Disruption of rafts attenuated platelet-derived growth factor (PDGF)-stimulated Akt activity in rafts without affecting that in nonraft regions. However, in insulin-like growth factor-1 (IGF)-1 stimulation, Akt signaling in nonraft regions is dependent on that in raft regions. As a result, cholesterol depletion diminishes Akt activity in both regions. Thus, Akt activities are differentially regulated in different membrane microdomains, and the overall activity of this oncogenic pathway is dependent on raft function. Given the increased abundance of lipid rafts in some cancer cells, the distinct Akt-activating characteristics of PDGF and IGF-1, in terms of both effectiveness and raft dependence, demonstrate the capabilities of different growth factor signaling pathways to transduce differential oncogenic signals across plasma membrane. PMID:18701703

  15. Aquaporin-4 orthogonal arrays of particles are the target for neuromyelitis optica autoantibodies.

    PubMed

    Nicchia, Grazia Paola; Mastrototaro, Mauro; Rossi, Andrea; Pisani, Francesco; Tortorella, Carla; Ruggieri, Maddalena; Lia, Anna; Trojano, Maria; Frigeri, Antonio; Svelto, Maria

    2009-10-01

    Neuromyelitis optica (NMO) is an inflammatory autoimmune demyelinating disease of the central nervous system (CNS) which in autoantibodies produced by patients with NMO (NMO-IgG) recognize a glial water channel protein, Aquaporin-4 (AQP4) expressed as two major isoforms, M1- and M23-AQP4, in which the plasma membrane form orthogonal arrays of particles (OAPs). AQP4-M23 is the OAP-forming isoform, whereas AQP4-M1 alone is unable to form OAPs. The function of AQP4 organization into OAPs in normal physiology is unknown; however, alteration in OAP assemblies is reported for several CNS pathological states. In this study, we demonstrate that in the CNS, NMO-IgG is able to pull down both M1- and M23-AQP4 but experiments performed using cells selectively transfected with M1- or M23-AQP4 and native tissues show NMO-IgG epitope to be intrinsic in AQP4 assemblies into OAPs. Other OAP-forming water-channel proteins, such as the lens Aquaporin-0 and the insect Aquaporin-cic, were not recognized by NMO-IgG, indicating an epitope characteristic of AQP4-OAPs. Finally, water transport measurements show that NMO-IgG treatment does not significantly affect AQP4 function. In conclusion, our results suggest for the first time that OAP assemblies are required for NMO-IgG to recognize AQP4.

  16. Chloroquine accumulation by purified plasma membranes from Plasmodium falciparum.

    PubMed

    Elandaloussi, Laurence M; Smith, Peter J

    2006-01-01

    Resistance of Plasmodium falciparum to chloroquine (CQ) has been associated with a decrease in CQ accumulation by parasitized erythrocytes. This study aimed at investigating the role of parasite plasma membranes (PPM) in the mechanism of CQ accumulation. CQ accumulation capabilities of membranes were determined using tritiated CQ. PPM isolated from chloroquine-sensitive parasites were found to accumulate less CQ than those isolated from chloroquine-resistant parasites. However, CQ accumulation was found to be ATP-independent suggesting that this accumulation results from binding rather than transport.

  17. Aquaporin water channels – from atomic structure to clinical medicine

    PubMed Central

    Agre, Peter; King, Landon S; Yasui, Masato; Guggino, Wm B; Ottersen, Ole Petter; Fujiyoshi, Yoshinori; Engel, Andreas; Nielsen, Søren

    2002-01-01

    The water permeability of biological membranes has been a longstanding problem in physiology, but the proteins responsible for this remained unknown until discovery of the aquaporin 1 (AQP1) water channel protein. AQP1 is selectively permeated by water driven by osmotic gradients. The atomic structure of human AQP1 has recently been defined. Each subunit of the tetramer contains an individual aqueous pore that permits single-file passage of water molecules but interrupts the hydrogen bonding needed for passage of protons. At least 10 mammalian aquaporins have been identified, and these are selectively permeated by water (aquaporins) or water plus glycerol (aquaglyceroporins). The sites of expression coincide closely with the clinical phenotypes – ranging from congenital cataracts to nephrogenic diabetes insipidus. More than 200 members of the aquaporin family have been found in plants, microbials, invertebrates and vertebrates, and their importance to the physiology of these organisms is being uncovered. PMID:12096044

  18. Modulation of Erythrocyte Plasma Membrane Redox System Activity by Curcumin

    PubMed Central

    Singh, Prabhakar; Kesharwani, Rajesh Kumar; Misra, Krishna; Rizvi, Syed Ibrahim

    2016-01-01

    Plasma membrane redox system (PMRS) is an electron transport chain system ubiquitously present throughout all cell types. It transfers electron from intracellular substrates to extracellular acceptors for regulation of redox status. Curcumin, isolated from Curcuma longa, has modulatory effects on cellular physiology due to its membrane interaction ability and antioxidant potential. The present study investigates the effect of curcumin on PMRS activity of erythrocytes isolated from Wistar rats in vitro and in vivo and validated through an in silico docking simulation study using Molegro Virtual Docker (MVD). Effects of curcumin were also evaluated on level of glutathione (GSH) and the oxidant potential of plasma measured in terms of plasma ferric equivalent oxidative potentials (PFEOP). Results show that curcumin significantly (p < 0.01) downregulated the PMRS activity in a dose-dependent manner. Molecular docking results suggest that curcumin interacts with amino acids at the active site cavity of cytochrome b5 reductase, a key constituent of PMRS. Curcumin also increased the GSH level in erythrocytes and plasma while simultaneously decreasing the oxidant potential (PFEOP) of plasma. Altered PMRS activity and redox status are associated with the pathophysiology of several health complications including aging and diabetes; hence, the above finding may explain part of the role of curcumin in health beneficial effects. PMID:26904287

  19. 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).

  20. 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.

  1. THE RELATIONS OF THE PLASMA MEMBRANE, VITELLINE MEMBRANE, AND JELLY IN THE EGG OF NEREIS LIMBATA

    PubMed Central

    Costello, Donald P.

    1949-01-01

    1. The problem of the relation of the plasma membrane to the extraneous coats and cortex of the Nereis egg is discussed in the light of the observations of Lillie, Chambers, and Novikoff. 2. Evidence obtained from experiments with the centrifuge, and by treating eggs with alkaline sodium chloride, indicates that the plasma membrane of the unfertilized egg is external to the jelly precursor granules of the cortex. 3. Experiments with alkaline sodium chloride indicate that the perivitelline space of the fertilized egg is extraovular after jelly extrusion is complete. 4. The cortical behavior (membrane elevation) of the Nereis egg in alkaline sodium chloride and the cortical response (jelly extrusion) following activation of the egg in normal fertilization or parthenogenesis are attributed largely to the properties of the jelly, and presumably, to its reactions with calcium and hydroxyl ions. PMID:18123313

  2. Plasma-membrane calcium pumps and hereditary deafness.

    PubMed

    Brini, M; Di Leva, F; Domi, T; Fedrizzi, L; Lim, D; Carafoli, E

    2007-11-01

    In mammals, four different genes encode four PMCA (plasma-membrane Ca(2+)-ATPase) isoforms. PMCA1 and 4 are expressed ubiquitously, and PMCA2 and 3 are expressed predominantly in the central nervous system. More than 30 variants are generated by mechanisms of alternative splicing. The physiological meaning of the existence of so many isoforms is not clear, but evidently it must be related to the cell-specific demands of Ca(2+) homoeostasis. Recent studies suggest that the alternatively spliced regions in PMCA are responsible for specific targeting to plasma membrane domains, and proteins that bind specifically to the pumps could contribute to further regulation of Ca(2+) control. In addition, the combination of proteins obtained by alternative splicing occurring at two different sites could be responsible for different functional characteristics of the pumps.

  3. Identification of the family of aquaporin genes and their expression in Upland cotton (Gossypium hirsutum L.)

    USDA-ARS?s Scientific Manuscript database

    One of the primary factors affecting both the quantity and quality of cotton production is water. A major facilitator of water movement through cell membranes of cotton and other plants are the aquaporin proteins. Aquaporin proteins are present as diverse forms in plants, where they function as tran...

  4. Aquaporins in Digestive System.

    PubMed

    Zhu, Shuai; Ran, Jianhua; Yang, Baoxue; Mei, Zhechuan

    2017-01-01

    In this chapter, we mainly discuss the expression and function of aquaporins (AQPs ) expressed in digestive system . AQPs in gastrointestinal tract include four members of aquaporin subfamily: AQP1, AQP4, AQP5 and AQP8, and a member of aquaglyceroporin subfamily: AQP3. In the digestive glands, especially the liver, we discuss three members of aquaporin subfamily: AQP1, AQP5 and AQP8, a member of aquaglyceroporin subfamily: AQP9. AQP3 is involved in the diarrhea and inflammatory bowel disease; AQP5 is relevant to gastric carcinoma cell proliferation and migration; AQP9 plays considerable role in glycerol metabolism , urea transport and hepatocellular carcinoma. Further investigation is necessary for specific locations and functions of AQPs in digestive system.

  5. Rotation of plasma membrane proteins measured by polarized fluorescence depletion

    NASA Astrophysics Data System (ADS)

    Barisas, B. George; Rahman, Noorul A.; Yoshida, Thomas M.; Roess, Deborah A.

    1990-05-01

    We have implemented a new laser microscopic method, polarized fluorescence depletion (PFD), for measuring the rotational dynamics of functional membrane proteins on individual, microscopically selected cells under physiological conditions. This method combines the long lifetimes of triplet-state probes with the sensitivity of fluorescence detection to measure macromolecular rotational correlation times from 10 microsec to > 1 ms. As examples, the rotational correlation time of Fc receptors (FcR) on the surface of 2H3 rat basophilic leukemia cells is 79.9 4.4 microsec at 4°C when labeled with eosin conjugates of IgE. This value is consistent with the known 100 kDa receptor size. When labeled with intact F4 anti-FcR monoclonal antibody, the rotational correlation time for FcER is increased about 2-fold to 170.8 +/- 6.5 microsec, consistent with receptor dimer formation on the plasma membrane and with the ability of this antibody to form FcER dimers on 2H3 cell surfaces. We have also examined the rotational diffusion of the luteinizing hormone receptor on plasma membranes of small ovine luteal cells. Luteinizing hormone receptors (LHR), when occupied by ovine luteinizing hormone (oLH), have a rotational correlation time of 20.5 +/- 0.1 microsec at 4°C. When occupied by human chorionic gonadotropin (hCG), LHR have a rotational correlation time of 46.2 +/- 0.4 microsec suggesting that binding of hCG triggers additional LHR interactions with plasma membrane proteins. Together these studies suggest the utility of PFD measurements in assessing molecular size and molecular association of membrane proteins on individual cells. Relative advantages of time- and frequency-domain implementations of PFD are also discussed.

  6. Plasma Membrane Lipids and Their Role in Fungal Virulence

    PubMed Central

    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 has 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. PMID:26703191

  7. Analysis of plasma membrane phosphoinositides from fusogenic carrot cells

    SciTech Connect

    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 solvent 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.

  8. Plasma membrane ATPase of red beet forms a phosphorylated intermediate.

    PubMed

    Briskin, D P; Poole, R J

    1983-03-01

    When a plasma membrane-enriched fraction isolated from red beet (Beta vulgaris L.) was incubated in the presence of 40 micromolar [gamma-(32)P] ATP, 40 micromolar MgSO(4) at pH 6.5, a rapidly turning over phosphorylated protein was formed. Phosphorylation of the protein was substrate-specific for ATP, sensitive to diethylstilbestrol and vanadate, but insensitive to azide. When the dephosphorylation reaction was specifically studied, KCl was found to increase the turnover of the phosphorylated protein consistent with its stimulatory effect upon plasma membrane ATPase. The protein-bound phosphate was found to be most stable at a pH between 2 and 3 and under cold temperature, suggesting that the protein phosphate bond was an acyl-phosphate. When the phosphorylated protein was analyzed with lithium dodecyl sulfate gel electrophoresis, a labeled polypeptide with a molecular weight of about 100,000 daltons was observed. Phosphorylation of this polypeptide was rapidly turning over and Mg-dependent. It is concluded that the phosphorylation observed represents a reaction intermediate of the red beet plasma membrane ATPase.

  9. 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.

  10. Inside job: ligand-receptor pharmacology beneath the plasma membrane.

    PubMed

    Babcock, Joseph J; Li, Min

    2013-07-01

    Most drugs acting on the cell surface receptors are membrane permeable and thus able to engage their target proteins in different subcellular compartments. However, these drugs' effects on cell surface receptors have historically been studied on the plasma membrane alone. Increasing evidence suggests that small molecules may also modulate their targeted receptors through membrane trafficking or organelle-localized signaling inside the cell. These additional modes of interaction have been reported for functionally diverse ligands of GPCRs, ion channels, and transporters. Such intracellular drug-target engagements affect cell surface expression. Concurrent intracellular and cell surface signaling may also increase the complexity and therapeutic opportunities of small molecule modulation. Here we discuss examples of ligand-receptor interactions that are present in both intra- and extracellular sites, and the potential therapeutic opportunities presented by this phenomenon.

  11. Super-resolution optical microscopy of lipid plasma membrane dynamics.

    PubMed

    Eggeling, Christian

    2015-01-01

    Plasma membrane dynamics are an important ruler of cellular activity, particularly through the interaction and diffusion dynamics of membrane-embedded proteins and lipids. FCS (fluorescence correlation spectroscopy) on an optical (confocal) microscope is a popular tool for investigating such dynamics. Unfortunately, its full applicability is constrained by the limited spatial resolution of a conventional optical microscope. The present chapter depicts the combination of optical super-resolution STED (stimulated emission depletion) microscopy with FCS, and why it is an important tool for investigating molecular membrane dynamics in living cells. Compared with conventional FCS, the STED-FCS approach demonstrates an improved possibility to distinguish free from anomalous molecular diffusion, and thus to give new insights into lipid-protein interactions and the traditional lipid 'raft' theory.

  12. TRPM7 facilitates cholinergic vesicle fusion with the plasma membrane.

    PubMed

    Brauchi, Sebastian; Krapivinsky, Grigory; Krapivinsky, Luba; Clapham, David E

    2008-06-17

    TRPM7, of the transient receptor potential (TRP) family, is both an ion channel and a kinase. Previously, we showed that TRPM7 is located in the membranes of acetylcholine (ACh)-secreting synaptic vesicles of sympathetic neurons, forms a molecular complex with proteins of the vesicular fusion machinery, and is critical for stimulated neurotransmitter release. Here, we targeted pHluorin to small synaptic-like vesicles (SSLV) in PC12 cells and demonstrate that it can serve as a single-vesicle plasma membrane fusion reporter. In PC12 cells, as in sympathetic neurons, TRPM7 is located in ACh-secreting SSLVs. TRPM7 knockdown by siRNA, or abolishing channel activity by expression of a dominant negative TRPM7 pore mutant, decreased the frequency of spontaneous and voltage-stimulated SSLV fusion events without affecting large dense core vesicle secretion. We conclude that the conductance of TRPM7 across the vesicle membrane is important in SSLV fusion.

  13. Role of plasma membrane transporters in muscle metabolism.

    PubMed Central

    Zorzano, A; Fandos, C; Palacín, M

    2000-01-01

    Muscle plays a major role in metabolism. Thus it is a major glucose-utilizing tissue in the absorptive state, and changes in muscle insulin-stimulated glucose uptake alter whole-body glucose disposal. In some conditions, muscle preferentially uses lipid substrates, such as fatty acids or ketone bodies. Furthermore, muscle is the main reservoir of amino acids and protein. The activity of many different plasma membrane transporters, such as glucose carriers and transporters of carnitine, creatine and amino acids, play a crucial role in muscle metabolism by catalysing the influx or the efflux of substrates across the cell surface. In some cases, the membrane transport process is subjected to intense regulatory control and may become a potential pharmacological target, as is the case with the glucose transporter GLUT4. The goal of this review is the molecular characterization of muscle membrane transporter proteins, as well as the analysis of their possible regulatory role. PMID:10903126

  14. Inside job: ligand-receptor pharmacology beneath the plasma membrane

    PubMed Central

    Babcock, Joseph J; Li, Min

    2013-01-01

    Most drugs acting on the cell surface receptors are membrane permeable and thus able to engage their target proteins in different subcellular compartments. However, these drugs' effects on cell surface receptors have historically been studied on the plasma membrane alone. Increasing evidence suggests that small molecules may also modulate their targeted receptors through membrane trafficking or organelle-localized signaling inside the cell. These additional modes of interaction have been reported for functionally diverse ligands of GPCRs, ion channels, and transporters. Such intracellular drug-target engagements affect cell surface expression. Concurrent intracellular and cell surface signaling may also increase the complexity and therapeutic opportunities of small molecule modulation. Here we discuss examples of ligand-receptor interactions that are present in both intra- and extracellular sites, and the potential therapeutic opportunities presented by this phenomenon. PMID:23685953

  15. Plasma membrane microdomains regulate turnover of transport proteins in yeast

    PubMed Central

    Grossmann, Guido; Malinsky, Jan; Stahlschmidt, Wiebke; Loibl, Martin; Weig-Meckl, Ina; Frommer, Wolf B.; Opekarová, Miroslava; Tanner, Widmar

    2008-01-01

    In this study, we investigate whether the stable segregation of proteins and lipids within the yeast plasma membrane serves a particular biological function. We show that 21 proteins cluster within or associate with the ergosterol-rich membrane compartment of Can1 (MCC). However, proteins of the endocytic machinery are excluded from MCC. In a screen, we identified 28 genes affecting MCC appearance and found that genes involved in lipid biosynthesis and vesicle transport are significantly overrepresented. Deletion of Pil1, a component of eisosomes, or of Nce102, an integral membrane protein of MCC, results in the dissipation of all MCC markers. These deletion mutants also show accelerated endocytosis of MCC-resident permeases Can1 and Fur4. Our data suggest that release from MCC makes these proteins accessible to the endocytic machinery. Addition of arginine to wild-type cells leads to a similar redistribution and increased turnover of Can1. Thus, MCC represents a protective area within the plasma membrane to control turnover of transport proteins. PMID:19064668

  16. Plasma membrane wounding and repair in pulmonary diseases.

    PubMed

    Cong, Xiaofei; Hubmayr, Rolf D; Li, Changgong; Zhao, Xiaoli

    2017-03-01

    Various pathophysiological conditions such as surfactant dysfunction, mechanical ventilation, inflammation, pathogen products, environmental exposures, and gastric acid aspiration stress lung cells, and the compromise of plasma membranes occurs as a result. The mechanisms necessary for cells to repair plasma membrane defects have been extensively investigated in the last two decades, and some of these key repair mechanisms are also shown to occur following lung cell injury. Because it was theorized that lung wounding and repair are involved in the pathogenesis of acute respiratory distress syndrome (ARDS) and idiopathic pulmonary fibrosis (IPF), in this review, we summarized the experimental evidence of lung cell injury in these two devastating syndromes and discuss relevant genetic, physical, and biological injury mechanisms, as well as mechanisms used by lung cells for cell survival and membrane repair. Finally, we discuss relevant signaling pathways that may be activated by chronic or repeated lung cell injury as an extension of our cell injury and repair focus in this review. We hope that a holistic view of injurious stimuli relevant for ARDS and IPF could lead to updated experimental models. In addition, parallel discussion of membrane repair mechanisms in lung cells and injury-activated signaling pathways would encourage research to bridge gaps in current knowledge. Indeed, deep understanding of lung cell wounding and repair, and discovery of relevant repair moieties for lung cells, should inspire the development of new therapies that are likely preventive and broadly effective for targeting injurious pulmonary diseases.

  17. Grapevine aquaporins: gating of a tonoplast intrinsic protein (TIP2;1) by cytosolic pH.

    PubMed

    Leitão, Luís; Prista, Catarina; Moura, Teresa F; Loureiro-Dias, Maria C; Soveral, Graça

    2012-01-01

    Grapevine (Vitis vinifera L.) is one of the oldest and most important perennial crops being considered as a fruit ligneous tree model system in which the water status appears crucial for high fruit and wine quality, controlling productivity and alcohol level. V. vinifera genome contains 28 genes coding for aquaporins, which acting in a concerted and regulated manner appear relevant for plant withstanding extremely unfavorable drought conditions essential for the quality of berries and wine. Several Vv aquaporins have been reported to be expressed in roots, shoots, berries and leaves with clear cultivar differences in their expression level, making their in vivo biochemical characterization a difficult task. In this work V. vinifera cv. Touriga nacional VvTnPIP1;1, VvTnPIP2;2 and VvTnTIP2;1 were expressed in yeast and water transport activity was characterized in intact cells of the transformants. The three aquaporins were localized in the yeast plasma membrane but only VvTnTIP2;1 expression enhanced the water permeability with a concomitant decrease of the activation energy of water transport. Acidification of yeast cytosol resulted in loss of VvTnTIP2;1 activity. Sequence analysis revealed the presence of a His(131) residue, unusual in TIPs. By site directed mutagenesis, replacement of this residue by aspartic acid or alanine resulted in loss of pH(in) dependence while replacement by lysine resulted in total loss of activity. In addition to characterization of VvTn aquaporins, these results shed light on the gating of a specific tonoplast aquaporin by cytosolic pH.

  18. Grapevine Aquaporins: Gating of a Tonoplast Intrinsic Protein (TIP2;1) by Cytosolic pH

    PubMed Central

    Leitão, Luís; Prista, Catarina; Moura, Teresa F.; Loureiro-Dias, Maria C.; Soveral, Graça

    2012-01-01

    Grapevine (Vitis vinifera L.) is one of the oldest and most important perennial crops being considered as a fruit ligneous tree model system in which the water status appears crucial for high fruit and wine quality, controlling productivity and alcohol level. V. vinifera genome contains 28 genes coding for aquaporins, which acting in a concerted and regulated manner appear relevant for plant withstanding extremely unfavorable drought conditions essential for the quality of berries and wine. Several Vv aquaporins have been reported to be expressed in roots, shoots, berries and leaves with clear cultivar differences in their expression level, making their in vivo biochemical characterization a difficult task. In this work V. vinifera cv. Touriga nacional VvTnPIP1;1, VvTnPIP2;2 and VvTnTIP2;1 were expressed in yeast and water transport activity was characterized in intact cells of the transformants. The three aquaporins were localized in the yeast plasma membrane but only VvTnTIP2;1 expression enhanced the water permeability with a concomitant decrease of the activation energy of water transport. Acidification of yeast cytosol resulted in loss of VvTnTIP2;1 activity. Sequence analysis revealed the presence of a His131 residue, unusual in TIPs. By site directed mutagenesis, replacement of this residue by aspartic acid or alanine resulted in loss of pHin dependence while replacement by lysine resulted in total loss of activity. In addition to characterization of VvTn aquaporins, these results shed light on the gating of a specific tonoplast aquaporin by cytosolic pH. PMID:22427995

  19. 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

  20. Plasma membrane domains enriched in cortical endoplasmic reticulum function as membrane protein trafficking hubs.

    PubMed

    Fox, Philip D; Haberkorn, Christopher J; Weigel, Aubrey V; Higgins, Jenny L; Akin, Elizabeth J; Kennedy, Matthew J; Krapf, Diego; Tamkun, Michael M

    2013-09-01

    In mammalian cells, the cortical endoplasmic reticulum (cER) is a network of tubules and cisterns that lie in close apposition to the plasma membrane (PM). We provide evidence that PM domains enriched in underlying cER function as trafficking hubs for insertion and removal of PM proteins in HEK 293 cells. By simultaneously visualizing cER and various transmembrane protein cargoes with total internal reflectance fluorescence microscopy, we demonstrate that the majority of exocytotic delivery events for a recycled membrane protein or for a membrane protein being delivered to the PM for the first time occur at regions enriched in cER. Likewise, we observed recurring clathrin clusters and functional endocytosis of PM proteins preferentially at the cER-enriched regions. Thus the cER network serves to organize the molecular machinery for both insertion and removal of cell surface proteins, highlighting a novel role for these unique cellular microdomains in membrane trafficking.

  1. Aquaporins in development -- a review.

    PubMed

    Liu, Huishu; Wintour, E Marelyn

    2005-05-11

    Water homeostasis during fetal development is of crucial physiologic importance. It depends upon maternal fetal fluid exchange at the placenta and fetal membranes, and some exchange between fetus and amniotic fluid can occur across the skin before full keratinization. Lungs only grow and develop normally with fluid secretion, and there is evidence that cerebral spinal fluid formation is important in normal brain development. The aquaporins are a growing family of molecular water channels, the ontogeny of which is starting to be explored. One question that is of particular importance is how well does the rodent (mouse, rat) fetus serve as a model for long-gestation mammals such as sheep and human? This is particularly important for organs such as the lung and the kidney, whose development before birth is very much less in rodents than in the long-gestation species.

  2. Selectivity of alkali cation influx across the plasma membrane of oat roots: cation specificity of the plasma membrane ATPase.

    PubMed

    Sze, H; Hodges, T K

    1977-04-01

    Influx of alkali cations (Li(+), Na(+), K(+), Rb(+), Cs(+)) across plasma membranes of cells of excised roots of Avena sativa cv. Goodfield was selective, but different, in the absence and in the presence of 1 mm CaSO(4). Ca(2+) reduced the influx rates of all of the alkali cations-especially Na(+) and Li(+). Transport selectivity changed as the external concentrations of the alkali cations increased.Plasma membrane ATPase, purified from Avena sativa roots, was differentially stimulated by alkali cations. This specificity, however, was not altered by Ca(2+) or the external cation concentrations. A close correspondence existed between the relative influx rates of K(+), Rb(+), and Cs(+) and the relative stimulation of the ATPase by these cations. A similar correspondence did not occur for Na(+) and Li(+).Selective cation transport in oat roots could result, in part, from the specificity of the plasma membrane ATPase, but other factors such as specific carriers or porters or differential diffusion rates must also be involved.

  3. 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.

  4. Magnetic bead isolation of neutrophil plasma membranes and quantification of membrane-associated guanine nucleotide binding proteins.

    PubMed

    Chang, Peter S; Absood, Afaf; Linderman, Jennifer J; Omann, Geneva M

    2004-02-15

    A protocol for isolation of neutrophil plasma membranes utilizing a plasma membrane marker antibody, anti-CD15, attached to superparamagnetic beads was developed. Cells were initially disrupted by nitrogen cavitation and then incubated with anti-CD15 antibody-conjugated superparamagnetic beads. The beads were then washed to remove unbound cellular debris and cytosol. Recovered plasma membranes were quantified by immunodetection of G(beta2) in Western blots. This membrane marker-based separation yielded highly pure plasma membranes. This protocol has advantages over standard density sedimentation protocols for isolating plasma membrane in that it is faster and easily accommodates cell numbers as low as 10(6). These methods were coupled with immunodetection methods and an adenosine 5(')-diphosphate-ribosylation assay to measure the amount of membrane-associated G(ialpha) proteins available for receptor coupling in neutrophils either stimulated with N-formyl peptides or treated to differing degrees with pertussis toxin. As expected, pertussis toxin treatment decreased the amount of membrane G protein available for signaling although total membrane G protein was not affected. In addition, activation of neutrophils with N-formyl peptides resulted in an approximately 50% decrease in G protein associated with the plasma membrane.

  5. Isolation of Synaptosomes, Synaptic Plasma Membranes, and Synaptic Junctional Complexes.

    PubMed

    Michaelis, Mary L; Jiang, Lei; Michaelis, Elias K

    2017-01-01

    Isolation of synaptic nerve terminals or synaptosomes provides an opportunity to study the process of neurotransmission at many levels and with a variety of approaches. For example, structural features of the synaptic terminals and the organelles within them, such as synaptic vesicles and mitochondria, have been elucidated with electron microscopy. The postsynaptic membranes are joined to the presynaptic "active zone" of transmitter release through cell adhesion molecules and remain attached throughout the isolation of synaptosomes. These "post synaptic densities" or "PSDs" contain the receptors for the transmitters released from the nerve terminals and can easily be seen with electron microscopy. Biochemical and cell biological studies with synaptosomes have revealed which proteins and lipids are most actively involved in synaptic release of neurotransmitters. The functional properties of the nerve terminals, such as responses to depolarization and the uptake or release of signaling molecules, have also been characterized through the use of fluorescent dyes, tagged transmitters, and transporter substrates. In addition, isolated synaptosomes can serve as the starting material for the isolation of relatively pure synaptic plasma membranes (SPMs) that are devoid of organelles from the internal environment of the nerve terminal, such as mitochondria and synaptic vesicles. The isolated SPMs can reseal and form vesicular structures in which transport of ions such as sodium and calcium, as well as solutes such as neurotransmitters can be studied. The PSDs also remain associated with the presynaptic membranes during isolation of SPM fractions, making it possible to isolate the synaptic junctional complexes (SJCs) devoid of the rest of the plasma membranes of the nerve terminals and postsynaptic membrane components. Isolated SJCs can be used to identify the proteins that constitute this highly specialized region of neurons. In this chapter, we describe the steps involved

  6. 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.

  7. Controlled Proteolysis Activates the Plasma Membrane Ca2+ Pump of Higher Plants (A Comparison with the Effect of Calmodulin in Plasma Membrane from Radish Seedlings).

    PubMed Central

    Rasi-Caldogno, F.; Carnelli, A.; De Michelis, M. I.

    1993-01-01

    The effects of calmodulin and of controlled trypsin treatments on the activity of the Ca2+ pump were investigated in plasma membrane purified from radish (Raphanus sativus L.) seedlings. Treatment of the plasma membrane with ethylenediaminetetra-acetate (EDTA), which removed about two-thirds of the plasma membrane-associated calmodulin, markedly increased the stimulation of the Ca2+ pump by calmodulin. In EDTA-treated plasma membrane, stimulation by calmodulin of the Ca2+ pump activity was maximal at low free Ca2+ (2-5 [mu]M) and decreased with the increase of free Ca2+ concentration. The Ca2+ pump activity was stimulated also by a controlled treatment of the plasma membrane with trypsin: the effect of trypsin treatment depended on the concentration of both trypsin and plasma membrane proteins and on the duration of incubation. Stimulation of the Ca2+ pump activity by trypsin treatment of the plasma membrane was similar to that induced by calmodulin both in extent and in dependence on the free Ca2+ concentration in the assay medium. Moreover, the Ca2+ pump of trypsin-treated plasma membrane was insensitive to further stimulation by calmodulin, suggesting that limited proteolysis preferentially cleaves a regulatory domain of the enzyme that is involved in its activation by calmodulin. PMID:12231945

  8. Incorporation of proteins into (Xenopus) oocytes by proteoliposome microinjection: functional characterization of a novel aquaporin.

    PubMed

    Le Cahérec, F; Bron, P; Verbavatz, J M; Garret, A; Morel, G; Cavalier, A; Bonnec, G; Thomas, D; Gouranton, J; Hubert, J F

    1996-06-01

    Xenopus laevis oocytes are widely used as an expression system for plasma membrane proteins, achieved by cytoplasmic microinjection of messenger RNA. In the present study, we propose an alternative system allowing functional insertion of exogenous proteins into the plasma membrane of Xenopus oocytes. We microinjected proteoliposome suspensions into the cytoplasm and then analyzed membrane protein function. The proteins used in this work were members of the MIP family: the human erythrocyte water channel aquaporin 1 (AQP1), the major intrinsic protein (MIP26) from bovine eye lens and a 25 kDa polypeptide (P25) from a water shunting complex found in the digestive tract of an homopteran sap-sucking insect (Cicadella viridis). Proteoliposomes containing either AQP1, MIP26, or P25 were injected into Xenopus oocytes. The subsequent insertion of these proteins into the plasma membrane of oocytes was demonstrated by immunocytochemistry. Oocytes microinjected with either AQP1 or P25-proteoliposomes exhibited significantly increased osmotic membrane water permeabilities (Pf = 3.16 +/- 026 and 4.03 +/- 0.26 x 10(-3) cm/second, respectively) compared to those measured for oocytes injected with liposomes alone or with MIP26-proteoliposomes (Pf = 1.39 +/- 0.07 and 1.44 +/- 0.10 x 10(-3) cm/second, respectively). These effects were inhibited by HgCl2 in a reversible manner. Arrhenius activation energies of water transfer were low when AQP1 or P25 were present in oocyte plasma membranes (Ea = 2.29 and 3.01 kcal/mol, respectively, versus Ea = 11.75 kcal/mol for liposome injected oocytes). The properties observed here for AQP1 are identical to those widely reported following AQP1 cRNA expression in oocytes. From the present study, we conclude that: (1) exogenous plasma membrane proteins incorporated into liposomes and microinjected into the cytoplasm of Xenopus oocytes are subsequently found in the plasma membrane of the oocytes in a functional state; and (2) in this system, the P25

  9. Artificial plasma membrane models based on lipidomic profiling.

    PubMed

    Essaid, Donia; Rosilio, Véronique; Daghildjian, Katia; Solgadi, Audrey; Vergnaud, Juliette; Kasselouri, Athena; Chaminade, Pierre

    2016-11-01

    Phospholipid monolayers are often described as membrane models for analyzing drug-lipid interactions. In many works, a single phosphatidylcholine is chosen, sometimes with one or two additional components. Drug penetration is studied at 30mN/m, a surface pressure considered as corresponding to the pressure in bilayers, independently of the density of lipid molecular packing. In this work, we have extracted, identified, and quantified the major lipids constituting the lipidome of plasma and mitochondrial membranes of retinoblastoma (Y79) and retinal pigment epithelium cells (ARPE-19), using liquid chromatography coupled to high-resolution mass spectrometry (LC-MS/MS). The results obtained from this lipidomic analysis were used in an attempt to build an artificial lipid monolayer with a composition mimicking that of the plasma membrane of Y79 cells, better than a single phospholipid. The variety and number of lipid classes and species in cell extracts monolayers exceeding by far those of the phospholipids chosen to mimic them, the π-A isotherms of model monolayers differed from those of lipid extracts in shape and apparent packing density. We propose a model monolayer based on the most abundant species identified in the extracts, with a surface compressional modulus at 30mN/m close to the one of the lipid extracts. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Hyaluronan production enhances shedding of plasma membrane-derived microvesicles.

    PubMed

    Rilla, Kirsi; Pasonen-Seppänen, Sanna; Deen, Ashik J; Koistinen, Ville V T; Wojciechowski, Sara; Oikari, Sanna; Kärnä, Riikka; Bart, Genevieve; Törrönen, Kari; Tammi, Raija H; Tammi, Markku I

    2013-08-01

    Many cell types secrete plasma membrane-bound microvesicles, suggested to play an important role in tissue morphogenesis, wound healing, and cancer spreading. However, the mechanisms of their formation have remained largely unknown. It was found that the tips of long microvilli induced in cells by overexpression of hyaluronan synthase 3 (HAS3) were detach into the culture medium as microvesicles. Moreover, several cell types with naturally active hyaluronan synthesis released high numbers of plasma membrane-derived vesicles, and inhibition of hyaluronan synthesis reduced their formation. The vesicles contained HAS, and were covered with a thick hyaluronan coat, a part of which was retained even after purification with high-speed centrifugation. HAS3 overexpressing MDCK cells cultured in a 3-D matrix as epithelial cysts released large amounts of HAS- and hyaluronan-positive vesicles from their basal surfaces into the extracellular matrix. As far as we know, hyaluronan synthesis is one of the first molecular mechanisms shown to stimulate the production of microvesicles. The microvesicles have a potential to deliver the hyaluronan synthase machinery and membrane and cytoplasmic materials to other cells, influencing tissue regeneration, inflammation and tumor progression.

  11. Analytical study of microsomes and isolated subcellular membranes from rat liver VIII. Subfractionation of preparations enriched with plasma membranes, outer mitochondrial membranes, or Golgi complex membranes

    PubMed Central

    1981-01-01

    Preparations enriched with plasmalemmal, outer mitochondrial, or Golgi complex membranes from rat liver were subfractionated by isopycnic centrifugation, without or after treatment with digitonin, to establish the subcellular distribution of a variety of enzymes. The typical plasmalemmal enzymes 5'-nucleotidase, alkaline phosphodiesterase I, and alkaline phosphatase were markedly shifted by digitonin toward higher densities in all three preparations. Three glycosyltransferases, highly purified in the Golgi fraction, were moderately shifted by digitonin in both this Golgi complex preparation and the microsomal fraction. The outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the out mitochondrial membrane preparation, in agreement wit its behavior in microsomes. With the exception of NADH cytochrome c reductase (which was concentrated in the outer mitochondrial membrane preparation), typical microsomal enzymes (glucose-6-phosphatase, esterase, and NADPH cytochrome c reductase) displayed low specific activities in the three preparations; except for part of the glucose-6-phosphatase activity in the plasma membrane preparation, their density distributions were insensitive to digitonin, as they were in microsomes. The influence of digitonin on equilibrium densities was correlated with its morphological effects. Digitonin induced pseudofenestrations in plasma membranes. In Golgi and outer mitochondrial membrane preparations, a few similarly altered membranes were detected in subfractions enriched with 5'-nucleotidase and alkaline phosphodiesterase I. The alterations of Golgi membranes were less obvious and seemingly restricted to some elements in the Golgi preparation. No morphological modification was detected in digitonin-treated outer mitochondrial membranes. These results indicate that each enzyme is associated with the same membrane

  12. Plant lipid environment and membrane enzymes: the case of the plasma membrane H+-ATPase.

    PubMed

    Morales-Cedillo, Francisco; González-Solís, Ariadna; Gutiérrez-Angoa, Lizbeth; Cano-Ramírez, Dora Luz; Gavilanes-Ruiz, Marina

    2015-04-01

    Several lipid classes constitute the universal matrix of the biological membranes. With their amphipathic nature, lipids not only build the continuous barrier that confers identity to every cell and organelle, but they are also active actors that modulate the activity of the proteins immersed in the lipid bilayer. The plasma membrane H(+)-ATPase, an enzyme from plant cells, is an excellent example of a transmembrane protein whose activity is influenced by the hydrophilic compartments at both sides of the membrane and by the hydrophobic domains of the lipid bilayer. As a result, an extensive documentation of the effect of numerous amphiphiles in the enzyme activity can be found. Detergents, membrane glycerolipids, and sterols can produce activation or inhibition of the enzyme activity. In some cases, these effects are associated with the lipids of the membrane bulk, but in others, a direct interaction of the lipid with the protein is involved. This review gives an account of reports related to the action of the membrane lipids on the H(+)-ATPase activity.

  13. Assembly of fission yeast eisosomes in the plasma membrane of budding yeast: import of foreign membrane microdomains.

    PubMed

    Vaskovicova, Katarina; Stradalova, Vendula; Efenberk, Ales; Opekarova, Miroslava; Malinsky, Jan

    2015-01-01

    Eisosomes are plasma membrane-associated protein complexes organizing the membrane compartment of Can1 (MCC), a membrane microdomain of specific structure and function in ascomycetous fungi. By heterologous expression of specific components of Schizosaccharomyces pombe eisosomes in Saccharomyces cerevisiae we reconstitute structures exhibiting the composition and morphology of S. pombe eisosome in the host plasma membrane. We show S. pombe protein Pil1 (SpPil1) to substitute the function of its S. cerevisiae homologue in building plasma membrane-associated assemblies recognized by inherent MCC/eisosome constituents Sur7 and Seg1. Our data indicate that binding of SpPil1 to the plasma membrane of S. cerevisiae also induces formation of furrow-like invaginations characteristic for MCC. To the best of our knowledge, this is the first report of interspecies transfer of a functional plasma membrane microdomain. In the described system, we identify a striking difference between eisosome stabilizer proteins Seg1 and SpSle1. While Seg1 recruits both Pil1 and SpPil1 to the plasma membrane, SpSle1 recognizes only its natural counterpart, SpPil1. In the presence of Pil1, SpSle1 is segregated outside the Pil1-organized eisosomes and forms independent microdomains in the host membrane. Copyright © 2014 Elsevier GmbH. All rights reserved.

  14. Approaches for plasma membrane wounding and assessment of lysosome-mediated repair responses

    PubMed Central

    Corrotte, M.; Castro-Gomes, T.; Koushik, A.B.; Andrews, N.W.

    2016-01-01

    Rapid plasma membrane repair is essential to restore cellular homeostasis and improve cell survival after injury. Several mechanisms for plasma membrane repair have been proposed, including formation of an intracellular vesicle patch, reduction of plasma membrane tension, lesion removal by endocytosis, and/or shedding of the wounded membrane. Under all conditions studied to date, plasma membrane repair is strictly dependent on the entry of calcium into cells, from the extracellular medium. Calcium-dependent exocytosis of lysosomes is an important early step in the plasma membrane repair process, and defects in plasma membrane repair have been observed in cells carrying mutations responsible for serious lysosomal diseases, such as Chediak–Higashi (Huynh, Roth, Ward, Kaplan, & Andrews, 2004) and Niemann–Pick Disease type A (Tam et al., 2010). A functional role for release of the lysosomal enzyme acid sphingomyelinase, which generates ceramide on the cell surface and triggers endocytosis, has been described (Corrotte et al., 2013; Tam et al., 2010). Therefore, procedures for measuring the extent of lysosomal fusion with the plasma membrane of wounded cells are important indicators of the cellular repair response. The importance of carefully selecting the methodology for experimental plasma membrane injury, in order not to adversely impact the membrane repair machinery, is becoming increasingly apparent. Here, we describe physiologically relevant methods to induce different types of cellular wounds, and sensitive assays to measure the ability of cells to secrete lysosomes and reseal their plasma membrane. PMID:25665445

  15. Effects of freezing and cold acclimation on the plasma membrane of isolated protoplasts, progress report

    SciTech Connect

    Steponkus, P L

    1993-01-01

    Our goal is to provide a mechanistic understanding of the cellular and molecular aspects of freezing injury and cold acclimation from a perspective of the structural and functional integrity of the plasma membrane -- the primary site of freezing injury in winter cereals. We have utilized protoplasts isolated from leaves of winter rye (Secale cereale L. cv Puma) to study the cryobehavior of the plasma membrane during a freeze/thaw cycle. The focus of our current studies is on lesions in the plasma membrane that result from severe freeze-induced dehydration and result in the alteration of the semipermeable characteristics of the plasma membrane so that the protoplasts are osmotically unresponsive. In protoplasts isolated from non-acclimated rye leaves (NA protoplasts), injury is associated with the formation of aparticulate domains in the plasma membrane, aparticulate lamellae subtending the plasma membrane, and lamellar-to-hexagonal II phase transitions in the plasma membrane and the subtending lamellae. However, lamellar-to-hexagonal II phase transitions are not observed following severe dehydration of protoplasts isolated from cold-acclimated rye leaves (ACC protoplasts). Rather, injury is associated with the fracture-jump lesion,'' which, in freeze-fracture electron microscopy studies, is manifested as localized deviations in the fracture face of the plasma membrane. The fracture plane jumps'' from the plasma membrane to either subtending aparticulate lamellae or aparticulate regions of various endomembranes (predominantly chloroplast envelopes) that are in close apposition with the plasma membrane.

  16. Localization and trafficking of aquaporin 2 in the kidney

    PubMed Central

    Matsuzaki, Toshiyuki; Tajika, Yuki; Ablimit, Abduxukur; Hasegawa, Takahiro

    2008-01-01

    Aquaporins (AQPs) are membrane proteins serving in the transfer of water and small solutes across cellular membranes. AQPs play a variety of roles in the body such as urine formation, prevention from dehydration in covering epithelia, water handling in the blood–brain barrier, secretion, conditioning of the sensory system, cell motility and metastasis, formation of cell junctions, and fat metabolism. The kidney plays a central role in water homeostasis in the body. At least seven isoforms, namely AQP1, AQP2, AQP3, AQP4, AQP6, AQP7, and AQP11, are expressed. Among them, AQP2, the anti-diuretic hormone (ADH)-regulated water channel, plays a critical role in water reabsorption. AQP2 is expressed in principal cells of connecting tubules and collecting ducts, where it is stored in Rab11-positive storage vesicles in the basal state. Upon ADH stimulation, AQP2 is translocated to the apical plasma membrane, where it serves in the influx of water. The translocation process is regulated through the phosphorylation of AQP2 by protein kinase A. As soon as the stimulation is terminated, AQP2 is retrieved to early endosomes, and then transferred back to the Rab 11-positive storage compartment. Some AQP2 is secreted via multivesicular bodies into the urine as exosomes. Actin plays an important role in the intracellular trafficking of AQP2. Recent findings have shed light on the molecular basis that controls the trafficking of AQP2. PMID:18566824

  17. Localization and trafficking of aquaporin 2 in the kidney.

    PubMed

    Takata, Kuniaki; Matsuzaki, Toshiyuki; Tajika, Yuki; Ablimit, Abduxukur; Hasegawa, Takahiro

    2008-08-01

    Aquaporins (AQPs) are membrane proteins serving in the transfer of water and small solutes across cellular membranes. AQPs play a variety of roles in the body such as urine formation, prevention from dehydration in covering epithelia, water handling in the blood-brain barrier, secretion, conditioning of the sensory system, cell motility and metastasis, formation of cell junctions, and fat metabolism. The kidney plays a central role in water homeostasis in the body. At least seven isoforms, namely AQP1, AQP2, AQP3, AQP4, AQP6, AQP7, and AQP11, are expressed. Among them, AQP2, the anti-diuretic hormone (ADH)-regulated water channel, plays a critical role in water reabsorption. AQP2 is expressed in principal cells of connecting tubules and collecting ducts, where it is stored in Rab11-positive storage vesicles in the basal state. Upon ADH stimulation, AQP2 is translocated to the apical plasma membrane, where it serves in the influx of water. The translocation process is regulated through the phosphorylation of AQP2 by protein kinase A. As soon as the stimulation is terminated, AQP2 is retrieved to early endosomes, and then transferred back to the Rab 11-positive storage compartment. Some AQP2 is secreted via multivesicular bodies into the urine as exosomes. Actin plays an important role in the intracellular trafficking of AQP2. Recent findings have shed light on the molecular basis that controls the trafficking of AQP2.

  18. 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.

  19. ABCC6 is a Basolateral Plasma Membrane Protein

    PubMed Central

    Pomozi, Viola; Le Saux, Olivier; Brampton, Christopher; Apana, Ailea; Iliás, Attila; Szeri, Flóra; Martin, Ludovic; Monostory, Katalin; Paku, Sándor; Sarkadi, Balázs; Szakács, Gergely; Váradi, András

    2013-01-01

    Rationale ABCC6 plays a crucial role in ectopic calcification; mutations of the gene cause pseudoxanthoma elasticum (PXE) and general arterial calcification of infancy (GACI). To elucidate the role of ABCC6 in cellular physiology and disease, it is crucial to establish the exact subcellular localization of the native ABCC6 protein. Objective In a recent paper in Circulation Research, ABCC6 was reported to localize to the mitochondria-associated membrane (MAM) and not the plasma membrane. Since the suggested mitochondrial localization is inconsistent with published data and the presumed role of ABCC6, we performed experiments to determine the cellular localization of ABCC6 in its physiological environment. Methods and Results We performed immunofluorescent labeling of frozen mouse and human liver sections as well as primary hepatocytes. We used several different antibodies recognizing human and mouse ABCC6. Our results unequivocally show that ABCC6 is in the basolateral membrane of hepatocytes and is not associated with the mitochondria, MAM or the ER. Conclusion Our findings support the model that ABCC6 is in the basolateral membrane, mediating the sinusoidal efflux of a metabolite from the hepatocytes to the systemic circulation. PMID:23625951

  20. Solid polymer electrolyte composite membrane comprising plasma etched porous support

    DOEpatents

    Liu, Han; LaConti, Anthony B.

    2010-10-05

    A solid polymer electrolyte composite membrane and method of manufacturing the same. According to one embodiment, the composite membrane comprises a rigid, non-electrically-conducting support, the support preferably being a sheet of polyimide having a thickness of about 7.5 to 15 microns. The support has a plurality of cylindrical pores extending perpendicularly between opposing top and bottom surfaces of the support. The pores, which preferably have a diameter of about 0.1 to 5 microns, are made by plasma etching and preferably are arranged in a defined pattern, for example, with fewer pores located in areas of high membrane stress and more pores located in areas of low membrane stress. The pores are filled with a first solid polymer electrolyte, such as a perfluorosulfonic acid (PFSA) polymer. A second solid polymer electrolyte, which may be the same as or different than the first solid polymer electrolyte, may be deposited over the top and/or bottom of the first solid polymer electrolyte.

  1. Purification and properties of 5'-nucleotidase from lymphocyte plasma membranes.

    PubMed

    Dornand, J; Bonnafous, J C; Mani, J C

    1978-07-03

    5'-Nucleotidase is purified from lymphocyte plasma membranes by two affinity chromatographies. The first one, on Lens culinaris lectin-Sepharose 4B yields a fraction of twelve lectin-binding glycoproteins (lectin-receptor fraction). The second one on 5'-AMP-Sepharose 4B leads to pure enzyme. This enzyme is a glycoprotein with a molecular weight of 130 000; it gives a single band in polyacrylamide/dodecylsulfate electrophoresis and displays a very high specific activity (2500-3000 mumol Pih-1mg-1). Some properties of purified 5'-nucleotidase are similar to those of membrane-bound enzyme: substrate specificity, temperature dependence, effects of ions and SH-blocking reagents. Others are completely different for the two systems and these differences result from an interaction between the enzyme molecule and other Lens culinaris lectin binding proteins.

  2. Plant Endoplasmic Reticulum-Plasma Membrane Contact Sites.

    PubMed

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

    2017-04-01

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

  3. The influence of membrane lipid structure on plasma membrane Ca2+ -ATPase activity.

    PubMed

    Tang, Daxin; Dean, William L; Borchman, Douglas; Paterson, Christopher A

    2006-03-01

    Lipid composition and Ca(2+)-ATPase activity both change with age and disease in many tissues. We explored relationships between lipid composition/structure and plasma membrane Ca(2+)-ATPase (PMCA) activity. PMCA was purified from human erythrocytes and was reconstituted into liposomes prepared from human ocular lens membrane lipids and synthetic lipids. Lens lipids were used in this study as a model for naturally ordered lipids, but the influence of lens lipids on PMCA function is especially relevant to the lens since calcium homeostasis is vital to lens clarity. Compared to fiber cell lipids, epithelial lipids exhibited an ordered to disordered phase transition temperature that was 12 degrees C lower. Reconstitution of PMCA into lipids was essential for maximal activity. PMCA activity was two to three times higher when the surrounding phosphatidylcholine molecules contained acyl chains that were ordered (stiff) compared to disordered (fluid) acyl chains. In a completely ordered lipid hydrocarbon chain environment, PMCA associates more strongly with the acidic lipid phosphatidylserine in comparison to phosphatidylcholine. PMCA associates much more strongly with phosphatidylcholine containing disordered hydrocarbon chains than ordered hydrocarbon chains. PMCA activity is influenced by membrane lipid composition and structure. The naturally high degree of lipid order in plasma membranes such as those found in the human lens may serve to support PMCA activity. The absence of PMCA activity in the cortical region of human lenses is apparently not due to a different lipid environment. Changes in lipid composition such as those observed with age or disease could potentially influence PMCA function.

  4. Density of newly synthesized plasma membrane proteins in intracellular membranes. I. Stereological studies

    PubMed Central

    1984-01-01

    As the spike proteins of Semliki Forest virus (SFV) pass from their site of synthesis in the endoplasmic reticulum (ER) to the cell surface, they must be concentrated and freed from endogenous proteins. To determine the magnitude of this sorting process we have measured the density of spike proteins in membranes of the intracellular transport pathway. In this first paper, using stereological procedures, we have estimated the surface areas of the ER, Golgi complex, and plasma membrane of infected and mock-infected baby hamster kidney cells. First, we estimated the mean cell volume in absolute units. This was done using a novel in situ method which is described in detail. Infection by SFV was found to have no effect on any of the parameters measured. In the accompanying paper ( Quinn , P., G. Griffiths, and G. Warren, 1984, J. Cell Biol., 2142-2147) these stereological estimates were combined with biochemical estimates of the amount of spike proteins in ER, Golgi complex, and plasma membrane to determine the density in the membranes of these compartments. PMID:6563037

  5. Cold induced changes in the water balance affect immunocytolocalization pattern of one of the aquaporins in the vascular system in the leaves of maize (Zea mays L.).

    PubMed

    Bilska-Kos, Anna; Szczepanik, Jarosław; Sowiński, Paweł

    2016-10-20

    Chilling stress is known to affect the water balance in plants, which often manifests itself in the decrease of the water potential in different organs. Relationships between chilling, assimilate transport and water balance are far from being understood. Although aquaporins play a key role in regulating water balance in plants, especially under stress conditions, the role of individual aquaporins in stress response remains unclear. In this report we show the specific localization within plasma membranes of one of the aquaporins (PIP2;3) in the leaves of two maize inbred lines differing in their chilling-sensitivity. This form of aquaporin has been also observed in thick-walled sieve elements - an additional type of sieve tubes of unclear function found only in monocotyledons. Moderate chilling (about 15°C) caused significant reduction of labelling in these cells accompanied by a steep decrease in the water potential in leaves of chilling-sensitive maize line. Our results suggest that both PIP2;3 and thick-walled sieve tubes may be an unknown element of the mechanism of the response of maize to cold stress. Copyright © 2016 Elsevier GmbH. All rights reserved.

  6. Alkaline ribonuclease and phosphodiesterase activity in rat liver plasma membranes

    PubMed Central

    Prospero, Terence D.; Burge, Malcolm L. E.; Norris, Kenneth A.; Hinton, Richard H.; Reid, Eric

    1973-01-01

    The ribonuclease and phosphodiesterase activities of rat liver plasma membranes, purified from the crude nuclear fraction by centrifugation in an A-XII zonal rotor and flotation, were examined and compared. The plasma membrane is responsible for between 65 and 90% of the phosphodiesterase activity of the cell and between 25 and 30% of the particulate ribonuclease activity measured at pH8.7 in the presence of 7.5mm-MgCl2. Both enzymes were most active between pH8.5 and 8.9. Close to the pH optimum, both enzymes were more active in Tris buffer than in Bicine or glycine buffer. Both plasma-membrane phosphodiesterase and ribonuclease were strongly activated by Mg2+, there being at least a 12-fold difference between the activity in the presence of Mg2+ and of EDTA. There is, however, a difference in the response of the enzymes to Mg2+ and EDTA in that the phosphodiesterase is fully activated by 1.0mm-MgCl2 and fully inhibited by 1.0mm-EDTA, whereas the ribonuclease requires 7.5mm-MgCl2 for full activation and 5mm-EDTA for full inhibition. Density-gradient centrifugation has indicated that on solubilization in Triton X-100 most of the ribonuclease activity is released into a small fragment of the same size as that containing the phosphodiesterase activity. The relationship between the two activities is discussed in view of these results. PMID:4353377

  7. The human erythrocyte plasma membrane: a Rosetta Stone for decoding membrane-cytoskeleton structure.

    PubMed

    Fowler, Velia M

    2013-01-01

    The mammalian erythrocyte, or red blood cell (RBC), is a unique experiment of nature: a cell with no intracellular organelles, nucleus or transcellular cytoskeleton, and a plasma membrane with uniform structure across its entire surface. By virtue of these specialized properties, the RBC membrane has provided a template for discovery of the fundamental actin filament network machine of the membrane skeleton, now known to confer mechanical resilience, anchor membrane proteins, and organize membrane domains in all cells. This chapter provides a historical perspective and critical analysis of the biochemistry, structure, and physiological functions of this actin filament network in RBCs. The core units of this network are nodes of ~35-37 nm-long actin filaments, interconnected by long strands of (α1β1)₂-spectrin tetramers, forming a 2D isotropic lattice with quasi-hexagonal symmetry. Actin filament length and stability is critical for network formation, relying upon filament capping at both ends: tropomodulin-1 at pointed ends and αβ-adducin at barbed ends. Tropomodulin-1 capping is essential for precise filament lengths, and is enhanced by tropomyosin, which binds along the short actin filaments. αβ-adducin capping recruits spectrins to sites near barbed ends, promoting network formation. Accessory proteins, 4.1R and dematin, also promote spectrin binding to actin and, with αβ-adducin, link to membrane proteins, targeting actin nodes to the membrane. Dissection of the molecular organization within the RBC membrane skeleton is one of the paramount achievements of cell biological research in the past century. Future studies will reveal the structure and dynamics of actin filament capping, mechanisms of precise length regulation, and spectrin-actin lattice symmetry. © 2013 Elsevier Inc. All rights reserved.

  8. Molecular and functional characterization of multiple aquaporin water channel proteins from the western tarnished plant bug, Lygus hesperus

    USDA-ARS?s Scientific Manuscript database

    Aquaporins (AQPs) are integral membrane channel proteins that facilitate the bidirectional transfer of water or other small solutes across biological membranes involved in numerous essential physiological processes. In arthropods, AQPs belong to several subfamilies, which contribute to osmoregulatio...

  9. Calcium Modulation of Plant Plasma Membrane-Bound Atpase Activities

    NASA Technical Reports Server (NTRS)

    Caldwell, C.

    1983-01-01

    The kinetic properties of barley enzyme are discussed and compared with those of other plants. Possibilities for calcium transport in the plasma membrane by proton pump and ATPase-dependent calcium pumps are explored. Topics covered include the ph phase of the enzyme; high affinity of barley for calcium; temperature dependence, activation enthalpy, and the types of ATPase catalytic sites. Attention is given to lipids which are both screened and bound by calcium. Studies show that barley has a calmodulin activated ATPase that is found in the presence of magnesium and calcium.

  10. Lipoproteins removed from serum and plasma by membrane filtration.

    PubMed

    Olson, W P; Faith, M W

    1978-01-01

    Tangential (crossflow) filtration of a plasma/serum mixture through 0.2 micrometer-poresize polycarbonate track-etch membrane filters (PC) at pressures less than 10 psi removes low density lipoproteins (LDL) and very low density lipoproteins (VLDL) but not high density lipoproteins (HDL) from the filtrate. At pressures greater than 10 psi all lipoproteins pass through the PC. Once the filters have been intruded with LDL and VLDL those lipoproteins continue to pass the filters despite subsequent reduction in differential pressure below 10 psi.

  11. Calcium Modulation of Plant Plasma Membrane-Bound Atpase Activities

    NASA Technical Reports Server (NTRS)

    Caldwell, C.

    1983-01-01

    The kinetic properties of barley enzyme are discussed and compared with those of other plants. Possibilities for calcium transport in the plasma membrane by proton pump and ATPase-dependent calcium pumps are explored. Topics covered include the ph phase of the enzyme; high affinity of barley for calcium; temperature dependence, activation enthalpy, and the types of ATPase catalytic sites. Attention is given to lipids which are both screened and bound by calcium. Studies show that barley has a calmodulin activated ATPase that is found in the presence of magnesium and calcium.

  12. A PIP1 aquaporin contributes to hydrostatic pressure-induced water transport in both the root and rosette of Arabidopsis.

    PubMed

    Postaire, Olivier; Tournaire-Roux, Colette; Grondin, Alexandre; Boursiac, Yann; Morillon, Raphaël; Schäffner, Anton R; Maurel, Christophe

    2010-03-01

    Aquaporins are channel proteins that facilitate the transport of water across plant cell membranes. In this work, we used a combination of pharmacological and reverse genetic approaches to investigate the overall significance of aquaporins for tissue water conductivity in Arabidopsis (Arabidopsis thaliana). We addressed the function in roots and leaves of AtPIP1;2, one of the most abundantly expressed isoforms of the plasma membrane intrinsic protein family. At variance with the water transport phenotype previously described in AtPIP2;2 knockout mutants, disruption of AtPIP1;2 reduced by 20% to 30% the root hydrostatic hydraulic conductivity but did not modify osmotic root water transport. These results document qualitatively distinct functions of different PIP isoforms in root water uptake. The hydraulic conductivity of excised rosettes (K(ros)) was measured by a novel pressure chamber technique. Exposure of Arabidopsis plants to darkness increased K(ros) by up to 90%. Mercury and azide, two aquaporin inhibitors with distinct modes of action, were able to induce similar inhibition of K(ros) by approximately 13% and approximately 25% in rosettes from plants grown in the light or under prolonged (11-18 h) darkness, respectively. Prolonged darkness enhanced the transcript abundance of several PIP genes, including AtPIP1;2. Mutant analysis showed that, under prolonged darkness conditions, AtPIP1;2 can contribute to up to approximately 20% of K(ros) and to the osmotic water permeability of isolated mesophyll protoplasts. Therefore, AtPIP1;2 can account for a significant portion of aquaporin-mediated leaf water transport. The overall work shows that AtPIP1;2 represents a key component of whole-plant hydraulics.

  13. Effects of freezing and cold acclimation on the plasma membrane of isolated protoplasts

    SciTech Connect

    Steponkus, P.L.

    1991-01-01

    This project focuses on lesions in the plasma membrane of protoplasts that occur during freezing to temperatures below {minus}5{degrees} which result in changes in the semipermeablity of the plasma membrane. This injury, referred to as loss of osmotic responsiveness, is associated with the formation of large, aparticulate domains in the plasma membrane, aparticulate lamellae subtending the plasma membrane, and lamellar-to-hexagonal{sub II} phase transitions in the plasma membrane and subtending lamellar. The goals of this project are to provide a mechanistic understanding of the mechanism by which freeze-induced dehydration effects the formation of aparticulate domains and lamellar-to-hexagonal{sub II} phase transitions and to determine the mechanisms by which cold acclimation and cryoprotectants preclude or diminish these ultrastructural changes. Our working hypothesis is the formation of aparticulate domains and lamellar-to-hexagon{sub II} phase transitions in the plasma membrane and subtending lamellae are manifestations of hydration-dependent bilayer-bilayer interactions.

  14. Modification of polysulfone porous hollow fiber membranes by air plasma treatment

    NASA Astrophysics Data System (ADS)

    Volkov, V. V.; Ibragimov, R. G.; Abdullin, I. Sh; Gallyamov, R. T.; Ovcharova, A. A.; Bildyukevich, A. V.

    2016-09-01

    Air plasma treatment was used to enhance the surface hydrophilic properties of the polysulfone porous hollow fiber membranes prepared via a dry-wet phase invertion technique in the free spinning mode in air. Membranes prepared had porous asymmetric structure with macroporous support on the shell side and fine-porous selective layer on the lumen side. The wettability of the inner membrane surfaces were checked by contact angle measurements and FTIR was used to compare the surfaces before and after plasma treatment. Membrane morphology was examined with confocal scanning laser microscopy (CSLM). Contact angle measurements confirm that air plasma treatment affords improvement in the wettability of polysulfone membranes and FTIR results show that air plasmas chemically modify the lumen side membrane surface, however, there is no significant change in membranes chemical structure after modification. CSLM data obtained, as well as gas permeability (He and CO2) measurements show that after plasma treatment pore etching occurs.

  15. Specific interaction of postsynaptic densities with membrane rafts isolated from synaptic plasma membranes.

    PubMed

    Liu, Qian; Yao, Wei-Dong; Suzuki, Tatsuo

    2013-06-01

    Postsynaptic membrane rafts are believed to play important roles in synaptic signaling, plasticity, and maintenance. We recently demonstrated the presence, at the electron microscopic level, of complexes consisting of membrane rafts and postsynaptic densities (PSDs) in detergent-resistant membranes (DRMs) prepared from synaptic plasma membranes (SPMs) ( Suzuki et al., 2011 , J Neurochem, 119, 64-77). To further explore these complexes, here we investigated the nature of the binding between purified SPM-DRMs and PSDs in vitro. In binding experiments, we used SPM-DRMs prepared after treating SPMs with n-octyl-β-d-glucoside, because at concentrations of 1.0% or higher it completely separates SPM-DRMs and PSDs, providing substantially PSD-free unique SPM-DRMs as well as DRM-free PSDs. PSD binding to PSD-free DRMs was identified by mass spectrometry, Western blotting, and electron microscopy. PSD proteins were not incorporated into SPMs, and significantly less PSD proteins were incorporated into DRMs prepared from liver membranes, providing in vitro evidence that binding of PSDs to DRMs is specific and suggestion of the presence of specific interacting molecules. These specific interactions may have important roles in synaptic development, function, and plasticity in vivo. In addition, the binding system we developed may be a good tool to search for binding molecules and binding mechanisms between PSDs and rafts.

  16. Ectopic overexpression of a novel Glycine soja stress-induced plasma membrane intrinsic protein increases sensitivity to salt and dehydration in transgenic Arabidopsis thaliana plants.

    PubMed

    Wang, Xi; Cai, Hua; Li, Yong; Zhu, Yanming; Ji, Wei; Bai, Xi; Zhu, Dan; Sun, Xiaoli

    2015-01-01

    Plasma membrane intrinsic proteins (PIPs) belong to the aquaporin family and facilitate water movement across plasma membranes. Existing data indicate that PIP genes are associated with the abilities of plants to tolerate certain stress conditions. A review of our Glycine soja expressed sequence tag (EST) dataset revealed that abiotic stress stimulated expression of a PIP, herein designated as GsPIP2;1 (GenBank_Accn: FJ825766). To understand the roles of this PIP in stress tolerance, we generated a coding sequence for GsPIP2;1 by in silico elongation and cloned the cDNA by 5'-RACE. Semiquantitative RT-PCR showed that GsPIP2;1 expression was stimulated in G. soja leaves by cold, salt, or dehydration stress, whereas the same stresses suppressed GsPIP2;1 expression in the roots. Transgenic Arabidopsis thaliana plants overexpressing GsPIP2;1 grew normally under unstressed and cold conditions, but exhibited depressed tolerance to salt and dehydration stresses. Moreover, greater changes in water potential were detected in the transgenic A. thaliana shoots, implying that GsPIP2;1 may negatively impact stress tolerance by regulating water potential. These results, deviating from those obtained in previous reports, provide new insights into the relationship between PIPs and abiotic stress tolerance in plants.

  17. PtdIns4P synthesis by PI4KIIIα at the plasma membrane and its impact on plasma membrane identity.

    PubMed

    Nakatsu, Fubito; Baskin, Jeremy M; Chung, Jeeyun; Tanner, Lukas B; Shui, Guanghou; Lee, Sang Yoon; Pirruccello, Michelle; Hao, Mingming; Ingolia, Nicholas T; Wenk, Markus R; De Camilli, Pietro

    2012-12-10

    Plasma membrane phosphatidylinositol (PI) 4-phosphate (PtdIns4P) has critical functions via both direct interactions and metabolic conversion to PI 4,5-bisphosphate (PtdIns(4,5)P₂) and other downstream metabolites. However, mechanisms that control this PtdIns4P pool in cells of higher eukaryotes remain elusive. PI4KIIIα, the enzyme thought to synthesize this PtdIns4P pool, is reported to localize in the ER, contrary to the plasma membrane localization of its yeast homologue, Stt4. In this paper, we show that PI4KIIIα was targeted to the plasma membrane as part of an evolutionarily conserved complex containing Efr3/rolling blackout, which we found was a palmitoylated peripheral membrane protein. PI4KIIIα knockout cells exhibited a profound reduction of plasma membrane PtdIns4P but surprisingly only a modest reduction of PtdIns(4,5)P₂ because of robust up-regulation of PtdIns4P 5-kinases. In these cells, however, much of the PtdIns(4,5)P₂ was localized intracellularly, rather than at the plasma membrane as in control cells, along with proteins typically restricted to this membrane, revealing a major contribution of PI4KIIIα to the definition of plasma membrane identity.

  18. 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

  19. 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

  20. Supported lipid bilayer membranes for water purification by reverse osmosis.

    PubMed

    Kaufman, Yair; Berman, Amir; Freger, Viatcheslav

    2010-05-18

    Some biological plasma membranes pass water with a permeability and selectivity largely exceeding those of commercial membranes for water desalination using specialized trans-membrane proteins aquaporins. However, highly selective transport of water through aquaporins is usually driven by an osmotic rather mechanical pressure, which is not as attractive from the engineering point of view. The feasibility of adopting biomimetic membranes for water purification driven by a mechanical pressure, i.e., filtration is explored in this paper. Toward this goal, it is proposed to use a commercial nanofiltration (NF) membrane as a support for biomimetic lipid bilayer membranes to render them robust enough to withstand the required pressures. It is shown in this paper for the first time that by properly tuning molecular interactions supported phospholipid bilayers (SPB) can be prepared on a commercial NF membrane. The presence of SPB on the surface was verified and quantified by several spectroscopic and microscopic techniques, which showed morphology close to the desired one with very few defects. As an ultimate test it is shown that hydraulic permeability of the SPB supported on the NF membrane (NTR-7450) approaches the values deduced from the typical osmotic permeabilities of intact continuous bilayers. This permeability was unaffected by the trans-membrane flow of water and by repeatedly releasing and reapplying a 10 bar pressure. Along with a parallel demonstration that aquaporins could be incorporated in a similar bilayer on mica, this demonstrates the feasibility of the proposed approach. The prepared SPB structure may be used as a platform for preparing biomimetic filtration membranes with superior performance based on aquaporins. The concept of SPBs on permeable substrates of the present type may also be useful in the future for studying transport of various molecules through trans-membrane proteins.

  1. Structure and Function of Thyroid Hormone Plasma Membrane Transporters

    PubMed Central

    Schweizer, Ulrich; Johannes, Jörg; Bayer, Dorothea; Braun, Doreen

    2014-01-01

    Thyroid hormones (TH) cross the plasma membrane with the help of transporter proteins. As charged amino acid derivatives, TH cannot simply diffuse across a lipid bilayer membrane, despite their notorious hydrophobicity. The identification of monocarboxylate transporter 8 (MCT8, SLC16A2) as a specific and very active TH transporter paved the way to the finding that mutations in the MCT8 gene cause a syndrome of psychomotor retardation in humans. The purpose of this review is to introduce the current model of transmembrane transport and highlight the diversity of TH transmembrane transporters. The interactions of TH with plasma transfer proteins, T3 receptors, and deiodinase are summarized. It is shown that proteins may bind TH owing to their hydrophobic character in hydrophobic cavities and/or by specific polar interaction with the phenolic hydroxyl, the aminopropionic acid moiety, and by weak polar interactions with the iodine atoms. These findings are compared with our understanding of how TH transporters interact with substrate. The presumed effects of mutations in MCT8 on protein folding and transport function are explained in light of the available homology model. PMID:25538896

  2. Structure and function of thyroid hormone plasma membrane transporters.

    PubMed

    Schweizer, Ulrich; Johannes, Jörg; Bayer, Dorothea; Braun, Doreen

    2014-09-01

    Thyroid hormones (TH) cross the plasma membrane with the help of transporter proteins. As charged amino acid derivatives, TH cannot simply diffuse across a lipid bilayer membrane, despite their notorious hydrophobicity. The identification of monocarboxylate transporter 8 (MCT8, SLC16A2) as a specific and very active TH transporter paved the way to the finding that mutations in the MCT8 gene cause a syndrome of psychomotor retardation in humans. The purpose of this review is to introduce the current model of transmembrane transport and highlight the diversity of TH transmembrane transporters. The interactions of TH with plasma transfer proteins, T3 receptors, and deiodinase are summarized. It is shown that proteins may bind TH owing to their hydrophobic character in hydrophobic cavities and/or by specific polar interaction with the phenolic hydroxyl, the aminopropionic acid moiety, and by weak polar interactions with the iodine atoms. These findings are compared with our understanding of how TH transporters interact with substrate. The presumed effects of mutations in MCT8 on protein folding and transport function are explained in light of the available homology model.

  3. Flat clathrin lattices: stable features of the plasma membrane

    PubMed Central

    Grove, Joe; Metcalf, Daniel J.; Knight, Alex E.; Wavre-Shapton, Silène T.; Sun, Tony; Protonotarios, Emmanouil D.; Griffin, Lewis D.; Lippincott-Schwartz, Jennifer; Marsh, Mark

    2014-01-01

    Clathrin-mediated endocytosis (CME) is a fundamental property of eukaryotic cells. Classical CME proceeds via the formation of clathrin-coated pits (CCPs) at the plasma membrane, which invaginate to form clathrin-coated vesicles, a process that is well understood. However, clathrin also assembles into flat clathrin lattices (FCLs); these structures remain poorly described, and their contribution to cell biology is unclear. We used quantitative imaging to provide the first comprehensive description of FCLs and explore their influence on plasma membrane organization. Ultrastructural analysis by electron and superresolution microscopy revealed two discrete populations of clathrin structures. CCPs were typified by their sphericity, small size, and homogeneity. FCLs were planar, large, and heterogeneous and present on both the dorsal and ventral surfaces of cells. Live microscopy demonstrated that CCPs are short lived and culminate in a peak of dynamin recruitment, consistent with classical CME. In contrast, FCLs were long lived, with sustained association with dynamin. We investigated the biological relevance of FCLs using the chemokine receptor CCR5 as a model system. Agonist activation leads to sustained recruitment of CCR5 to FCLs. Quantitative molecular imaging indicated that FCLs partitioned receptors at the cell surface. Our observations suggest that FCLs provide stable platforms for the recruitment of endocytic cargo. PMID:25165141

  4. Carboxylic Acids Plasma Membrane Transporters in Saccharomyces cerevisiae.

    PubMed

    Casal, Margarida; Queirós, Odília; Talaia, Gabriel; Ribas, David; Paiva, Sandra

    2016-01-01

    This chapter covers the functionally characterized plasma membrane carboxylic acids transporters Jen1, Ady2, Fps1 and Pdr12 in the yeast Saccharomyces cerevisiae, addressing also their homologues in other microorganisms, as filamentous fungi and bacteria. Carboxylic acids can either be transported into the cells, to be used as nutrients, or extruded in response to acid stress conditions. The secondary active transporters Jen1 and Ady2 can mediate the uptake of the anionic form of these substrates by a H(+)-symport mechanism. The undissociated form of carboxylic acids is lipid-soluble, crossing the plasma membrane by simple diffusion. Furthermore, acetic acid can also be transported by facilitated diffusion via Fps1 channel. At the cytoplasmic physiological pH, the anionic form of the acid prevails and it can be exported by the Pdr12 pump. This review will highlight the mechanisms involving carboxylic acids transporters, and the way they operate according to the yeast cell response to environmental changes, as carbon source availability, extracellular pH and acid stress conditions.

  5. Influence of decavanadate on rat synaptic plasma membrane ATPases activity.

    PubMed

    Krstić, Danijela; Colović, Mirjana; Bosnjaković-Pavlović, Nada; Spasojević-De Bire, Anne; Vasić, Vesna

    2009-09-01

    The in vitro influence of decameric vanadate species on Na+/K+-ATPase, plasma membrane Ca2+-ATPase (PMCA)-calcium pump and ecto-ATPase activity, using rat synaptic plasma membrane (SPM) as model system was investigated, whereas the commercial porcine cerebral cortex Na+/K+-ATPase served as a reference. The thermal behaviour of the synthesized decavanadate (V10) has been studied by differential scanning calorimetry and thermogravimetric analysis, while the type of polyvanadate anion was identified using the IR spectroscopy. The concentration-dependent responses to V10 of all enzymes were obtained. The half-maximum inhibitory concentration (IC50) of the enzyme activity was achieved at (4.74 +/- 1.15) x 10(-7) mol/l for SPM Na+/K+-ATPase, (1.30 +/- 0.10) x 10(-6) mol/l for commercial Na+/K+-ATPase and (3.13 +/- 1.70) x 10(-8) mol/l for Ca2+-ATPase, while ecto-ATPase is significantly less sensitive toward V10 (IC50 = (1.05 +/- 0.10) x 10(-4) mol/l) than investigated P-type ATPases. Kinetic analysis showed that V10 inhibited Na+/K+-ATPase by reducing the maximum enzymatic velocity and apparent affinity for ATP (increasing K(m) value), implying a mixed mode of interaction between V10 and P-type ATPases.

  6. Plasma membrane-localized transporter for aluminum in rice

    PubMed Central

    Xia, Jixing; Yamaji, Naoki; Kasai, Tomonari; Ma, Jian Feng

    2010-01-01

    Aluminum (Al) is the most abundant metal in the Earth's crust, but its trivalent ionic form is highly toxic to all organisms at low concentrations. How Al enters cells has not been elucidated in any organisms. Herein, we report a transporter, Nrat1 (Nramp aluminum transporter 1), specific for trivalent Al ion in rice. Nrat1 belongs to the Nramp (natural resistance-associated macrophage protein) family, but shares a low similarity with other Nramp members. When expressed in yeast, Nrat1 transports trivalent Al ion, but not other divalent ions, such as manganese, iron, and cadmium, or the Al–citrate complex. Nrat1 is localized at the plasma membranes of all cells of root tips except epidermal cells. Knockout of Nrat1 resulted in decreased Al uptake, increased Al binding to cell wall, and enhanced Al sensitivity, but did not affect the tolerance to other metals. Expression of Nrat1 is up-regulated by Al in the roots and regulated by a C2H2 zinc finger transcription factor (ART1). We therefore concluded that Nrat1 is a plasma membrane-localized transporter for trivalent Al, which is required for a prior step of final Al detoxification through sequestration of Al into vacuoles. PMID:20937890

  7. Plasma membrane-localized transporter for aluminum in rice.

    PubMed

    Xia, Jixing; Yamaji, Naoki; Kasai, Tomonari; Ma, Jian Feng

    2010-10-26

    Aluminum (Al) is the most abundant metal in the Earth's crust, but its trivalent ionic form is highly toxic to all organisms at low concentrations. How Al enters cells has not been elucidated in any organisms. Herein, we report a transporter, Nrat1 (Nramp aluminum transporter 1), specific for trivalent Al ion in rice. Nrat1 belongs to the Nramp (natural resistance-associated macrophage protein) family, but shares a low similarity with other Nramp members. When expressed in yeast, Nrat1 transports trivalent Al ion, but not other divalent ions, such as manganese, iron, and cadmium, or the Al-citrate complex. Nrat1 is localized at the plasma membranes of all cells of root tips except epidermal cells. Knockout of Nrat1 resulted in decreased Al uptake, increased Al binding to cell wall, and enhanced Al sensitivity, but did not affect the tolerance to other metals. Expression of Nrat1 is up-regulated by Al in the roots and regulated by a C2H2 zinc finger transcription factor (ART1). We therefore concluded that Nrat1 is a plasma membrane-localized transporter for trivalent Al, which is required for a prior step of final Al detoxification through sequestration of Al into vacuoles.

  8. Fluorescent castasterone reveals BRI1 signaling from the plasma membrane.

    PubMed

    Irani, Niloufer G; Di Rubbo, Simone; Mylle, Evelien; Van den Begin, Jos; Schneider-Pizoń, Joanna; Hniliková, Jaroslava; Šíša, Miroslav; Buyst, Dieter; Vilarrasa-Blasi, Josep; Szatmári, Anna-Mária; Van Damme, Daniël; Mishev, Kiril; Codreanu, Mirela-Corina; Kohout, Ladislav; Strnad, Miroslav; Caño-Delgado, Ana I; Friml, Jiří; Madder, Annemieke; Russinova, Eugenia

    2012-05-06

    Receptor-mediated endocytosis is an integral part of signal transduction as it mediates signal attenuation and provides spatial and temporal dimensions to signaling events. One of the best-studied leucine-rich repeat receptor-like kinases in plants, BRASSINOSTEROID INSENSITIVE 1 (BRI1), perceives its ligand, the brassinosteroid (BR) hormone, at the cell surface and is constitutively endocytosed. However, the importance of endocytosis for BR signaling remains unclear. Here we developed a bioactive, fluorescent BR analog, Alexa Fluor 647-castasterone (AFCS), and visualized the endocytosis of BRI1-AFCS complexes in living Arabidopsis thaliana cells. Impairment of endocytosis dependent on clathrin and the guanine nucleotide exchange factor for ARF GTPases (ARF-GEF) GNOM enhanced BR signaling by retaining active BRI1-ligand complexes at the plasma membrane. Increasing the trans-Golgi network/early endosome pool of BRI1-BR complexes did not affect BR signaling. Our findings provide what is to our knowledge the first visualization of receptor-ligand complexes in plants and reveal clathrin- and ARF-GEF-dependent endocytic regulation of BR signaling from the plasma membrane.

  9. A Plasma Membrane Association Module in Yeast Amino Acid Transporters*

    PubMed Central

    Popov-Čeleketić, Dušan; Bianchi, Frans; Ruiz, Stephanie J.; Meutiawati, Febrina; Poolman, Bert

    2016-01-01

    Amino acid permeases (AAPs) in the plasma membrane (PM) of Saccharomyces cerevisiae are responsible for the uptake of amino acids and involved in regulation of their cellular levels. Here, we report on a strong and complex module for PM association found in the C-terminal tail of AAPs. Using in silico analyses and mutational studies we found that the C-terminal sequences of Gap1, Bap2, Hip1, Tat1, Tat2, Mmp1, Sam3, Agp1, and Gnp1 are about 50 residues long, associate with the PM, and have features that discriminate them from the termini of organellar amino acid transporters. We show that this sequence (named PMasseq) contains an amphipathic α-helix and the FWC signature, which is palmitoylated by palmitoyltransferase Pfa4. Variations of PMasseq, found in different AAPs, lead to different mobilities and localization patterns, whereas the disruption of the sequence has an adverse effect on cell viability. We propose that PMasseq modulates the function and localization of AAPs along the PM. PMasseq is one of the most complex protein signals for plasma membrane association across species and can be used as a delivery vehicle for the PM. PMID:27226538

  10. A Plasma Membrane Association Module in Yeast Amino Acid Transporters.

    PubMed

    Popov-Čeleketić, Dušan; Bianchi, Frans; Ruiz, Stephanie J; Meutiawati, Febrina; Poolman, Bert

    2016-07-29

    Amino acid permeases (AAPs) in the plasma membrane (PM) of Saccharomyces cerevisiae are responsible for the uptake of amino acids and involved in regulation of their cellular levels. Here, we report on a strong and complex module for PM association found in the C-terminal tail of AAPs. Using in silico analyses and mutational studies we found that the C-terminal sequences of Gap1, Bap2, Hip1, Tat1, Tat2, Mmp1, Sam3, Agp1, and Gnp1 are about 50 residues long, associate with the PM, and have features that discriminate them from the termini of organellar amino acid transporters. We show that this sequence (named PMasseq) contains an amphipathic α-helix and the FWC signature, which is palmitoylated by palmitoyltransferase Pfa4. Variations of PMasseq, found in different AAPs, lead to different mobilities and localization patterns, whereas the disruption of the sequence has an adverse effect on cell viability. We propose that PMasseq modulates the function and localization of AAPs along the PM. PMasseq is one of the most complex protein signals for plasma membrane association across species and can be used as a delivery vehicle for the PM. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Regulation of Ras signaling and function by plasma membrane microdomains.

    PubMed

    Goldfinger, Lawrence E; Michael, James V

    2017-02-07

    Together H-, N- and KRAS mutations are major contributors to ~30% of all human cancers. Thus, Ras inhibition remains an important anti-cancer strategy. The molecular mechanisms of isotypic Ras oncogenesis are still not completely understood. Monopharmacological therapeutics have not been successful in the clinic. These disappointing outcomes have led to attempts to target elements downstream of Ras, mainly targeting either the Phosphatidylinositol 3-Kinase (PI3K) or Mitogen-Activated Protein Kinase (MAPK) pathways. While several such approaches are moderately effective, recent efforts have focused on preclinical evaluation of combination therapies to improve efficacies. This review will detail current understanding of the contributions of plasma membrane microdomain targeting of Ras to mitogenic and tumorigenic signaling and tumor progression. Moreover, this review will outline novel approaches to target Ras in cancers, including targeting schemes for new drug development, as well as putative re-purposing of drugs in current use to take advantage of blunting Ras signaling by interfering with Ras plasma membrane microdomain targeting and retention.

  12. Aquaporins in the Skin.

    PubMed

    Patel, Ravi; Kevin Heard, L; Chen, Xunsheng; Bollag, Wendy B

    2017-01-01

    The skin is the largest organ of the body, serving as an important barrier between the internal milieu and the external environment. The skin is also one of the first lines of defense against microbial infection and other hazards, and thus, the skin has important immune functions . This organ is composed of many cell types, including immune-active dendritic cells (epidermal Langerhans cells and dermal dendritic cells), connective tissue-generating dermal fibroblasts and pigment-producing melanocytes. Comprising the outer skin layer are the epidermal keratinocytes, the predominant cell of this layer, the epidermis , which provides both a mechanical barrier and a water -permeability barrier. Recent data suggest that aquaporins, a family of barrel-shaped proteins surrounding internal pores that allow the passage of water and, in some family members, small solutes such as glycerol , play critical roles in regulating various skin parameters. The involvement of different aquaporin family members in skin function is discussed.

  13. Organized living: formation mechanisms and functions of plasma membrane domains in yeast.

    PubMed

    Ziółkowska, Natasza E; Christiano, Romain; Walther, Tobias C

    2012-03-01

    Plasma membrane proteins and lipids organize into lateral domains of specific composition. Domain formation is achieved by a combination of lipid-lipid and lipid-protein interactions, membrane-binding protein scaffolds and protein fences. The resulting domains function in membrane protein turnover and homeostasis, as well as in cell signaling. We review the mechanisms generating plasma membrane domains and the functional consequences of this organization, focusing on recent findings from research on the yeast model system.

  14. Presence and localization of aquaporin-6 in rat parotid acinar cells.

    PubMed

    Matsuki-Fukushima, Miwako; Hashimoto, Sadamitsu; Shimono, Masaki; Satoh, Keitaro; Fujita-Yoshigaki, Junko; Sugiya, Hiroshi

    2008-04-01

    Aquaporins (AQPs) are integral membrane proteins that function as channels for the transfer of water and small solutes across membranes. In mammalian cells, 13 isoforms (AQP0-12) have been identified, and these exhibit unique patterns of expression in various cell types and tissues. Among these isoforms, AQP6 is considered to function not as water channel, but as an anion channel. We investigated the presence and localization of AQP6 in rat parotid acinar cells. AQP6 mRNA was detected in these cells by using reverse transcription/polymerase chain reaction, and Western blotting analysis identified a protein band that reacted with an anti-AQP6 antibody in the membrane fraction and secretory granule membrane. In order to localize AQP6, we used the anti-AQP6 antibody for histological immunodetection. Under confocal microscopy, we observed positive immunoreactions near the tight junctions of parotid acinar cells. Immunolabeling of ultrathin cryosections detected AQP6 near tight junctions and around secretory granule membranes. Immunoelectron microscopy confirmed the presence of AQP6 in the membranes of isolated secretory granules. These results suggest that AQP6 participates in water and anion transport in plasma membranes near tight junctions and secretory granule membranes in rat parotid acinar cells.

  15. 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.

  16. The plasma membrane: Penultimate regulator of ADAM sheddase function.

    PubMed

    Reiss, Karina; Bhakdi, Sucharit

    2017-11-01

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

  17. Fluconazole treatment hyperpolarizes the plasma membrane of Candida cells.

    PubMed

    Elicharova, Hana; Sychrova, Hana

    2013-11-01

    Five pathogenic Candida species were compared in terms of their osmotolerance, tolerance to toxic sodium and lithium cations, and resistance to fluconazole. The species not only differed, in general, in their tolerance to high osmotic pressure (C. albicans and C. parapsilosis being the most osmotolerant) but exhibited distinct sensitivities to toxic sodium and lithium cations, with C. parapsilosis and C. tropicalis being very tolerant but C. krusei and C. dubliniensis sensitive to LiCl. The treatment of both fluconazole-susceptible (C. albicans and C. parapsilosis) and fluconazole-resistant (C. dubliniensis, C. krusei and C. tropicalis) growing cells with subinhibitory concentrations of fluconazole resulted in substantially elevated intracellular Na(+) levels. Using a diS-C3(3) assay, for the first time, to monitor the relative membrane potential (ΔΨ) of Candida cells, we show that the fluconazole treatment of growing cells of all five species results in a substantial hyperpolarization of their plasma membranes, which is responsible for an increased non-specific transport of toxic alkali metal cations and other cationic drugs (e.g., hygromycin B). Thus, the combination of relatively low doses of fluconazole and drugs, whose import into the tested Candida strains is driven by the cell membrane potential, might be especially potent in terms of its ability to inhibit the growth of or even kill various Candida species.

  18. 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

  19. Decreases in Plasma Membrane Ca2+-ATPase in Brain Synaptic Membrane Rafts from Aged Rats

    PubMed Central

    Jiang, Lei; Bechtel, Misty D.; Galeva, Nadezhda A.; Williams, Todd D.; Michaelis, Elias K.; Michaelis, Mary L.

    2012-01-01

    Precise regulation of free intracellular Ca2+ concentrations [Ca2+]i is critical for normal neuronal function, and alterations in Ca2+ homeostasis are associated with brain aging and neurodegenerative diseases. One of the most important proteins controlling [Ca2+]i is the plasma membrane Ca2+-ATPase (PMCA), the high affinity transporter that fine tunes the cytosolic nanomolar levels of Ca2+. We previously found that PMCA protein in synaptic plasma membranes (SPMs) is decreased with advancing age and the decrease in enzyme activity is much greater than that in protein levels. In the present study, we isolated raft and non-raft fractions from rat brain SPMs and used quantitative mass spectrometry to show that the specialized lipid microdomains in SPMs, the rafts, contain 60% of total PMCA, comprised of all four isoforms. The raft PMCA pool had the highest specific activity and this decreased progressively with age. The reduction in PMCA protein could not account for the dramatic activity loss. Addition of excess CaM to the assay did not restore PMCA activity to that in young brains. Analysis of the major raft lipids revealed a slight age-related increase in cholesterol levels and such increases might enhance membrane lipid order and prevent further loss of PMCA activity. PMID:22889001

  20. 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.

  1. Bi-functionality of Opisthorchis viverrini aquaporins.

    PubMed

    Geadkaew, Amornrat; von Bülow, Julia; Beitz, Eric; Tesana, Smarn; Vichasri Grams, Suksiri; Grams, Rudi

    2015-01-01

    Aquaporins (AQP) are essential mediators of water regulation in all living organisms and members of the major intrinsic protein (MIP) superfamily of integral membrane proteins. They are potential vehicles or targets for chemotherapy, e.g. in Trypanosoma brucei melarsoprol and pentamidine uptake is facilitated by TbAQP-2. Transcriptome data suggests that there are at least three active aquaporins in the human liver fluke, Opisthorchis viverrini, OvAQP-1, 2 and 3, and crude RNA silencing of OvAQP-1 and 2 has recently been shown to affect parasite swelling in destilled water. In the present work we demonstrate that OvAQP-3 is a major water-conducting channel of the parasite, that it can be detected from the newly excysted juvenile to the adult stage and that it is present in major tissues of the parasite. Furthermore, a comparative functional characterization of the three parasite AQPs was performed by using Xenopus oocyte swelling and yeast phenotypic assays. OvAQP-1, OvAQP-2, and OvAQP-3 were found to conduct water and glycerol while only the latter two were also able to conduct urea. In addition, all OvAQPs were found to transport ammonia and methylamine. Our findings demonstrate that the sequence-based classification into orthodox aquaporins and glycerol-conducting aquaglyceroporins is not functionally conserved in the parasite and implicate a broder range of functions for these channels.

  2. 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.

  3. Differential association of rat liver heparan sulfate proteoglycans in membranes of the Golgi apparatus and the plasma membrane

    SciTech Connect

    Brandan, E.; Hirschberg, C.B.

    1989-06-25

    Heparan sulfate proteoglycans (HSPG) of rat liver are associated with the plasma membrane in a hydrophobic intrinsic and a hydrophilic extrinsic form. We were interested in determining whether or not these two forms could be detected in the Golgi apparatus, the subcellular site of addition of oligosaccharides and sulfate to HSPG. In vivo and in vitro radiolabeled HSPG from rat liver Golgi apparatus membranes could only be solubilized with detergents that disrupt the membrane lipid bilayer, suggesting that they are solely associated via hydrophobic interactions. Both forms of HSPG were detected in plasma membranes of rat liver and isolated rat hepatocytes. The detergent-solubilized HSPG bound to octyl-Sepharose columns, whereas the hydrophilic form did not; this latter form, however, was released from the membrane by heparin. The hydrophobic anchor of HSPG in the Golgi and plasma membranes was insensitive to treatment with phosphatidylinositol-specific phospholipase C under conditions in which alkaline phosphatase was sensitive; this suggests that the hydrophobic anchor of HSPG is the core protein itself. Preliminary experiments suggest that the subcellular site of processing of the hydrophobic to the hydrophilic form of HSPG is the plasma membrane. A specific processing activity, probably a protease of the plasma membrane not present in serum or the endoplasmic reticulum membrane, converted hydrophobic HSPG of the Golgi membrane to the hydrophilic form. In addition, pulse-chase experiments with (35S)Na2SO4 in rats demonstrated that at short times, the bulk of the radiolabeled cellular HSPG was in the Golgi apparatus; later on, the bulk of the radioactivity was found in the plasma membrane, the only subcellular site where the hydrophilic form of HSPG was detected.

  4. Inhibition of the aquaporin 3 water channel increases the sensitivity of prostate cancer cells to cryotherapy

    PubMed Central

    Ismail, M; Bokaee, S; Davies, J; Harrington, K J; Pandha, H

    2009-01-01

    Aquaporins (AQPs) are intrinsic membrane proteins that facilitate selective water and small solute movement across the plasma membrane. In this study, we investigate the role of inhibiting AQPs in sensitising prostate cancer cells to cryotherapy. PC-3 and DU145 prostate cancer cells were cooled to 0, −5 and −10°C. The expression of AQP3 in response to freezing was determined using real-time quantitative polymerase chain reaction (RT–qPCR) and western blot analysis. Aquaporins were inhibited using mercuric chloride (HgCl2) and small interfering RNA (siRNA) duplex, and cell survival was assessed using a colorimetric assay. There was a significant increase in AQP3 expression in response to freezing. Cells treated with AQP3 siRNA were more sensitive to cryoinjury compared with control cells (P<0.001). Inhibition of the AQPs by HgCl2 also increased the sensitivity of both cell lines to cryoinjury and there was a complete loss of cell viability at −10°C (P<0.01). In conclusion, we have shown that AQP3 is involved directly in cryoinjury. Inhibition of AQP3 increases the sensitivity of prostate cancer cells to freezing. This strategy may be exploited in the clinic to improve the efficacy of prostate cryotherapy. PMID:19513079

  5. AqF026 Is a Pharmacologic Agonist of the Water Channel Aquaporin-1

    PubMed Central

    Morelle, Johann; Cnops, Yvette; Verbavatz, Jean-Marc; Campbell, Ewan M.; Beckett, Elizabeth A.H.; Booker, Grant W.; Flynn, Gary

    2013-01-01

    Aquaporin-1 (AQP1) facilitates the osmotic transport of water across the capillary endothelium, among other cell types, and thereby has a substantial role in ultrafiltration during peritoneal dialysis. At present, pharmacologic agents that enhance AQP1-mediated water transport, which would be expected to increase the efficiency of peritoneal dialysis, are not available. Here, we describe AqF026, an aquaporin agonist that is a chemical derivative of the arylsulfonamide compound furosemide. In the Xenopus laevis oocyte system, extracellular AqF026 potentiated the channel activity of human AQP1 by >20% but had no effect on channel activity of AQP4. We found that the intracellular binding site for AQP1 involves loop D, a region associated with channel gating. In a mouse model of peritoneal dialysis, AqF026 enhanced the osmotic transport of water across the peritoneal membrane but did not affect the osmotic gradient, the transport of small solutes, or the localization and expression of AQP1 on the plasma membrane. Furthermore, AqF026 did not potentiate water transport in Aqp1-null mice, suggesting that indirect mechanisms involving other channels or transporters were unlikely. Last, in a mouse gastric antrum preparation, AqF026 did not affect the Na-K-Cl cotransporter NKCC1. In summary, AqF026 directly and specifically potentiates AQP1-mediated water transport, suggesting that it deserves additional investigation for applications such as peritoneal dialysis or clinical situations associated with defective water handling. PMID:23744886

  6. Aquaporin Biology and Nervous System

    PubMed Central

    Barbara, Buffoli

    2010-01-01

    Our understanding of the movement of water through cell membranes has been greatly advanced by the discovery of a family of water-specific, membrane-channel proteins: the Aquaporins (AQPs). These proteins are present in organisms at all levels of life, and their unique permeability characteristics and distribution in numerous tissues indicate diverse roles in the regulation of water homeostasis. Phenotype analysis of AQP knock-out mice has confirmed the predicted role of AQPs in osmotically driven transepithelial fluid transport, as occurs in the urinary concentrating mechanism and glandular fluid secretion. Regarding their expression in nervous system, there are evidences suggesting that AQPs are differentially expressed in the peripheral versus central nervous system and that channel-mediated water transport mechanisms may be involved in cerebrospinal fluid formation, neuronal signal transduction and information processing. Moreover, a number of recent studies have revealed the importance of mammalian AQPs in both physiological and pathophysiological mechanisms and have suggested that pharmacological modulation of AQP expression and activity may provide new tools for the treatment of variety of human disorders in which water and small solute transport may be involved. For all the AQPs, new contributions to physiological functions are likely to be discovered with ongoing work in this rapidly expanding field of research. PMID:21119880

  7. Phosphatidic acid phosphatase and phospholipdase A activities in plasma membranes from fusing muscle cells.

    PubMed

    Kent, C; Vagelos, P R

    1976-06-17

    Plasma membrane from fusing embryonic muscle cells were assayed for phospholipase A activity to determine if this enzyme plays a role in cell fusion. The membranes were assayed under a variety of conditions with phosphatidylcholine as the substrate and no phospholipase A activity was found. The plasma membranes did contain a phosphatidic acid phosphatase which was optimally active in the presence of Triton X-100 and glycerol. The enzyme activity was constant from pH 5.2 to 7.0, and did not require divalent cations. Over 97% of the phosphatidic acid phosphatase activity was in the particulate fraction. The subcellular distribution of the phosphatidic acid phosphatase was the same as the distributions of the plasma membrane markers, (Na+ + k+)-ATPase and the acetylcholine receptor, which indicates that this phosphatase is located exclusively in the plasma membranes. There was no detectable difference in the phosphatidic acid phosphatase activities of plasma membranes from fusing and non-fusing cells.

  8. Characterization of Membrane Protein Interactions in Plasma Membrane Derived Vesicles with Quantitative Imaging FRET

    PubMed Central

    Sarabipour, Sarvenaz; Del Piccolo, Nuala; Hristova, Kalina

    2016-01-01

    CONSPECTUS Here we describe an experimental tool, termed Quantitative Imaging Förster Resonance Energy Transfer (QI-FRET), which enables the quantitative characterization of membrane protein interactions. The QI-FRET methodology allows us to acquire binding curves and calculate association constants for complex membrane proteins in the native plasma membrane environment. The method utilizes FRET detection, and thus requires that the proteins of interest are labeled with florescent proteins, either FRET donors or FRET acceptors. Since plasma membranes of cells have complex topologies precluding the acquisition of two-dimensional binding curves, the FRET measurements are performed in plasma membrane derived vesicles which bud off cells as a result of chemical or osmotic stress. The results overviewed here are acquired in vesicles produced with an osmotic vesiculation buffer developed in our laboratory, which does not utilize harsh chemicals. The concentrations of the donor-labeled and the acceptor-labeled proteins are determined, along with the FRET efficiencies, in each vesicle. The experiments utilize transient transfection, such that a wide variety of concentrations is sampled. Then, data from hundreds of vesicles are combined to yield dimerization curves. Here we discuss recent findings about the dimerization of receptor tyrosine kinases (RTKs), membrane proteins that control cell growth and differentiation via lateral dimerization in the plasma membrane. We focus on the dimerization of fibroblast growth factor receptor 3 (FGFR3), an RTK that plays a critically important role in skeletal development. We study the role of different FGFR3 domains in FGFR3 dimerization in the absence of ligand, and we show that FGFR3 extracellular domains inhibit unliganded dimerization, while contacts between the juxtamembrane domains, which connect the transmembrane domains to the kinase domains, stabilize the unliganded FGFR3 dimers. Since FGFR3 has been documented to harbor

  9. Voltage- and Tension-Dependent Lipid Mobility in the Outer Hair Cell Plasma Membrane

    NASA Astrophysics Data System (ADS)

    Oghalai, John S.; Zhao, Hong-Bo; Kutz, J. Walter; Brownell, William E.

    2000-01-01

    The mechanism responsible for electromotility of outer hair cells in the ear is unknown but is thought to reside within the plasma membrane. Lipid lateral diffusion in the outer hair cell plasma membrane is a sigmoidal function of transmembrane potential and bathing media osmolality. Cell depolarization or hyposmotic challenge shorten the cell and reduce membrane fluidity by half. Changing the membrane tension with amphipathic drugs results in similar reductions. These dynamic changes in membrane fluidity represent the modulation of membrane tension by lipid-protein interactions. The voltage dependence may be associated with the force-generating motors that contribute to the exquisite sensitivity of mammalian hearing.

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

    PubMed Central

    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. PMID:24381579

  11. 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.

  12. Isolation and characterization of the plasma membrane from the yeast Pichia pastoris.

    PubMed

    Grillitsch, Karlheinz; Tarazona, Pablo; Klug, Lisa; Wriessnegger, Tamara; Zellnig, Günther; Leitner, Erich; Feussner, Ivo; Daum, Günther

    2014-07-01

    Despite similarities of cellular membranes in all eukaryotes, every compartment displays characteristic and often unique features which are important for the functions of the specific organelles. In the present study, we biochemically characterized the plasma membrane of the methylotrophic yeast Pichia pastoris with emphasis on the lipids which form the matrix of this compartment. Prerequisite for this effort was the design of a standardized and reliable isolation protocol of the plasma membrane at high purity. Analysis of isolated plasma membrane samples from P. pastoris revealed an increase of phosphatidylserine and a decrease of phosphatidylcholine compared to bulk membranes. The amount of saturated fatty acids in the plasma membrane was higher than in total cell extracts. Ergosterol, the final product of the yeast sterol biosynthetic pathway, was found to be enriched in plasma membrane fractions, although markedly lower than in Saccharomyces cerevisiae. A further characteristic feature of the plasma membrane from P. pastoris was the enrichment of inositol phosphorylceramides over neutral sphingolipids, which accumulated in internal membranes. The detailed analysis of the P. pastoris plasma membrane is discussed in the light of cell biological features of this microorganism especially as a microbial cell factory for heterologous protein production. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Plasma membrane calcium ATPase (PMCA4): A housekeeper for RT-PCR relative quantification of polytopic membrane proteins

    PubMed Central

    Calcagno, Anna Maria; Chewning, Katherine J; Wu, Chung-Pu; Ambudkar, Suresh V

    2006-01-01

    Background Although relative quantification of real-time RT-PCR data can provide valuable information, one limitation remains the selection of an appropriate reference gene. No one gene has emerged as a universal reference gene and much debate surrounds some of the more commonly used reference genes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). At this time, no gene encoding for a plasma membrane protein serves as a reference gene, and relative quantification of plasma membrane proteins is performed with genes encoding soluble proteins, which differ greatly in quantity and in targeting and trafficking from plasma membrane proteins. In this work, our aim was to identify a housekeeping gene, ideally one that codes for a plasma membrane protein, whose expression remains the same regardless of drug treatment and across a wide range of tissues to be used for relative quantification of real-time RT-PCR data for ATP binding cassette (ABC) plasma membrane transporters. Results In studies evaluating the expression levels of two commonly used reference genes coding for soluble proteins and two genes coding for membrane proteins, one plasma membrane protein, plasma membrane calcium-ATPase 4 (PMCA4), was comparable to the two reference genes already in use. In addition, PMCA4 expression shows little variation across eight drug-treated cell lines and was found to be superior to GAPDH and HPRT1, commonly used reference genes. Finally, we show PMCA4 used as a reference gene for normalizing ABC transporter expression in a drug-resistant lung carcinoma cell line. Conclusion We have found that PMCA4 is a good housekeeping gene for normalization of gene expression for polytopic membrane proteins including transporters and receptors. PMID:16978418

  14. One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A1

    PubMed Central

    Lee, Yu-Chen; Block, Gregory; Chen, Huiwen; Folch-Puy, Emma; Foronjy, Robert; Jalili, Roxana; Jendresen, Christian Bille; Kimura, Masashi; Kraft, Edward; Lindemose, Søren; Lu, Jin; McLain, Teri; Nutt, Leta; Ramon-Garcia, Santiago; Smith, Joseph; Spivak, Aaron; Wang, Michael L.; Zanic, Marija; Lin, Sue-Hwa

    2008-01-01

    We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic-bead separation to obtain highly purified plasma membrane proteins from crude membrane preparations or cell lines. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. When these ConA magnetic beads were used to enrich plasma membranes from a crude membrane preparation, this procedure resulted in 3.7-fold enrichment of plasma membrane marker 5′-nucleotidase activity with 70% recovery of the activity in the crude membrane fraction of rat liver. In agreement with the results of 5′-nucleotidase activity, immunoblotting with antibodies specific for a rat liver plasma membrane protein, CEACAM1, indicated that CEACAM1 was enriched about threefold relative to that of the original membranes. In similar experiments, this method produced 13-fold enrichment of 5′-nucleotidase activity with 45% recovery of the activity from a total cell lysate of PC-3 cells and 7.1-fold enrichment of 5′-nucleotidase activity with 33% recovery of the activity from a total cell lysate of HeLa cells. These results suggest that this one-step purification method can be used to isolate total plasma membrane proteins from tissue or cells for the identification of membrane biomarkers. PMID:18765283

  15. Very high aquaporin-1 facilitated water permeability in mouse gallbladder

    PubMed Central

    Li, Lihua; Zhang, Hua; Ma, Tonghui; Verkman, A. S.

    2009-01-01

    Water transport across gallbladder epithelium is driven by osmotic gradients generated from active salt absorption and secretion. Aquaporin (AQP) water channels have been proposed to facilitate transepithelial water transport in gallbladder and to modulate bile composition. We found strong AQP1 immunofluorescence at the apical membrane of mouse gallbladder epithelium. Transepithelial osmotic water permeability (Pf) was measured in freshly isolated gallbladder sacs from the kinetics of luminal calcein self-quenching in response to an osmotic gradient. Pf was very high (0.12 cm/s) in gallbladders from wild-type mice, cAMP independent, and independent of osmotic gradient size and direction. Although gallbladders from AQP1 knockout mice had similar size and morphology to those from wild-type mice, their Pf was reduced by ∼10-fold. Apical plasma membrane water permeability was greatly reduced in AQP1-deficient gallbladders, as measured by cytoplasmic calcein quenching in perfluorocarbon-filled, inverted gallbladder sacs. However, neither bile osmolality nor bile salt concentration differed in gallbladders from wild-type vs. AQP1 knockout mice. Our data indicate constitutively high water permeability in mouse gallbladder epithelium involving transcellular water transport through AQP1. The similar bile salt concentration in gallbladders from AQP1 knockout mice argues against a physiologically important role for AQP1 in mouse gallbladder. PMID:19179619

  16. Osmotic water permeability diversification in primary trophoblast cultures from aquaporin 1-deficient pregnant mice.

    PubMed

    Sha, Xiao-Yan; Liu, Hui-Shu; Ma, Tong-Hui

    2015-09-01

    Aquaporins (AQP) are water channel proteins, and some play an important role in maternal-fetal fluid exchange. The present study aimed to measure the osmotic water permeability in primary cultures of trophoblast cells from AQP1-deficient (AQP1(-/-) ) pregnant mice and to define the quantitative role of AQP1 in water transport across the trophoblast plasma membrane. Trophoblast cells were obtained from placental tissue cell culture of AQP1(-/-) pregnant mice and were characterized by cytokeratin 7 immunostaining. The expression of the AQP1 gene in trophoblast cells of wild-type (AQP1(+/+) ) mice was confirmed by immunofluorescence. The osmotic water permeability of trophoblast plasma membranes was measured by a calcein fluorescence quenching method in response to osmotic gradients. A primary cell culture system for trophoblasts was successfully established. Immunofluorescence showed the expression of AQP1 in the trophoblast cell membrane of AQP1(+/+) mice. The osmotic water permeability of AQP1(-/-) trophoblast cells was significantly lower than that in AQP1(+/+) trophoblast cells, in response to both hypotonic and hypertonic challenges. The results suggest an important role of AQP1-mediated plasma membrane water permeability in maternal-fetal fluid balance and also provide a potential direction for the identification of therapeutic targets for the treatment of abnormalities in amniotic fluid volume. © 2015 Japan Society of Obstetrics and Gynecology.

  17. Expression and immunolocalization of aquaporin-6 (Aqp6) in the rat inner ear.

    PubMed

    Taguchi, Daizo; Takeda, Taizo; Kakigi, Akinobu; Okada, Teruhiko; Nishioka, Rie; Kitano, Hiroya

    2008-08-01

    Since aquaporin-6 (Aqp6) protein was located in the membrane of intracellular vesicles of the stria vascularis, endolymphatic sac, and vestibule, Aqp6 might be involved in some distinct physiological function of acid-base metabolism and water balance in endolymphatic fluid homeostasis. However, its lack of expression on the plasma membrane indicates that Aqp6 does not have a direct role in water flux via the plasma membrane. To evaluate the expression and immunolocalization of Aqp6 in the rat inner ear. Wistar rats were used. Aqp6 mRNA expression in the rat inner ear was investigated in the vestibulum as well as in the cochlea and endolymphatic sac using the reverse transcription-polymerase chain reaction (RT-PCR) method, and detailed immunolocalization of Aqp6 in the rat inner ear was investigated using immunohistochemical methods including immunofluorescence microscopy and immunoelectron microscopy. We obtained novel data showing that not just Aqp6 mRNA but also Aqp6 protein is expressed in the cochlea, endolymphatic sac, and vestibule. Immunoelectron microscopic studies revealed that the immunolabelled gold was diffusely seen in the intracellular area of the stria vascularis, endolymphatic sac, and vestibule, but never in the plasma membranes.

  18. Cyclic AMP is sufficient for triggering the exocytic recruitment of aquaporin-2 in renal epithelial cells

    PubMed Central

    Lorenz, Dorothea; Krylov, Andrey; Hahm, Daniel; Hagen, Volker; Rosenthal, Walter; Pohl, Peter; Maric, Kenan

    2003-01-01

    The initial response of renal epithelial cells to the antidiuretic hormone arginine vasopressin (AVP) is an increase in cyclic AMP. By applying immunofluorescence, cell membrane capacitance and transepithelial water flux measurements we show that cAMP alone is sufficient to elicit the antidiuretic cellular response in primary cultured epithelial cells from renal inner medulla, namely the transport of aquaporin-2 (AQP2)-bearing vesicles to, and their subsequent fusion with, the plasma membrane (AQP2 shuttle). The AQP2 shuttle is evoked neither by AVP-independent Ca2+ increases nor by AVP-induced Ca2+ increases. However, clamping cytosolic Ca2+ concentrations below resting levels at 25 nM inhibited exocytosis. Exocytosis was confined to a slow monophasic response, and readily releasable vesicles were missing. Analysis of endocytic capacitance steps revealed that cAMP does not decelerate the retrieval of AQP2 from the plasma membrane. Our data suggest that cAMP initiates an early step, namely the transport of AQP2-bearing vesicles towards the plasma membrane, and do not support a regulatory function for Ca2+ in the AQP2 shuttle. PMID:12524527

  19. Towards a membrane proteome in Drosophila: a method for the isolation of plasma membrane

    PubMed Central

    2010-01-01

    Background The plasma membrane (PM) is a compartment of significant interest because cell surface proteins influence the way in which a cell interacts with its neighbours and its extracellular environment. However, PM is hard to isolate because of its low abundance. Aqueous two-phase affinity purification (2PAP), based on PEG/Dextran two-phase fractionation and lectin affinity for PM-derived microsomes, is an emerging method for the isolation of high purity plasma membranes from several vertebrate sources. In contrast, PM isolation techniques in important invertebrate genetic model systems, such as Drosophila melanogaster, have relied upon enrichment by density gradient centrifugation. To facilitate genetic investigation of activities contributing to the content of the PM sub-proteome, we sought to adapt 2PAP to this invertebrate model to provide a robust PM isolation technique for Drosophila. Results We show that 2PAP alone does not completely remove contaminating endoplasmic reticulum and mitochondrial membrane. However, a novel combination of density gradient centrifugation plus 2PAP results in a robust PM preparation. To demonstrate the utility of this technique we isolated PM from fly heads and successfully identified 432 proteins using MudPIT, of which 37% are integral membrane proteins from all compartments. Of the 432 proteins, 22% have been previously assigned to the PM compartment, and a further 34% are currently unassigned to any compartment and represent candidates for assignment to the PM. The remainder have previous assignments to other compartments. Conclusion A combination of density gradient centrifugation and 2PAP results in a robust, high purity PM preparation from Drosophila, something neither technique can achieve on its own. This novel preparation should lay the groundwork for the proteomic investigation of the PM in different genetic backgrounds in Drosophila. Our results also identify two key steps in this procedure: The optimization of

  20. 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.

  1. A Comparative Spin-Label Study of Isolated Plasma Membranes and Plasma Membranes of Whole Cells and Protoplasts from Cold-Hardened and Nonhardened Winter Rye

    PubMed Central

    Windle, John J.

    1988-01-01

    Lipid-lipid and lipid-protein interactions in the plasma membranes of whole cells and protoplasts and an isolated plasma membrane fraction from winter rye (Secale cereale L. cv Puma) have been studied by spin labeling. Spectra were recorded between −40°C and 40°C using the freely diffusing spin-label, 16-doxyl stearic acid, as a midbilayer membrane probe. The probe was reduced by the whole cells and protoplasts and reoxidized by external potassium ferricyanide. The reoxidized probe was assumed to be localized in the plasma membrane. The spectra consisted of the superposition of a narrow and a broad component indicating that both fluid and immobilized lipids were present in the plasma membrane. The two components were separated by digital subtraction of the immobilized component. Temperature profiles of the membranes were developed using the percentage of immobilized lipid present at each temperature and the separation between the outermost hyperfine lines for the fluid lipid component. Lipid immobilization was attributed to lipid-protein interactions, lipid-cell wall interactions, and temperature-induced lipid phase transitions to the gel-state. Temperature profiles were compared for both cold-hardened and nonhardened protoplasts, plasma membranes, and plasma membrane lipids, respectively. Although cold-hardening extended the range of lipid fluidity by 5°C, it had no effect on lipid-protein interactions or activation energies of lipid mobility. Differences were found, however, between the temperature profiles for the different samples, suggesting that alterations in the plasma membrane occurred as a consequence of the isolation methods used. PMID:16666471

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

    PubMed

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

    2016-08-01

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

  3. Involvement of aquaporin channels in water extrusion from biosilica during maturation of sponge siliceous spicules.

    PubMed

    Wang, Xiaohong; Müller, Werner E G

    2015-08-01

    Aquaporins are a family of small, pore-forming, integral cell membrane proteins. This ancient protein family functions as water channels and is found in all kingdoms (including archaea, eubacteria, fungi, plants, and animals). We discovered that in sponges aquaporin plays a novel role during the maturation of spicules, their skeletal elements. Spicules are synthesized enzymatically via silicatein following a polycondensation reaction. During this process, a 1:1 stoichiometric release of water per one Si-O-Si bond formed is produced. The product of silicatein, biosilica, is a fluffy, soft material that must be hardened in order to function as a solid rod. Using the model of the demosponge species Suberites domuncula Olivi, 1792, which expresses aquaporin, cDNA was cloned and the protein was heterologously expressed. The sponge aquaporin is grouped with the type 8 aquaporins. The function of the sponge aquaporin can be blocked by Mn-sulfate (MnSO4) and mercury chloride (HgCl2). Microscopic and functional studies suggest that aquaporin is involved in removal of the reaction water at the site where siliceous spicules are formed. Another molecule that is likely to be involved in biosilica maturation is the mucin/nidogen-like polypeptide. cDNA has also been cloned from S. domuncula. Experimental studies suggest that water extrusion/suctioning from biosilica after enzymatic synthesis during spicule formation involves both aquaporin-mediated water channeling and "polymerization-induced phase separation" facilitated by the mucin/nidogen-like polypeptide.

  4. Glycosidases in the plasma membrane of Ceratitis capitata spermatozoa.

    PubMed

    Intra, Jari; De Caro, Daniela; Perotti, Maria-Elisa; Pasini, Maria Enrica

    2011-02-01

    Fruit flies in the family Tephritidae are rated among the world's most destructive agricultural pests. The Mediterranean fruit fly Ceratitis capitata is emerging as a model organism to study the fertilization in Insects. Three integral proteins with glycosidase activity are present in the plasma membrane of spermatozoa. The glycosidases have been purified and characterized. We have demonstrated the presence of three enzymes, a β-N-acetylhexosaminidase, an α-mannosidase and an α-l-fucosidase. The molecular mass of the native enzymes estimated by gel filtration was 160 kDa for β-N-acetylhexosaminidase, 310 kDa for α-mannosidase and 140 kDa for α-l-fucosidase. SDS-PAGE showed that β-N-acetylhexosaminidase is a dimer of a single protein of 73 kDa, α-mannosidase consists of six subunits with different molecular weights and α-l-fucosidase is a dimer made up by two different monomers. Characterization of the purified enzymes included glycosylation pattern, pI, optimal pH, substrate preference, kinetic properties and thermal stability. Soluble forms similar to the sperm associated glycosidases are present. Polyclonal antibodies raised against synthetic peptides designed from the predicted products of the Drosophila melanogaster genes encoding β-N-acetylhexosaminidase and α-l-fucosidase were used. Immunofluorescence labelling of spermatozoa showed that the enzymes are present in the sperm plasma membrane overlying the acrosome and the tail. This work represents the first report on the characterization in C. capitata of sperm proteins that are potentially involved in primary gamete recognition.

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

    SciTech Connect

    Frisz, Jessica F.; Lou, Kaiyan; Klitzing, Haley A.; Hanafin, William P.; Lizunov, Vladimir; Wilson, Robert L.; Carpenter, Kevin J.; Kim, Raehyun; Hutcheon, Ian D.; Zimmerberg, Joshua; Weber, Peter K.; Kraft, Mary L.

    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 diameters 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.

  6. 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

  7. 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

  8. Subcellular localization of selectively permeable aquaporins in the male germ line of a marine teleost reveals spatial redistribution in activated spermatozoa.

    PubMed

    Chauvigné, François; Boj, Mónica; Vilella, Sebastiano; Finn, Roderick Nigel; Cerdà, Joan

    2013-08-01

    In oviparous vertebrates such as the marine teleost gilthead seabream, water and fluid homeostasis associated with testicular physiology and the external activation of spermatozoa is potentially mediated by multiple aquaporins. To test this hypothesis, we isolated five novel members of the aquaporin superfamily from gilthead seabream and developed paralog-specific antibodies to localize the cellular sites of protein expression in the male reproductive tract. Together with phylogenetic classification, functional characterization of four of the newly isolated paralogs, Aqp0a, -7, -8b, and -9b, demonstrated that they were water permeable, while Aqp8b was also permeable to urea, and Aqp7 and -9b were permeable to glycerol and urea. Immunolocalization experiments indicated that up to seven paralogous aquaporins are differentially expressed in the seabream testis: Aqp0a and -9b in Sertoli and Leydig cells, respectively; Aqp1ab, -7, and -10b from spermatogonia to spermatozoa; and Aqp1aa and -8b in spermatids and sperm. In the efferent duct, only Aqp10b was found in the luminal epithelium. Ejaculated spermatozoa showed a segregated spatial distribution of five aquaporins: Aqp1aa and -7 in the entire flagellum or the head, respectively, and Aqp1ab, -8b, and -10b both in the head and the anterior tail. The combination of immunofluorescence microscopy and biochemical fractionation of spermatozoa indicated that Aqp10b and phosphorylated Aqp1ab are rapidly translocated to the head plasma membrane upon activation, whereas Aqp8b accumulates in the mitochondrion of the spermatozoa. In contrast, Aqp1aa and -7 remained unchanged. These data reveal that aquaporin expression in the teleost testis shares conserved features of the mammalian system, and they suggest that the piscine channels may play different roles in water and solute transport during spermatogenesis, sperm maturation and nutrition, and the initiation and maintenance of sperm motility.

  9. Drought, Abscisic Acid and Transpiration Rate Effects on the Regulation of PIP Aquaporin Gene Expression and Abundance in Phaseolus vulgaris Plants

    PubMed Central

    AROCA, RICARDO; FERRANTE, ANTONIO; VERNIERI, PAOLO; CHRISPEELS, MAARTEN J.

    2006-01-01

    • Background and Aims Drought causes a decline of root hydraulic conductance, which aside from embolisms, is governed ultimately by aquaporins. Multiple factors probably regulate aquaporin expression, abundance and activity in leaf and root tissues during drought; among these are the leaf transpiration rate, leaf water status, abscisic acid (ABA) and soil water content. Here a study is made of how these factors could influence the response of aquaporin to drought. • Methods Three plasma membrane intrinsic proteins (PIPs) or aquaporins were cloned from Phaseolus vulgaris plants and their expression was analysed after 4 d of water deprivation and also 1 d after re-watering. The effects of ABA and of methotrexate (MTX), an inhibitor of stomatal opening, on gene expression and protein abundance were also analysed. Protein abundance was examined using antibodies against PIP1 and PIP2 aquaporins. At the same time, root hydraulic conductance (L), transpiration rate, leaf water status and ABA tissue concentration were measured. • Key Results None of the treatments (drought, ABA or MTX) changed the leaf water status or tissue ABA concentration. The three treatments caused a decline in the transpiration rate and raised PVPIP2;1 gene expression and PIP1 protein abundance in the leaves. In the roots, only the drought treatment raised the expression of the three PIP genes examined, while at the same time diminishing PIP2 protein abundance and L. On the other hand, ABA raised both root PIP1 protein abundance and L. • Conclusions The rise of PvPIP2;1 gene expression and PIP1 protein abundance in the leaves of P. vulgaris plants subjected to drought was correlated with a decline in the transpiration rate. At the same time, the increase in the expression of the three PIP genes examined caused by drought and the decline of PIP2 protein abundance in the root tissues were not correlated with any of the parameters measured. PMID:17028296

  10. Water Uptake along the Length of Grapevine Fine Roots: Developmental Anatomy, Tissue-Specific Aquaporin Expression, and Pathways of Water Transport1[W][OPEN

    PubMed Central

    Gambetta, Gregory A.; Fei, Jiong; Rost, Thomas L.; Knipfer, Thorsten; Matthews, Mark A.; Shackel, Ken A.; Walker, M. Andrew; McElrone, Andrew J.

    2013-01-01

    To better understand water uptake patterns in root systems of woody perennial crops, we detailed the developmental anatomy and hydraulic physiology along the length of grapevine (Vitis berlandieri × Vitis rupestris) fine roots from the tip to secondary growth zones. Our characterization included the localization of suberized structures and aquaporin gene expression and the determination of hydraulic conductivity (Lpr) and aquaporin protein activity (via chemical inhibition) in different root zones under both osmotic and hydrostatic pressure gradients. Tissue-specific messenger RNA levels of the plasma membrane aquaporin isogenes (VvPIPs) were quantified using laser-capture microdissection and quantitative polymerase chain reaction. Our results highlight dramatic changes in structure and function along the length of grapevine fine roots. Although the root tip lacked suberization altogether, a suberized exodermis and endodermis developed in the maturation zone, which gave way to the secondary growth zone containing a multilayer suberized periderm. Longitudinally, VvPIP isogenes exhibited strong peaks of expression in the root tip that decreased precipitously along the root length in a pattern similar to Arabidopsis (Arabidopsis thaliana) roots. In the radial orientation, expression was always greatest in interior tissues (i.e. stele, endodermis, and/or vascular tissues) for all root zones. High Lpr and aquaporin protein activity were associated with peak VvPIP expression levels in the root tip. This suggests that aquaporins play a limited role in controlling water uptake in secondary growth zones, which contradicts existing theoretical predictions. Despite having significantly lower Lpr, woody roots can constitute the vast majority of the root system surface area in mature vines and thus provide for significant water uptake potential. PMID:24047863

  11. Changes in Air CO2 Concentration Differentially Alter Transcript Levels of NtAQP1 and NtPIP2;1 Aquaporin Genes in Tobacco Leaves

    PubMed Central

    Secchi, Francesca; Schubert, Andrea; Lovisolo, Claudio

    2016-01-01

    The aquaporin specific control on water versus carbon pathways in leaves is pivotal in controlling gas exchange and leaf hydraulics. We investigated whether Nicotiana tabacum aquaporin 1 (NtAQP1) and Nicotiana tabacum plasma membrane intrinsic protein 2;1 (NtPIP2;1) gene expression varies in tobacco leaves subjected to treatments with different CO2 concentrations (ranging from 0 to 800 ppm), inducing changes in photosynthesis, stomatal regulation and water evaporation from the leaf. Changes in air CO2 concentration ([CO2]) affected net photosynthesis (Pn) and leaf substomatal [CO2] (Ci). Pn was slightly negative at 0 ppm air CO2; it was one-third that of ambient controls at 200 ppm, and not different from controls at 800 ppm. Leaves fed with 800 ppm [CO2] showed one-third reduced stomatal conductance (gs) and transpiration (E), and their gs was in turn slightly lower than in 200 ppm– and in 0 ppm–treated leaves. The 800 ppm air [CO2] strongly impaired both NtAQP1 and NtPIP2;1 gene expression, whereas 0 ppm air [CO2], a concentration below any in vivo possible conditions and specifically chosen to maximize the gene expression alteration, increased only the NtAQP1 transcript level. We propose that NtAQP1 expression, an aquaporin devoted to CO2 transport, positively responds to CO2 scarcity in the air in the whole range 0–800 ppm. On the contrary, expression of NtPIP2;1, an aquaporin not devoted to CO2 transport, is related to water balance in the leaf, and changes in parallel with gs. These observations fit in a model where upregulation of leaf aquaporins is activated at low Ci, while downregulation occurs when high Ci saturates photosynthesis and causes stomatal closure. PMID:27089333

  12. Water uptake along the length of grapevine fine roots: developmental anatomy, tissue-specific aquaporin expression, and pathways of water transport.

    PubMed

    Gambetta, Gregory A; Fei, Jiong; Rost, Thomas L; Knipfer, Thorsten; Matthews, Mark A; Shackel, Ken A; Walker, M Andrew; McElrone, Andrew J

    2013-11-01

    To better understand water uptake patterns in root systems of woody perennial crops, we detailed the developmental anatomy and hydraulic physiology along the length of grapevine (Vitis berlandieri × Vitis rupestris) fine roots from the tip to secondary growth zones. Our characterization included the localization of suberized structures and aquaporin gene expression and the determination of hydraulic conductivity (Lpr) and aquaporin protein activity (via chemical inhibition) in different root zones under both osmotic and hydrostatic pressure gradients. Tissue-specific messenger RNA levels of the plasma membrane aquaporin isogenes (VvPIPs) were quantified using laser-capture microdissection and quantitative polymerase chain reaction. Our results highlight dramatic changes in structure and function along the length of grapevine fine roots. Although the root tip lacked suberization altogether, a suberized exodermis and endodermis developed in the maturation zone, which gave way to the secondary growth zone containing a multilayer suberized periderm. Longitudinally, VvPIP isogenes exhibited strong peaks of expression in the root tip that decreased precipitously along the root length in a pattern similar to Arabidopsis (Arabidopsis thaliana) roots. In the radial orientation, expression was always greatest in interior tissues (i.e. stele, endodermis, and/or vascular tissues) for all root zones. High Lpr and aquaporin protein activity were associated with peak VvPIP expression levels in the root tip. This suggests that aquaporins play a limited role in controlling water uptake in secondary growth zones, which contradicts existing theoretical predictions. Despite having significantly lower Lpr, woody roots can constitute the vast majority of the root system surface area in mature vines and thus provide for significant water uptake potential.

  13. Intrarenal localization of the plasma membrane ATP channel pannexin1.

    PubMed

    Hanner, Fiona; Lam, Lisa; Nguyen, Mien T X; Yu, Alan; Peti-Peterdi, János

    2012-11-15

    In the renal tubules, ATP released from epithelial cells stimulates purinergic receptors, regulating salt and water reabsorption. However, the mechanisms by which ATP is released into the tubular lumen are multifaceted. Pannexin1 (Panx1) is a newly identified. ubiquitously expressed protein that forms connexin-like channels in the plasma membrane, which have been demonstrated to function as a mechanosensitive ATP conduit. Here, we report on the localization of Panx1 in the mouse kidney. Using immunofluorescence, strong Panx1 expression was observed in renal tubules, including proximal tubules, thin descending limbs, and collecting ducts, along their apical cell membranes. In the renal vasculature, Panx1 expression was localized to vascular smooth muscle cells in renal arteries, including the afferent and efferent arterioles. Additionally, we tested whether Panx1 channels expressed in renal epithelial cells facilitate luminal ATP release by measuring the ATP content of urine samples freshly collected from wild-type and Panx1(-/-) mice. Urinary ATP levels were reduced by 30% in Panx1(-/-) compared with wild-type mice. These results suggest that Panx1 channels in the kidney may regulate ATP release and via purinergic signaling may participate in the control of renal epithelial fluid and electrolyte transport and vascular functions.

  14. Intrarenal localization of the plasma membrane ATP channel pannexin1

    PubMed Central

    Hanner, Fiona; Lam, Lisa; Nguyen, Mien T. X.; Yu, Alan

    2012-01-01

    In the renal tubules, ATP released from epithelial cells stimulates purinergic receptors, regulating salt and water reabsorption. However, the mechanisms by which ATP is released into the tubular lumen are multifaceted. Pannexin1 (Panx1) is a newly identified. ubiquitously expressed protein that forms connexin-like channels in the plasma membrane, which have been demonstrated to function as a mechanosensitive ATP conduit. Here, we report on the localization of Panx1 in the mouse kidney. Using immunofluorescence, strong Panx1 expression was observed in renal tubules, including proximal tubules, thin descending limbs, and collecting ducts, along their apical cell membranes. In the renal vasculature, Panx1 expression was localized to vascular smooth muscle cells in renal arteries, including the afferent and efferent arterioles. Additionally, we tested whether Panx1 channels expressed in renal epithelial cells facilitate luminal ATP release by measuring the ATP content of urine samples freshly collected from wild-type and Panx1−/− mice. Urinary ATP levels were reduced by 30% in Panx1−/− compared with wild-type mice. These results suggest that Panx1 channels in the kidney may regulate ATP release and via purinergic signaling may participate in the control of renal epithelial fluid and electrolyte transport and vascular functions. PMID:22952282

  15. Yeast mutants affecting possible quality control of plasma membrane proteins.

    PubMed

    Li, Y; Kane, T; Tipper, C; Spatrick, P; Jenness, D D

    1999-05-01

    Mutations gef1, stp22, STP26, and STP27 in Saccharomyces cerevisiae were identified as suppressors of the temperature-sensitive alpha-factor receptor (mutation ste2-3) and arginine permease (mutation can1(ts)). These suppressors inhibited the elimination of misfolded receptors (synthesized at 34 degrees C) as well as damaged surface receptors (shifted from 22 to 34 degrees C). The stp22 mutation (allelic to vps23 [M. Babst and S. Emr, personal communication] and the STP26 mutation also caused missorting of carboxypeptidase Y, and ste2-3 was suppressed by mutations vps1, vps8, vps10, and vps28 but not by mutation vps3. In the stp22 mutant, both the mutant and the wild-type receptors (tagged with green fluorescent protein [GFP]) accumulated within an endosome-like compartment and were excluded from the vacuole. GFP-tagged Stp22p also accumulated in this compartment. Upon reaching the vacuole, cytoplasmic domains of both mutant and wild-type receptors appeared within the vacuolar lumen. Stp22p and Gef1p are similar to tumor susceptibility protein TSG101 and voltage-gated chloride channel, respectively. These results identify potential elements of plasma membrane quality control and indicate that cytoplasmic domains of membrane proteins are translocated into the vacuolar lumen.

  16. Photodynamic activity of substituted zinc trisulfophthalocyanines: role of plasma membrane damage.

    PubMed

    Cauchon, Nicole; Nader, Moni; Bkaily, Ghassan; van Lier, Johan E; Hunting, Darel

    2006-01-01

    We recently reported that variations in cellular phototoxicity among a series of alkynyl-substituted zinc trisulfophthalocyanines (ZnPcS3Cn) correlates with their hydrophobicity, with the most amphiphilic derivatives showing the highest cell uptake and phototoxicity. In this study we address the role of the plasma membrane in the photodynamic response as it relates to the overall hydrophobicity of the photosensitizer. The membrane tracker dye 1-[4(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), which is incorporated into plasma membranes by endocytosis, was used to establish plasma membrane uptake by EMT-6 cells of the ZnPcS3C, by colocalization, and TMA-DPH membrane uptake rates after photodynamic therapy were used to quantify membrane damage. TMA-DPH colocalization patterns show plasma membrane uptake of the photosensitizers after short 1 h incubation periods. TMA-DPH plasma membrane uptake rates after illumination of the photosensitizer-treated cells show a parabolic relationship with photosensitizer hydrophobicity that correlates well with the phototoxicity of the ZnPcS3C,. After a 1 h incubation period, overall phototoxicity correlates closely with the postillumination rate of TMA-DPH incorporation into the cell membrane, suggesting a major role of plasma membrane damage in the overall PDT effect. In contrast, after a 24 h incubation, phototoxicity shows a stronger but imperfect correlation with total cellular photosensitizer uptake rather than TMA-DPH membrane uptake, suggesting a partial shift in the cellular damage responsible for photosensitization from the plasma membrane to intracellular targets. We conclude that plasma membrane localization of the amphiphilic ZnPcS3C6-C9 is a major factor in their overall photodynamic activity.

  17. β-Glucan Synthetases of Plasma Membrane and Golgi Apparatus from Onion Stem 1

    PubMed Central

    Van Der Woude, William J.; Lembi, Carole A.; Morré, D. James; Kindinger, Jaunita I.; Ordin, Lawrence

    1974-01-01

    Biosynthesis of glucans occurred in cell-free fractions isolated from onion stem (Allium cepa L.) enriched in either dictyosomes or plasma membranes. β-1,3- and β-1, 4-Glucans were synthesized in differing proportions and at different rates as the concentration of uridine diphosphoglucose or the proportion of dictyosomes or plasma membrane varied. At low (1.5 μm) UDP-glucose concentrations synthesis of alkali-insoluble glucan was correlated with abundance of dicytosomes; most of the substrate utilized by plasma membrane was for glycolipid synthesis. At high (1 mm) UDP-glucose concentration, the synthesis of alkali-insoluble glucans correlated with the abundance of plasma membrane. Substrate enhancement of β-1, 4-glucan synthesis in dictyosome fractions was less than proportional to increases in substrate concentration. In contrast, β-1, 4-glucan synthesis by plasma membrane was more than proportionately increased. At high substrate concentrations the synthesis of β-1, 3-glucans predominated in both dictyosome and plasma membrane fractions. The results show that the capacity to synthesize glucans resides in both Golgi apparatus and plasma membranes of onion stem, but that the plasma membrane has the greatest capacity for synthesis of alkali-insoluble glucans at high UDP-glucose concentrations. Images PMID:16658884

  18. Rapid Response of the Yeast Plasma Membrane Proteome to Salt Stress*

    PubMed Central

    Szopinska, Aleksandra; Degand, Hervé; Hochstenbach, Jean-François; Nader, Joseph; Morsomme, Pierre

    2011-01-01

    The plasma membrane separates the cell from the external environment and plays an important role in the stress response of the cell. In this study, we compared plasma membrane proteome modifications of yeast cells exposed to mild (0.4 m NaCl) or high (1 m NaCl) salt stress for 10, 30, or 90 min. Plasma membrane-enriched fractions were isolated, purified, and subjected to iTRAQ labeling for quantitative analysis. In total, 88–109 plasma membrane proteins were identified and quantified. The quantitative analysis revealed significant changes in the abundance of several plasma membrane proteins. Mild salt stress caused an increase in abundance of 12 plasma membrane proteins, including known salt-responsive proteins, as well as new targets. Interestingly, 20 plasma membrane proteins, including the P-type H+-ATPase Pma1, ABC transporters, glucose and amino acid transporters, t-SNAREs, and proteins involved in cell wall biogenesis showed a significant and rapid decrease in abundance in response to both 0.4 m and 1 m NaCl. We propose that rapid protein internalization occurs as a response to hyper-osmotic and/or ionic shock, which might affect plasma membrane morphology and ionic homeostasis. This rapid response might help the cell to survive until the transcriptional response takes place. PMID:21825281

  19. Enzymes of phosphoinositide synthesis in secretory vesicles destined for the plasma membrane in Saccharomyces cerevisiae.

    PubMed

    Kinney, A J; Carman, G M

    1990-07-01

    CDP-diacylglycerol synthase, phosphatidylinositol synthase, and phosphatidylinositol kinase activities were associated with post-Golgi apparatus secretory vesicles destined for the plasma membrane of Saccharomyces cerevisiae. These results suggest that the plasma membrane is capable of synthesizing both CDP-diacylglycerol and phosphatidylinositol as well as phosphorylating phosphatidylinositol.

  20. Detection of boar sperm plasma membrane protein using Rhodamine 640; implications for cryobiology and physiology

    USDA-ARS?s Scientific Manuscript database

    Rhodamine 640 (R640) was used to detect changes in boar sperm plasma membrane protein (PMP) during cryopreservation; a poorly understood phenomenon. The protocol was adapted for boar sperm so that semen samples (n = 17) could be analyzed for PMP (R640 positive) and plasma membrane integrity (PMI; Y...

  1. Actin dynamics provides membrane tension to merge fusing vesicles into the plasma membrane

    PubMed Central

    Wen, Peter J.; Grenklo, Staffan; Arpino, Gianvito; Tan, Xinyu; Liao, Hsien-Shun; Heureaux, Johanna; Peng, Shi-Yong; Chiang, Hsueh-Cheng; Hamid, Edaeni; Zhao, Wei-Dong; Shin, Wonchul; Näreoja, Tuomas; Evergren, Emma; Jin, Yinghui; Karlsson, Roger; Ebert, Steven N.; Jin, Albert; Liu, Allen P.; Shupliakov, Oleg; Wu, Ling-Gang

    2016-01-01

    Vesicle fusion is executed via formation of an Ω-shaped structure (Ω-profile), followed by closure (kiss-and-run) or merging of the Ω-profile into the plasma membrane (full fusion). Although Ω-profile closure limits release but recycles vesicles economically, Ω-profile merging facilitates release but couples to classical endocytosis for recycling. Despite its crucial role in determining exocytosis/endocytosis modes, how Ω-profile merging is mediated is poorly understood in endocrine cells and neurons containing small ∼30–300 nm vesicles. Here, using confocal and super-resolution STED imaging, force measurements, pharmacology and gene knockout, we show that dynamic assembly of filamentous actin, involving ATP hydrolysis, N-WASP and formin, mediates Ω-profile merging by providing sufficient plasma membrane tension to shrink the Ω-profile in neuroendocrine chromaffin cells containing ∼300 nm vesicles. Actin-directed compounds also induce Ω-profile accumulation at lamprey synaptic active zones, suggesting that actin may mediate Ω-profile merging at synapses. These results uncover molecular and biophysical mechanisms underlying Ω-profile merging. PMID:27576662

  2. Analysis of aquaporins from the euryhaline barnacle Balanus improvisus reveals differential expression in response to changes in salinity.

    PubMed

    Lind, Ulrika; Järvå, Michael; Alm Rosenblad, Magnus; Pingitore, Piero; Karlsson, Emil; Wrange, Anna-Lisa; Kamdal, Emelie; Sundell, Kristina; André, Carl; Jonsson, Per R; Havenhand, Jon; Eriksson, Leif A; Hedfalk, Kristina; Blomberg, Anders

    2017-01-01

    Barnacles are sessile macro-invertebrates, found along rocky shores in coastal areas worldwide. The euryhaline bay barnacle Balanus improvisus (Darwin, 1854) (= Amphibalanus improvisus) can tolerate a wide range of salinities, but the molecular mechanisms underlying the osmoregulatory capacity of this truly brackish species are not well understood. Aquaporins are pore-forming integral membrane proteins that facilitate transport of water, small solutes and ions through cellular membranes, and that have been shown to be important for osmoregulation in many organisms. The knowledge of the function of aquaporins in crustaceans is, however, limited and nothing is known about them in barnacles. We here present the repertoire of aquaporins from a thecostracan crustacean, the barnacle B. improvisus, based on genome and transcriptome sequencing. Our analyses reveal that B. improvisus contains eight genes for aquaporins. Phylogenetic analysis showed that they represented members of the classical water aquaporins (Aqp1, Aqp2), the aquaglyceroporins (Glp1, Glp2), the unorthodox aquaporin (Aqp12) and the arthropod-specific big brain aquaporin (Bib). Interestingly, we also found two big brain-like proteins (BibL1 and BibL2) constituting a new group of aquaporins not yet described in arthropods. In addition, we found that the two water-specific aquaporins were expressed as C-terminal splice variants. Heterologous expression of some of the aquaporins followed by functional characterization showed that Aqp1 transported water and Glp2 water and glycerol, agreeing with the predictions of substrate specificity based on 3D modeling and phylogeny. To investigate a possible role for the B. improvisus aquaporins in osmoregulation, mRNA expression changes in adult barnacles were analysed after long-term acclimation to different salinities. The most pronounced expression difference was seen for AQP1 with a substantial (>100-fold) decrease in the mantle tissue in low salinity (3 PSU

  3. Analysis of aquaporins from the euryhaline barnacle Balanus improvisus reveals differential expression in response to changes in salinity

    PubMed Central

    Järvå, Michael; Alm Rosenblad, Magnus; Pingitore, Piero; Karlsson, Emil; Wrange, Anna-Lisa; Kamdal, Emelie; Sundell, Kristina; André, Carl; Jonsson, Per R.; Havenhand, Jon; Eriksson, Leif A.; Hedfalk, Kristina; Blomberg, Anders

    2017-01-01

    Barnacles are sessile macro-invertebrates, found along rocky shores in coastal areas worldwide. The euryhaline bay barnacle Balanus improvisus (Darwin, 1854) (= Amphibalanus improvisus) can tolerate a wide range of salinities, but the molecular mechanisms underlying the osmoregulatory capacity of this truly brackish species are not well understood. Aquaporins are pore-forming integral membrane proteins that facilitate transport of water, small solutes and ions through cellular membranes, and that have been shown to be important for osmoregulation in many organisms. The knowledge of the function of aquaporins in crustaceans is, however, limited and nothing is known about them in barnacles. We here present the repertoire of aquaporins from a thecostracan crustacean, the barnacle B. improvisus, based on genome and transcriptome sequencing. Our analyses reveal that B. improvisus contains eight genes for aquaporins. Phylogenetic analysis showed that they represented members of the classical water aquaporins (Aqp1, Aqp2), the aquaglyceroporins (Glp1, Glp2), the unorthodox aquaporin (Aqp12) and the arthropod-specific big brain aquaporin (Bib). Interestingly, we also found two big brain-like proteins (BibL1 and BibL2) constituting a new group of aquaporins not yet described in arthropods. In addition, we found that the two water-specific aquaporins were expressed as C-terminal splice variants. Heterologous expression of some of the aquaporins followed by functional characterization showed that Aqp1 transported water and Glp2 water and glycerol, agreeing with the predictions of substrate specificity based on 3D modeling and phylogeny. To investigate a possible role for the B. improvisus aquaporins in osmoregulation, mRNA expression changes in adult barnacles were analysed after long-term acclimation to different salinities. The most pronounced expression difference was seen for AQP1 with a substantial (>100-fold) decrease in the mantle tissue in low salinity (3 PSU

  4. Modulation of Plasma Membrane Ca2+-ATPase by Neutral Phospholipids

    PubMed Central

    Pignataro, María Florencia; Dodes-Traian, Martín M.; González-Flecha, F. Luis; Sica, Mauricio; Mangialavori, Irene C.; Rossi, Juan Pablo F. C.

    2015-01-01

    The effects of lipids on membrane proteins are likely to be complex and unique for each membrane protein. Here we studied different detergent/phosphatidylcholine reconstitution media and tested their effects on plasma membrane Ca2+ pump (PMCA). We found that Ca2+-ATPase activity shows a biphasic behavior with respect to the detergent/phosphatidylcholine ratio. Moreover, the maximal Ca2+-ATPase activity largely depends on the length and the unsaturation degree of the hydrocarbon chain. Using static light scattering and fluorescence correlation spectroscopy, we monitored the changes in hydrodynamic radius of detergent/phosphatidylcholine particles during the micelle-vesicle transition. We found that, when PMCA is reconstituted in mixed micelles, neutral phospholipids increase the enzyme turnover. The biophysical changes associated with the transition from mixed micelles to bicelles increase the time of residence of the phosphorylated intermediate (EP), decreasing the enzyme turnover. Molecular dynamics simulations analysis of the interactions between PMCA and the phospholipid bilayer in which it is embedded show that in the 1,2-dioleoyl-sn-glycero-3-phosphocholine bilayer, charged residues of the protein are trapped in the hydrophobic core. Conversely, in the 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer, the overall hydrophobic-hydrophilic requirements of the protein surface are fulfilled the best, reducing the thermodynamic cost of exposing charged residues to the hydrophobic core. The apparent mismatch produced by a 1,2-dioleoyl-sn-glycero-3-phosphocholine thicker bilayer could be a structural foundation to explain its functional effect on PMCA. PMID:25605721

  5. 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.

  6. Sonoporation-induced depolarization of plasma membrane potential: analysis of heterogeneous impact.

    PubMed

    Qin, Peng; Xu, Lin; Hu, Yaxin; Zhong, Wenjing; Cai, Ping; Du, Lianfang; Jin, Lifang; Yu, Alfred C H

    2014-05-01

    Disrupting plasma membrane integrity would inevitably promote anomalous ion fluxes across the membrane and thereby upset the trans-membranous potential. In this article, we report new findings on how sonoporation as a physical membrane perforation strategy would lead to different forms of plasma membrane potential disruption. Our investigation was conducted with a customized fluorescence imaging platform that enabled live monitoring of plasma membrane potential in relation to individual sonoporation events triggered on HeLa cervical cancer cells. Sonovue microbubbles were used as sonoporation agents (added at a 4:3 cell-to-bubble ratio), and they were activated by 1-MHz pulsed ultrasound with 0.35-MPa peak negative pressure, 20-cycle pulse duration, 20-Hz pulse repetition frequency and 1-s total exposure duration. Results indicate that the plasma membrane potential response was heterogeneous among sonoporated cells: (i) membrane potential of irreversibly sonoporated cells was permanently depolarized; (ii) reversibly sonoporated cells exhibited either transient or sustained membrane depolarization; (iii) intact cells adjacent to sonoporated ones underwent transitory membrane depolarization. These findings effectively serve to substantiate the causal relationship between sonoporation and plasma membrane potential. Copyright © 2014 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

  7. Red wine activates plasma membrane redox system in human erythrocytes.

    PubMed

    Tedesco, Idolo; Moccia, Stefania; Volpe, Silvestro; Alfieri, Giovanna; Strollo, Daniela; Bilotto, Stefania; Spagnuolo, Carmela; Di Renzo, Massimo; Aquino, Rita P; Russo, Gian Luigi

    2016-01-01

    In the present study, we report that polyphenols present in red wine obtained by a controlled microvinification process are able to protect human erythrocytes from oxidative stress and to activate Plasma Membrane Redox System (PMRS). Human plasma obtained from healthy subjects was incubated in the presence of whole red wine at a concentration corresponding to 9.13-73 μg/ml gallic acid equivalents to verify the capacity to protect against hypochlorous acid (HOCl)-induced plasma oxidation and to minimize chloramine formation. Red wine reduced hemolysis and chloramine formation induced by HOCl of 40 and 35%, respectively. PMRS present on human erythrocytes transfers electrons from intracellular molecules to extracellular electron acceptors. We demonstrated that whole red wine activated PMRS activity in human erythrocytes isolated from donors in a dose-dependent manner with a maximum at about 70-100 μg/ml gallic acid equivalents. We also showed that red wine increased glutathione (GSH) levels and erythrocytic antioxidant capacity, measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) quenching assay. Furthermore, we reported that GSH played a crucial role in regulating PMRS activity in erythrocytes. In fact, the effect of iodoacetamide, an alkylating agent that induces depletion of intracellular GSH, was completely counteracted by red wine. Bioactive compounds present in red wine, such as gallic acid, resveratrol, catechin, and quercetin were unable to activate PMRS when tested at the concentrations normally present in aged red wines. On the contrary, the increase of PMRS activity was associated with the anthocyanin fraction, suggesting the capacity of this class of compounds to positively modulate PMRS enzymatic activity.

  8. 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

  9. Aquaporins in Cardiovascular System.

    PubMed

    Tie, Lu; Wang, Di; Shi, Yundi; Li, Xuejun

    2017-01-01

    Recent studies have shown that some aquaporins (AQPs ), including AQP1, AQP4, AQP7 and AQP9, are expressed in endothelial cells, vascular smooth muscle cells and heart of cardiovascular system. These AQPs are involved in the cardiovascular function and in pathological process of related diseases, such as cerebral ischemia , congestion heart failure , hypertension and angiogenesis. Therefore, it is important to understand the accurate association between AQPs and cardiovascular system, which may provide novel approaches to prevent and treat related diseases. Here we will discuss the expression and physiological function of AQPs in cardiovascular system and summarize recent researches on AQPs related cardiovascular diseases.

  10. Electrogenic Transport of Protons Driven by the Plasma Membrane ATPase in Membrane Vesicles from Radish 1

    PubMed Central

    Rasi-Caldogno, Franca; Pugliarello, Maria Chiara; De Michelis, Maria Ida

    1985-01-01

    Mg:ATP-dependent H+ pumping has been studied in microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings by monitoring both intravesicular acidification and the building up of an inside positive membrane potential difference (Δ ψ). ΔpH was measured as the decrease of absorbance of Acridine orange and Δ ψ as the shift of absorbance of bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol. Both Mg:ATP-dependent Δ pH and Δ ψ generation are completely inhibited by vanadate and insensitive to oligomycin; moreover, Δ pH generation is not inhibited by NO3−. These findings indicate that this membrane preparation is virtually devoid of mitochondrial and tonoplast H+-ATPases. Both intravesicular acidification and Δ ψ generation are influenced by anions: Δ pH increases and Δ ψ decreases following the sequence SO42−, Cl−, Br−, NO3−. ATP-dependent H+ pumping strictly requires Mg2+. It is very specific for ATP (apparent Km 0.76 millimolar) compared to GTP, UTP, CTP, ITP. Δ pH generation is inhibited by CuSO4 and diethylstilbestrol as well as vanadate. Δ pH generation is specificially stimulated by K+ (+ 80%) and to a lesser extent by Na+ and choline (+28% and +14%, respectively). The characteristics of H+ pumping in these microsomal vesicles closely resemble those described for the plasma membrane ATPase partially purified from several plant materials. PMID:16664008

  11. Enhancement of RNA Polymerase Activity by a Factor Released by Auxin from Plasma Membrane*

    PubMed Central

    Hardin, James W.; Cherry, Joe H.; Morré, D. James; Lembi, Carole A.

    1972-01-01

    Using recently developed techniques for solubilization of RNA polymerase from soybean chromatin and isolation of plasma membrane fractions from plants we can show the presence of a transcriptional factor specifically released from the membranes by auxin, 2,4-dichlorophenoxyacetic acid. The nonauxin, 3,5-dichlorophenoxyacetic acid, does not release the factor, but subsequent exposure of the membranes to auxin results in its release. Factor activity could not be demonstrated in fractions devoid of plasma membranes. The presence of a regulatory factor for RNA polymerase associated with plant plasma membrane and specifically released by auxin provides a mechanism whereby both rapid growth responses and delayed nuclear changes could be derived from a common auxin receptor site associated with plasma membrane. Images PMID:4508307

  12. Effect of calmodulin antagonists on calcium pump of ram spermatozoa plasma membrane.

    PubMed

    Breitbart, H; Rubinstein, S

    1988-01-01

    Plasma membranes isolated from ram spermatozoa contain calmodulin, which represents approximately 0.03% of the total sperm calmodulin and 0.025% of the membrane protein. When membranes were isolated in the presence of ethylene glycol (beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), the amount of calmodulin associated with the plasma membranes was reduced by only 20%. The ATP-dependent calcium transport activity of the isolated plasma membranes is not enhanced by adding calmodulin and not inhibited by the calmodulin antagonists trifluoperazinc (TFP), compound 48/80, or calmidazolium. In fact, there is an enhancement of calcium uptake by the calmodulin antagonists and this enhancement can be blocked by the Ca2+-channel blocker D-600. It is suggested that the ATP-dependent calcium transport activity in the plasma membrane of ram spermatozoa is not regulated by calmodulin.

  13. 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.

  14. Aquaporin-1 facilitates pressure-driven water flow across the aortic endothelium

    PubMed Central

    Nguyen, Tieuvi; Toussaint, Jimmy; Xue, Yan; Raval, Chirag; Cancel, Limary; Russell, Stewart; Shou, Yixin; Sedes, Omer; Sun, Yu; Yakobov, Roman; Tarbell, John M.; Jan, Kung-ming

    2015-01-01

    Aquaporin-1, a ubiquitous water channel membrane protein, is a major contributor to cell membrane osmotic water permeability. Arteries are the physiological system where hydrostatic dominates osmotic pressure differences. In the present study, we show that the walls of large conduit arteries constitute the first example where hydrostatic pressure drives aquaporin-1-mediated transcellular/transendothelial flow. We studied cultured aortic endothelial cell monolayers and excised whole aortas of male Sprague-Dawley rats with intact and inhibited aquaporin-1 activity and with normal and knocked down aquaporin-1 expression. We subjected these systems to transmural hydrostatic pressure differences at zero osmotic pressure differences. Impaired aquaporin-1 endothelia consistently showed reduced engineering flow metrics (transendothelial water flux and hydraulic conductivity). In vitro experiments with tracers that only cross the endothelium paracellularly showed that changes in junctional transport cannot explain these reductions. Percent reductions in whole aortic wall hydraulic conductivity with either chemical blocking or knockdown of aquaporin-1 differed at low and high transmural pressures. This observation highlights how aquaporin-1 expression likely directly influences aortic wall mechanics by changing the critical transmural pressure at which its sparse subendothelial intima compresses. Such compression increases transwall flow resistance. Our endothelial and historic erythrocyte membrane aquaporin density estimates were consistent. In conclusion, aquaporin-1 significantly contributes to hydrostatic pressure-driven water transport across aortic endothelial monolayers, both in culture and in whole rat aortas. This transport, and parallel junctional flow, can dilute solutes that entered the wall paracellularly or through endothelial monolayer disruptions. Lower atherogenic precursor solute concentrations may slow their intimal entrainment kinetics. PMID:25659484

  15. Aquaporin-1 facilitates pressure-driven water flow across the aortic endothelium.

    PubMed

    Nguyen, Tieuvi; Toussaint, Jimmy; Xue, Yan; Raval, Chirag; Cancel, Limary; Russell, Stewart; Shou, Yixin; Sedes, Omer; Sun, Yu; Yakobov, Roman; Tarbell, John M; Jan, Kung-ming; Rumschitzki, David S

    2015-05-01

    Aquaporin-1, a ubiquitous water channel membrane protein, is a major contributor to cell membrane osmotic water permeability. Arteries are the physiological system where hydrostatic dominates osmotic pressure differences. In the present study, we show that the walls of large conduit arteries constitute the first example where hydrostatic pressure drives aquaporin-1-mediated transcellular/transendothelial flow. We studied cultured aortic endothelial cell monolayers and excised whole aortas of male Sprague-Dawley rats with intact and inhibited aquaporin-1 activity and with normal and knocked down aquaporin-1 expression. We subjected these systems to transmural hydrostatic pressure differences at zero osmotic pressure differences. Impaired aquaporin-1 endothelia consistently showed reduced engineering flow metrics (transendothelial water flux and hydraulic conductivity). In vitro experiments with tracers that only cross the endothelium paracellularly showed that changes in junctional transport cannot explain these reductions. Percent reductions in whole aortic wall hydraulic conductivity with either chemical blocking or knockdown of aquaporin-1 differed at low and high transmural pressures. This observation highlights how aquaporin-1 expression likely directly influences aortic wall mechanics by changing the critical transmural pressure at which its sparse subendothelial intima compresses. Such compression increases transwall flow resistance. Our endothelial and historic erythrocyte membrane aquaporin density estimates were consistent. In conclusion, aquaporin-1 significantly contributes to hydrostatic pressure-driven water transport across aortic endothelial monolayers, both in culture and in whole rat aortas. This transport, and parallel junctional flow, can dilute solutes that entered the wall paracellularly or through endothelial monolayer disruptions. Lower atherogenic precursor solute concentrations may slow their intimal entrainment kinetics. Copyright © 2015

  16. [Use of platelet poor plasma for elimination of Schneider's membrane defects arising during sinus lifting].

    PubMed

    Ivanov, S Iu; Iarmukova, N F; Muraev, A A; Migura, S A

    2010-01-01

    Topografical anatomical features of maxillary sinus structure create complexities for carrying out the sinus lifting operation. With Schneider's membrane defect from 3 to 6 mm the technique of its elimination with use of platelet poor plasma is offered. Defects in mucous membrane from 3 to 6 mm arise during operation in 7,9% of cases. In such cases bone window is expanding, mucous membrane is mobilizing and the defect is covered with platelet poor plasma received from patient's blood.

  17. Graft polymerization and plasma treatment of polymer membranes for fouling reduction: a review.

    PubMed

    Kochkodan, Victor M; Sharma, Virender K

    2012-01-01

    This article presents a review of recent developments in surface modification of polymer membranes via graft polymerization and plasma treatment for reduction of fouling with organic compounds and microorganisms in pressure driven membrane processes. The factors affecting membrane fouling, such as membrane hydrophilicity, charge and surface roughness are discussed. The recent studies in which the reduction of organic fouling and biofouling by the modification of the membrane surface via ultraviolet/redox initiated surface grafting of hydrophilic polymers and low temperature plasma treatment are reviewed.

  18. Characterization of aquaporin-6 as a nitrate channel in mammalian cells. Requirement of pore-lining residue threonine 63.

    PubMed

    Ikeda, Masahiro; Beitz, Eric; Kozono, David; Guggino, William B; Agre, Peter; Yasui, Masato

    2002-10-18

    Aquaporins (AQP) were originally regarded as plasma membrane channels that are freely permeated by water or small uncharged solutes but not by ions. Unlike other aquaporins, AQP6 overexpressed in Xenopus laevis oocytes was previously found to exhibit Hg2+ or pH-activated ion conductance. AQP6 could not be analyzed electrophysiologically in mammalian cells, however, because the protein is restricted to intracellular vesicles. Here we report that addition of a green fluorescence protein (GFP) tag to the N terminus of rat AQP6 (GFP-AQP6) redirects the protein to the plasma membranes of transfected mammalian cells. This permitted measurement of rapid, reversible, pH-induced anion currents by GFP-AQP6 in human embryonic kidney 293 cells. Surprisingly, anion selectivity relative to Cl- revealed high nitrate permeability even at pH 7.4; P(NO3)/P(Cl) > 9.8. Site-directed mutation of a pore-lining threonine to isoleucine at position 63 at the midpoint of the channel reduced NO3-/Cl- selectivity. Moreover, no anomalous mole-fraction behavior was observed with NO3-/Cl- mixtures, suggesting a single ion-binding pore in each subunit. Our studies indicate that AQP6 exhibits a new form of anion permeation with marked specificity for nitrate conferred by a specific pore-lining residue, observations that imply that the primary role of AQP6 may be in cellular regulation rather than simple fluid transport.

  19. Osmotic water permeability of plasma and vacuolar membranes in protoplasts I: high osmotic water permeability in radish (Raphanus sativus) root cells as measured by a new method.

    PubMed

    Murai-Hatano, Mari; Kuwagata, Tsuneo

    2007-03-01

    Intra- and transcellular water movements in plants are regulated by the water permeability of the plasma membrane (PM) and vacuolar membrane (VM) in plant cells. In the present study, we investigated the osmotic water permeability of both PM (P ( f1)) and VM (P ( f2)), as well as the bulk osmotic water permeability of a protoplast (P ( f(bulk))) isolated from radish (Raphanus sativus) roots. The values of P ( f(bulk)) and P ( f2) were determined from the swelling/shrinking rate of protoplasts and isolated vacuoles under hypo- or hypertonic conditions. In order to minimize the effect of unstirred layer, we monitored dropping or rising protoplasts (vacuoles) in sorbitol solutions as they swelled or shrunk. P ( f1) was calculated from P ( f(bulk)) and P ( f2) by using the 'three-compartment model', which describes the theoretical relationship between P ( f1), P ( f2) and P ( f(bulk)) (Kuwagata and Murai-Hatano in J Plant Res, 2007). The time-dependent changes in the volume of protoplasts and isolated vacuoles fitted well to the theoretical curves, and solute permeation of PM and VM was able to be neglected for measuring the osmotic water permeability. High osmotic water permeability of more than 500 mum s(-1), indicating high activity of aquaporins (water channels), was observed in both PM and VM in radish root cells. This method has the advantage that P ( f1) and P ( f2) can be measured accurately in individual higher plant cells.

  20. Characterization of auxin-binding proteins from zucchini plasma membrane

    NASA Technical Reports Server (NTRS)

    Hicks, G. R.; Rice, M. S.; Lomax, T. L.

    1993-01-01

    We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may

  1. Characterization of auxin-binding proteins from zucchini plasma membrane

    NASA Technical Reports Server (NTRS)

    Hicks, G. R.; Rice, M. S.; Lomax, T. L.

    1993-01-01

    We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may

  2. Transport of cholesterol from the endoplasmic reticulum to the plasma membrane is constitutive in CaCo-2 cells and differs from the transport of plasma membrane cholesterol to the endoplasmic reticulum.

    PubMed

    Field, F J; Born, E; Murthy, S; Mathur, S N

    1998-02-01

    The transport of newly synthesized cholesterol from its site of synthesis, the endoplasmic reticulum, to the plasma membrane was studied in CaCo-2 cells. The appearance of newly synthesized cholesterol on the cell surface was rapid. By 30 min, 50% of the total labeled cholesterol was observed in the plasma membrane. The arrival of cholesterol at the plasma membrane was independent of new protein synthesis, a functional Golgi apparatus, or microtubular function. Progesterone, verapamil, and trifluoperazine, inhibitors of p-glycoprotein which are known to inhibit cholesterol transport from the plasma membrane to the endoplasmic reticulum, reduced the amount of newly synthesized cholesterol reaching the plasma membrane. The p-glycoprotein inhibitors, however, caused the accumulation of sterol intermediates in the plasma membrane, suggesting that sterol trafficking to the plasma membrane remained intact, but that trafficking from the plasma membrane to the endoplasmic reticulum was disrupted. In contrast, nigericin, another potent inhibitor of cholesterol movement from the plasma membrane to the endoplasmic reticulum, did not alter the transport of newly synthesized cholesterol to the plasma membrane. Moreover, promoting cholesterol transport from the plasma membrane to the endoplasmic reticulum by sphingomyelin hydrolysis or by micellar cholesterol influx did not alter the percent of newly synthesized cholesterol transported to the plasma membrane. Likewise, preventing plasma membrane cholesterol from reaching the endoplasmic reticulum by incubating cells with lysophosphatidylcholine, filipin, or digitonin did not alter the arrival of newly synthesized cholesterol to the plasma membrane. The results suggest that the amount of cholesterol moving to the plasma membrane from the endoplasmic reticulum is constitutive and regulated at the level of cholesterol synthesis and not at the level of the transport process. The pathways of cholesterol transport to and from the

  3. Factors Determining the Oxygen Permeability of Biological Membranes: Oxygen Transport Across Eye Lens Fiber-Cell Plasma Membranes.

    PubMed

    Subczynski, Witold Karol; Widomska, Justyna; Mainali, Laxman

    2017-01-01

    Electron paramagnetic resonance (EPR) spin-label oximetry allows the oxygen permeability coefficient to be evaluated across homogeneous lipid bilayer membranes and, in some cases, across coexisting membrane domains without their physical separation. The most pronounced effect on oxygen permeability is observed for cholesterol, which additionally induces the formation of membrane domains. In intact biological membranes, integral proteins induce the formation of boundary and trapped lipid domains with a low oxygen permeability. The effective oxygen permeability coefficient across the intact biological membrane is affected not only by the oxygen permeability coefficients evaluated for each lipid domain but also by the surface area occupied by these domains in the membrane. All these factors observed in fiber cell plasma membranes of clear human eye lenses are reviewed here.

  4. 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

  5. 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.

  6. Fluorescence interference contrast based approach to study real time interaction of melittin with plasma membranes

    NASA Astrophysics Data System (ADS)

    Gupta, Sharad; Gui, Dong; Zandi, Roya; Gill, Sarjeet; Mohideen, Umar

    2014-03-01

    Melittin is an anti-bacterial and hemolytic toxic peptide found in bee venom. Cell lysis behavior of peptides has been widely investigated, but the exact interaction mechanism of lytic peptides with lipid membranes and its constituents has not been understood completely. In this paper we study the melittin interaction with lipid plasma membranes in real time using non-invasive and non-contact fluorescence interference contrast microscopy (FLIC). Particularly the interaction of melittin with plasma membranes was studied in a controlled molecular environment, where these plasma membrane were composed of saturated lipid, 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) and unsaturated lipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC) with and without cholesterol. We found out that melittin starts to form nanometer size pores in the plasma membranes shortly after interacting with membranes. But the addition of cholesterol in plasma membrane slows down the pore formation process. Our results show that inclusion of cholesterol to the