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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. PMID:24452376

  2. 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. PMID:27022872

  3. Plasma Membrane Intrinsic Proteins from Maize Cluster in Two Sequence Subgroups with Differential Aquaporin Activity1

    PubMed Central

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

    2000-01-01

    The transport of water through membranes is regulated in part by aquaporins or water channel proteins. These proteins are members of the larger family of major intrinsic proteins (MIPs). Plant aquaporins are categorized as either tonoplast intrinsic proteins (TIPs) or plasma membrane intrinsic proteins (PIPs). Sequence analysis shows that PIPs form several subclasses. We report on the characterization of three maize (Zea mays) PIPs belonging to the PIP1 and PIP2 subfamilies (ZmPIP1a, ZmPIP1b, and ZmPIP2a). The ZmPIP2a clone has normal aquaporin activity in Xenopus laevis oocytes. ZmPIP1a and ZmPIP1b have no activity, and a review of the literature shows that most PIP1 proteins identified in other plants have no or very low activity in oocytes. Arabidopsis PIP1 proteins are the only exception. Control experiments show that this lack of activity of maize PIP1 proteins is not caused by their failure to arrive at the plasma membrane of the oocytes. ZmPIP1b also does not appear to facilitate the transport of any of the small solutes tried (glycerol, choline, ethanol, urea, and amino acids). These results are discussed in relationship to the function and regulation of the PIP family of aquaporins. PMID:10759498

  4. 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. PMID:26351149

  5. Plasma Membrane Abundance of Human Aquaporin 5 Is Dynamically Regulated by Multiple Pathways.

    PubMed

    Kitchen, Philip; Öberg, Fredrik; Sjöhamn, Jennie; Hedfalk, Kristina; Bill, Roslyn M; Conner, Alex C; Conner, Matthew T; Törnroth-Horsefield, Susanna

    2015-01-01

    Aquaporin membrane protein channels mediate cellular water flow. Human aquaporin 5 (AQP5) is highly expressed in the respiratory system and secretory glands where it facilitates the osmotically-driven generation of pulmonary secretions, saliva, sweat and tears. Dysfunctional trafficking of AQP5 has been implicated in several human disease states, including Sjögren's syndrome, bronchitis and cystic fibrosis. In order to investigate how the plasma membrane expression levels of AQP5 are regulated, we studied real-time translocation of GFP-tagged AQP5 in HEK293 cells. We show that AQP5 plasma membrane abundance in transfected HEK293 cells is rapidly and reversibly regulated by at least three independent mechanisms involving phosphorylation at Ser156, protein kinase A activity and extracellular tonicity. The crystal structure of a Ser156 phosphomimetic mutant indicates that its involvement in regulating AQP5 membrane abundance is not mediated by a conformational change of the carboxy-terminus. We suggest that together these pathways regulate cellular water flow. PMID:26569106

  6. Plasma Membrane Abundance of Human Aquaporin 5 Is Dynamically Regulated by Multiple Pathways

    PubMed Central

    Kitchen, Philip; Öberg, Fredrik; Sjöhamn, Jennie; Hedfalk, Kristina; Bill, Roslyn M.; Conner, Alex C.; Conner, Matthew T.; Törnroth-Horsefield, Susanna

    2015-01-01

    Aquaporin membrane protein channels mediate cellular water flow. Human aquaporin 5 (AQP5) is highly expressed in the respiratory system and secretory glands where it facilitates the osmotically-driven generation of pulmonary secretions, saliva, sweat and tears. Dysfunctional trafficking of AQP5 has been implicated in several human disease states, including Sjögren’s syndrome, bronchitis and cystic fibrosis. In order to investigate how the plasma membrane expression levels of AQP5 are regulated, we studied real-time translocation of GFP-tagged AQP5 in HEK293 cells. We show that AQP5 plasma membrane abundance in transfected HEK293 cells is rapidly and reversibly regulated by at least three independent mechanisms involving phosphorylation at Ser156, protein kinase A activity and extracellular tonicity. The crystal structure of a Ser156 phosphomimetic mutant indicates that its involvement in regulating AQP5 membrane abundance is not mediated by a conformational change of the carboxy-terminus. We suggest that together these pathways regulate cellular water flow. PMID:26569106

  7. 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. PMID:25277091

  8. Water transport activity of the plasma membrane aquaporin PM28A is regulated by phosphorylation.

    PubMed Central

    Johansson, I; Karlsson, M; Shukla, V K; Chrispeels, M J; Larsson, C; Kjellbom, P

    1998-01-01

    PM28A is a major intrinsic protein of the spinach leaf plasma membrane and the major phosphoprotein. Phosphorylation of PM28A is dependent in vivo on the apoplastic water potential and in vitro on submicromolar concentrations of Ca2+. Here, we demonstrate that PM28A is an aquaporin and that its water channel activity is regulated by phosphorylation. Wild-type and mutant forms of PM28A, in which putative phosphorylation sites had been knocked out, were expressed in Xenopus oocytes, and the resulting increase in osmotic water permeability was measured in the presence or absence of an inhibitor of protein kinases (K252a) or of an inhibitor of protein phosphatases (okadaic acid). The results indicate that the water channel activity of PM28A is regulated by phosphorylation of two serine residues, Ser-115 in the first cytoplasmic loop and Ser-274 in the C-terminal region. Labeling of spinach leaves with 32P-orthophosphate and subsequent sequencing of PM28A-derived peptides demonstrated that Ser-274 is phosphorylated in vivo, whereas phosphorylation of Ser-115, a residue conserved among all plant plasma membrane aquaporins, could not be demonstrated. This identifies Ser-274 of PM28A as the amino acid residue being phosphorylated in vivo in response to increasing apoplastic water potential and dephosphorylated in response to decreasing water potential. Taken together, our results suggest an active role for PM28A in maintaining cellular water balance. PMID:9501117

  9. A proteomic study reveals novel insights into the diversity of aquaporin forms expressed in the plasma membrane of plant roots.

    PubMed Central

    Santoni, Véronique; Vinh, Joëlle; Pflieger, Delphine; Sommerer, Nicolas; Maurel, Christophe

    2003-01-01

    Aquaporins are channel proteins that facilitate the diffusion of water across cell membranes. The genome of Arabidopsis thaliana encodes 35 full-length aquaporin homologues. Thirteen of them belong to the plasma membrane intrinsic protein (PIP) subfamily and predominantly sit at the plasma membrane (PM). In the present work we combine separations of membrane proteins (by one- and two-dimensional gel electrophoresis) with identification by MS (matrix-assisted laser-desorption ionization-time-of-flight and electrospray-ionization tandem MS) to take an inventory of aquaporin isoforms expressed in the PM of Arabidopsis thaliana roots. Our analysis provides direct evidence for the expression of five PIPs (PIP1;1, PIP1;5, PIP2;1, PIP2;2 and PIP2;7) in the root PM and suggests the presence of at least three other PIP isoforms. In addition, we show that the same PIP isoform can be present under several forms with distinct isoelectric points. More specifically, we identify phosphorylated aquaporins in the PIP1 and PIP2 subgroups and suggest the existence of other post-translational modifications. Their identification should provide clues to reveal novel molecular mechanisms for aquaporin regulation. PMID:12678916

  10. 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. PMID:27625676

  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. Expression and characterization of plasma membrane aquaporins in stomatal complexes of Zea mays.

    PubMed

    Heinen, Robert B; Bienert, Gerd Patrick; Cohen, David; Chevalier, Adrien S; Uehlein, Norbert; Hachez, Charles; Kaldenhoff, Ralf; Le Thiec, Didier; Chaumont, François

    2014-10-01

    Stomata, the microscopic pores on the surface of the aerial parts of plants, are bordered by two specialized cells, known as guard cells, which control the stomatal aperture according to endogenous and environmental signals. Like most movements occurring in plants, the opening and closing of stomata are based on hydraulic forces. During opening, the activation of plasma membrane and tonoplast transporters results in solute accumulation in the guard cells. To re-establish the perturbed osmotic equilibrium, water follows the solutes into the cells, leading to their swelling. Numerous studies have contributed to the understanding of the mechanism and regulation of stomatal movements. However, despite the importance of transmembrane water flow during this process, only a few studies have provided evidence for the involvement of water channels, called aquaporins. Here, we microdissected Zea mays stomatal complexes and showed that members of the aquaporin plasma membrane intrinsic protein (PIP) subfamily are expressed in these complexes and that their mRNA expression generally follows a diurnal pattern. The substrate specificity of two of the expressed ZmPIPs, ZmPIP1;5 and ZmPIP1;6, was investigated by heterologous expression in Xenopus oocytes and yeast cells. Our data show that both isoforms facilitate transmembrane water diffusion in the presence of the ZmPIP2;1 isoform. In addition, both display CO2 permeability comparable to that of the CO2 diffusion facilitator NtAQP1. These data indicate that ZmPIPs may have various physiological roles in stomatal complexes. PMID:25082269

  13. 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

  14. 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

  15. Arabidopsis SNAREs SYP61 and SYP121 coordinate the trafficking of plasma membrane aquaporin PIP2;7 to modulate the cell membrane water permeability.

    PubMed

    Hachez, Charles; Laloux, Timothée; Reinhardt, Hagen; Cavez, Damien; Degand, Hervé; Grefen, Christopher; De Rycke, Riet; Inzé, Dirk; Blatt, Michael R; Russinova, Eugenia; Chaumont, François

    2014-07-01

    Plant plasma membrane intrinsic proteins (PIPs) are aquaporins that facilitate the passive movement of water and small neutral solutes through biological membranes. Here, we report that post-Golgi trafficking of PIP2;7 in Arabidopsis thaliana involves specific interactions with two syntaxin proteins, namely, the Qc-SNARE SYP61 and the Qa-SNARE SYP121, that the proper delivery of PIP2;7 to the plasma membrane depends on the activity of the two SNAREs, and that the SNAREs colocalize and physically interact. These findings are indicative of an important role for SYP61 and SYP121, possibly forming a SNARE complex. Our data support a model in which direct interactions between specific SNARE proteins and PIP aquaporins modulate their post-Golgi trafficking and thus contribute to the fine-tuning of the water permeability of the plasma membrane. PMID:25082856

  16. 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

  17. Characterization and expression of plasma and tonoplast membrane aquaporins in primed seed of Brassica napus during germination under stress conditions.

    PubMed

    Gao, Y P; Young, L; Bonham-Smith, P; Gusta, L V

    1999-07-01

    Two aquaporin genes were isolated from a cDNA library of canola (Brassica napus L.). The first aquaporin, BnPIP1 of 1094 bp, encoding a putative polypeptide of 287 amino acids with a predicted molecular mass of 30.4 kDa and a pI of 7.8, belongs to the family of plasma membrane intrinsic protein (PIPs) aquaporins. The B. napus aquaporin showed 85-94% identity to the Arabidopsis thaliana PIPs. ABA priming of seed induced high levels of BnPIP1 transcript which remained after subsequent re-drying of the seed. The second aquaporin, Bny-TIP2 of 1020 bp, encoded a putative polypeptide of 253 amino acids with a predicted molecular mass of 25.8 kDa and a pI of 5.8. Bngamma-TIP2 showed 83-90% identity to gamma-TIP genes from a variety of plant species. Bngamma-TIP2 was expressed only when radicle protrusion occurred in either untreated or primed seeds. Seeds primed with PEG or ABA germinated earlier and showed a higher final percentage of germination than unprimed seed, particularly under salt and osmotic stresses at low temperature. Transcripts of both BnPIP1 and Bngamma-TIP2 genes were present earlier during germination of primed seeds than non-primed seed. From these results, we conclude that BnPIP1 is related to the water transportation required for enzymatic metabolism of storage nutrients at the early stages of canola seed germination whereas Bngamma-TIP2 expression is related to cell growth associated with radicle protrusion. Priming induced the expression of BnPIP1 but had no effect on Bngamma-TIP2. PMID:10480387

  18. Single Mutations in the Transmembrane Domains of Maize Plasma Membrane Aquaporins Affect the Activity of Monomers within a Heterotetramer.

    PubMed

    Berny, Marie C; Gilis, Dimitri; Rooman, Marianne; Chaumont, François

    2016-07-01

    Aquaporins are channels facilitating the diffusion of water and/or small uncharged solutes across biological membranes. They assemble as homotetramers but some of them also form heterotetramers, especially in plants. In Zea mays, aquaporins belonging to the plasma membrane intrinsic protein (PIP) subfamily are clustered into two groups, PIP1 and PIP2, which exhibit different water-channel activities when expressed in Xenopus oocytes. When PIP1 and PIP2 isoforms are co-expressed, they physically interact to modulate their subcellular localization and channel activity. Here, we demonstrated by affinity chromatography purification that, when co-expressed in Xenopus oocytes, the maize PIP1;2 and PIP2;5 isoforms assemble as homo- and heterodimers within heterotetramers. We built the 3D structure of such heterotetramers by comparative modeling on the basis of the spinach SoPIP2;1 X-ray structure and identified amino acid residues in the transmembrane domains which putatively interact at the interfaces between monomers. Their roles in the water-channel activity, subcellular localization, protein abundance, and physical interaction were investigated by mutagenesis. We highlighted single-residue substitutions that either inactivated PIP2;5 or activated PIP1;2 without affecting their interaction. Interestingly, the Phe220Ala mutation in the transmembrane domain 5 of PIP1;2 activated its water-channel activity and, at the same time, inactivated PIP2;5 within a heterotetramer. Altogether, these data contribute to a better understanding of the interaction mechanisms between PIP isoforms and the role of heterotetramerization on their water-channel activity. PMID:27109604

  19. 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

  20. A New LxxxA Motif in the Transmembrane Helix3 of Maize Aquaporins Belonging to the Plasma Membrane Intrinsic Protein PIP2 Group Is Required for Their Trafficking to the Plasma Membrane1[W][OPEN

    PubMed Central

    Chevalier, Adrien S.; Bienert, Gerd Patrick; Chaumont, François

    2014-01-01

    Aquaporins play important roles in maintaining plant water status under challenging environments. The regulation of aquaporin density in cell membranes is essential to control transcellular water flows. This work focuses on the maize (Zea mays) plasma membrane intrinsic protein (ZmPIP) aquaporin subfamily, which is divided into two sequence-related groups (ZmPIP1s and ZmPIP2s). When expressed alone in mesophyll protoplasts, ZmPIP2s are efficiently targeted to the plasma membrane, whereas ZmPIP1s are retained in the endoplasmic reticulum (ER). A protein domain-swapping approach was utilized to demonstrate that the transmembrane domain3 (TM3), together with the previously identified N-terminal ER export diacidic motif, account for the differential localization of these proteins. In addition to protoplasts, leaf epidermal cells transiently transformed by biolistic particle delivery were used to confirm and refine these results. By generating artificial proteins consisting of a single transmembrane domain, we demonstrated that the TM3 of ZmPIP1;2 or ZmPIP2;5 discriminates between ER and plasma membrane localization, respectively. More specifically, a new LxxxA motif in the TM3 of ZmPIP2;5, which is highly conserved in plant PIP2s, was shown to regulate its anterograde routing along the secretory pathway, particularly its export from the ER. PMID:24989232

  1. Salt stress induces internalization of plasma membrane aquaporin into the vacuole in Arabidopsis thaliana.

    PubMed

    Ueda, Masamichi; Tsutsumi, Nobuhiro; Fujimoto, Masaru

    2016-06-10

    Salt stress is a major environmental stress for plants, causing hyperosmotic, ionic and drought-like stresses. Plasma membrane intrinsic protein 2;1 (PIP2;1), which forms a water channel that regulates water flux thorough the plasma membrane (PM), is constitutively trafficked between the PM and the trans-Golgi network (TGN) in Arabidopsis thaliana. Salt stress is known to relocalize PIP2;1 to intracellular compartments, probably to decrease the water permeability of the root. However, the destination of internalized PIP2;1 and the mechanism by which PIP2;1 is internalized remain unclear. Here, we examined the effects of salt stress and inhibitors of endocytosis on the intracellular localization of green fluorescent protein-fused PIP2;1 (GFP-PIP2;1) in Arabidopsis thaliana root epidermal cells. Salt stress decreased the fluorescence of GFP-PIP2;1 at the PM and increased it in the vacuolar lumen as shown by staining of the vacuolar membrane. The internalization of PIP2;1 was suppressed by an inhibitor of clathrin-mediated endocytosis and by inhibitors of two kinases that appear to have roles in salt stress, phosphatidylinositol 3-kinase (PI3K) and phosphatidylinositol 4-kinase (PI4K). Inhibiting PI4K suppressed the salt-induced endocytosis of GFP-PIP2;1 at the PM, whereas inhibiting PI3K suppressed the trafficking of GFP-PIP2;1 after its internalization. These results suggest that salt stress induces the internalization of PIP2;1 from the PM to the vacuolar lumen, and that these processes are dependent on clathrin, PI3K and PI4K. PMID:27163638

  2. Plant aquaporins: membrane channels with multiple integrated functions.

    PubMed

    Maurel, Christophe; Verdoucq, Lionel; Luu, Doan-Trung; Santoni, Véronique

    2008-01-01

    Aquaporins are channel proteins present in the plasma and intracellular membranes of plant cells, where they facilitate the transport of water and/or small neutral solutes (urea, boric acid, silicic acid) or gases (ammonia, carbon dioxide). Recent progress was made in understanding the molecular bases of aquaporin transport selectivity and gating. The present review examines how a wide range of selectivity profiles and regulation properties allows aquaporins to be integrated in numerous functions, throughout plant development, and during adaptations to variable living conditions. Although they play a central role in water relations of roots, leaves, seeds, and flowers, aquaporins have also been linked to plant mineral nutrition and carbon and nitrogen fixation. PMID:18444909

  3. 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. PMID:24434310

  4. 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

  5. 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

  6. FRET imaging in living maize cells reveals that plasma membrane aquaporins interact to regulate their subcellular localization.

    PubMed

    Zelazny, Enric; Borst, Jan Willem; Muylaert, Mélanie; Batoko, Henri; Hemminga, Marcus A; Chaumont, François

    2007-07-24

    Zea mays plasma membrane intrinsic proteins (ZmPIPs) fall into two groups, ZmPIP1s and ZmPIP2s, that exhibit different water channel activities when expressed in Xenopus oocytes. ZmPIP1s are inactive, whereas ZmPIP2s induce a marked increase in the membrane osmotic water permeability coefficient, P(f). We previously showed that, in Xenopus oocytes, ZmPIP1;2 and ZmPIP2;1 interact to increase the cell P(f). Here, we report the localization and interaction of ZmPIP1s and ZmPIP2s in living maize cells. ZmPIPs were fused to monomeric yellow fluorescent protein and/or monomeric cyan fluorescent protein and expressed transiently in maize mesophyll protoplasts. When expressed alone, ZmPIP1 fusion proteins were retained in the endoplasmic reticulum, whereas ZmPIP2s were found in the plasma membrane. Interestingly, when coexpressed with ZmPIP2s, ZmPIP1s were relocalized to the plasma membrane. Using FRET/fluorescence lifetime imaging microscopy, we demonstrated that this relocalization results from interaction between ZmPIP1s and ZmPIP2s. Immunoprecipitation experiments provided additional evidence for the association of ZmPIP1;2 and ZmPIP2;1 in maize roots and suspension cells. These data suggest that PIP1-PIP2 interaction is required for in planta PIP1 trafficking to the plasma membrane to modulate plasma membrane permeability. PMID:17636130

  7. All-trans retinoic acid increases expression of aquaporin-5 and plasma membrane water permeability via transactivation of Sp1 in mouse lung epithelial cells.

    PubMed

    Nomura, Johji; Horie, Ichiro; Seto, Mayumi; Nagai, Kazufumi; Hisatsune, Akinori; Miyata, Takeshi; Isohama, Yoichiro

    2006-12-29

    Aquaporin-5 (AQP5) is a water-selective channel protein that is expressed in lacrimal glands, salivary glands, and distal lung. Several studies using AQP5 knockout mice have revealed that AQP5 plays an important role in maintaining water homeostasis in the lung. We report here that all-trans retinoic acid (atRA) increases plasma membrane water permeability, AQP5 mRNA and protein expression, and AQP5 promoter activity in MLE-12 cells. The promoter activation induced by atRA was diminished by mutation at the Sp1/Sp3 binding element (SBE), suggesting that the SBE mediates the effects of atRA. In addition, atRA increased the binding of Sp1 to the SBE without changing the levels of Sp1 in the nucleus. Taken together, our data indicate that atRA increases AQP5 expression through transactivation of Sp1, leading to an increase in plasma membrane water permeability. PMID:17097063

  8. 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. PMID:23745761

  9. Two water-specific aquaporins at the apical and basal plasma membranes of insect epithelia: molecular basis for water recycling through the cryptonephric rectal complex of lepidopteran larvae.

    PubMed

    Azuma, Masaaki; Nagae, Tomone; Maruyama, Mariya; Kataoka, Naoya; Miyake, Seiji

    2012-04-01

    Larval lepidopteran and coleopteran insects have evolved a specialised cryptonephric system in the hindgut in which water is constantly and rapidly taken up before defecation. In the silkworm, Bombyx mori, the movement of water through the epithelia within the cryptonephric rectal complex is likely facilitated by the two aquaporins, AQP-Bom1 and AQP-Bom3. Both are functionally water-specific and are predominantly expressed in the hindgut (colon and rectum). Phylogenetically, AQP-Bom1 and AQP-Bom3 belong to the DRIP (Drosophila integral protein) and PRIP (Pyrocoelia rufa integral protein) subfamilies, respectively, of the insect AQP clade. In immunoblot analyses using antipeptide antibodies for each Bombyx AQP, the predicted molecular mass for the respective AQPs were around 25 kDa, and further indicated that both tended to be oligomerised as a homotetramer (∼110 kDa). AQP-Bom1 [DRIP] was exclusively expressed at the apical plasma membrane of colonic and rectal epithelial cells, whereas AQP-Bom3 [PRIP] was expressed at the basal plasma membrane of these cells. This polarised localisation of DRIP/PRIP was also observed in the outer cryptonephric Malpighian tubules (outer cMT) and in the six tubules just outside the cryptonephric rectal complex (rectal lead MT). In the rectal epithelia, water is transported from the rectal lumen to the perinephric space and then deposited into the lumen of the outer cMT; the water then goes through the tubular lumen to exit the complex and is finally transported across the rectal lead MT. We conclude that rectal water retrieval into the haemocoele occurs at the very limited region of the water-permeable sites in MT epithelia after passing the rectal and cMT epithelia and that the high osmotic permeability is due to the presence of two distinct water-specific AQPs (DRIP and PRIP) in the epithelial cells of lepidopteran hindgut. PMID:22285686

  10. 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. PMID:26823528

  11. The Role of Plasma Membrane Aquaporins in Regulating the Bundle Sheath-Mesophyll Continuum and Leaf Hydraulics1[C][W][OPEN

    PubMed Central

    Sade, Nir; Shatil-Cohen, Arava; Attia, Ziv; Maurel, Christophe; Boursiac, Yann; Kelly, Gilor; Granot, David; Yaaran, Adi; Lerner, Stephen; Moshelion, Menachem

    2014-01-01

    Our understanding of the cellular role of aquaporins (AQPs) in the regulation of whole-plant hydraulics, in general, and extravascular, radial hydraulic conductance in leaves (Kleaf), in particular, is still fairly limited. We hypothesized that the AQPs of the vascular bundle sheath (BS) cells regulate Kleaf. To examine this hypothesis, AQP genes were silenced using artificial microRNAs that were expressed constitutively or specifically targeted to the BS. MicroRNA sequences were designed to target all five AQP genes from the PLASMA MEMBRANE-INTRINSIC PROTEIN1 (PIP1) subfamily. Our results show that the constitutively silenced PIP1 (35S promoter) plants had decreased PIP1 transcript and protein levels and decreased mesophyll and BS osmotic water permeability (Pf), mesophyll conductance of CO2, photosynthesis, Kleaf, transpiration, and shoot biomass. Plants in which the PIP1 subfamily was silenced only in the BS (SCARECROW:microRNA plants) exhibited decreased mesophyll and BS Pf and decreased Kleaf but no decreases in the rest of the parameters listed above, with the net result of increased shoot biomass. We excluded the possibility of SCARECROW promoter activity in the mesophyll. Hence, the fact that SCARECROW:microRNA mesophyll exhibited reduced Pf, but not reduced mesophyll conductance of CO2, suggests that the BS-mesophyll hydraulic continuum acts as a feed-forward control signal. The role of AQPs in the hierarchy of the hydraulic signal pathway controlling leaf water status under normal and limited-water conditions is discussed. PMID:25266632

  12. Acute Hypertonicity Alters Aquaporin-2 Trafficking and Induces a MAPK-dependent Accumulation at the Plasma Membrane of Renal Epithelial Cells*

    PubMed Central

    Hasler, Udo; Nunes, Paula; Bouley, Richard; Lu, Hua A. J.; Matsuzaki, Toshiyuki; Brown, Dennis

    2008-01-01

    The unique phenotype of renal medullary cells allows them to survive and functionally adapt to changes of interstitial osmolality/tonicity. We investigated the effects of acute hypertonic challenge on AQP2 (aquaporin-2) water channel trafficking. In the absence of vasopressin, hypertonicity alone induced rapid (<10 min) plasma membrane accumulation of AQP2 in rat kidney collecting duct principal cells in situ, and in several kidney epithelial lines. Confocal microscopy revealed that AQP2 also accumulated in the trans-Golgi network (TGN) following hypertonic challenge. AQP2 mutants that mimic the Ser256-phosphorylated and -nonphosphorylated state accumulated at the cell surface and TGN, respectively. Hypertonicity did not induce a change in cytosolic cAMP concentration, but inhibition of either calmodulin or cAMP-dependent protein kinase A activity blunted the hypertonicity-induced increase of AQP2 cell surface expression. Hypertonicity increased p38, ERK1/2, and JNK MAPK activity. Inhibiting MAPK activity abolished hypertonicity-induced accumulation of AQP2 at the cell surface but did not affect either vasopressin-dependent AQP2 trafficking or hypertonicity-induced AQP2 accumulation in the TGN. Finally, increased AQP2 cell surface expression induced by hypertonicity largely resulted from a reduction in endocytosis but not from an increase in exocytosis. These data indicate that acute hypertonicity profoundly alters AQP2 trafficking and that hypertonicity-induced AQP2 accumulation at the cell surface depends on MAP kinase activity. This may have important implications on adaptational processes governing transcellular water flux and/or cell survival under extreme conditions of hypertonicity. PMID:18664568

  13. Recruitment of human aquaporin 3 to internal membranes in the Plasmodium falciparum infected erythrocyte.

    PubMed

    Bietz, Sven; Montilla, Irine; Külzer, Simone; Przyborski, Jude M; Lingelbach, Klaus

    2009-09-01

    The molecular mechanisms underlying the formation of the parasitophorous vacuolar membrane in Plasmodium falciparum infected erythrocytes are incompletely understood, and the protein composition of this membrane is still enigmatic. Although the differentiated mammalian erythrocyte lacks the machinery required for endocytosis, some reports have described a localisation of host cell membrane proteins at the parasitophorous vacuolar membrane. Aquaporin 3 is an abundant plasma membrane protein of various cells, including mammalian erythrocytes where it is found in distinct oligomeric states. Here we show that human aquaporin 3 is internalized into infected erythrocytes, presumably during or soon after invasion. It is integrated into the PVM where it is organized in novel oligomeric states which are not found in non-infected cells. PMID:19393693

  14. Aquaporins.

    PubMed

    Echevarría, M; Ilundáin, A A

    1998-06-01

    Aquaporins (AQP) are members of the major intrinsic protein superfamily of integral membrane proteins, which function as specialized water channels to facilitate the passage of water through the cell membrane in animals, plants and bacterias. Ten AQP homologues named from 0 to 9 have been clones so far in mammals. They are widely distributed and more than one AQP could be present in the same cell. Most of the AQPs are only permeable to water and impermeable to small organic and inorganic molecules, except for AQP 3, 7 and 9 which are also permeable to urea and glycerol. From the hydrophobicity profile all AQPs seem to have six transmembrane domains with five connecting loops and with the amino and carboxyl termini in the cytoplasm. They are synthesized as monomers, but there is evidence suggesting that AQPs are formed in the membrane as tetrameric units, each of which has four water pores. The primary amino acid sequence contains putative phosphorylation sites for protein kinasses A and/or C or casein kinase II, and the expression and membrane protein abundance of some AQPs are known to be under hormonal regulation. The human genes for several AQPs have been cloned and an increasing number of disturbances associated to abnormal functioning of these proteins had been identified. PMID:9858131

  15. 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

  16. 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

    2015-01-01

    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

  17. Single-Molecule Analysis of PIP2;1 Dynamics and Partitioning Reveals Multiple Modes of Arabidopsis Plasma Membrane Aquaporin Regulation[C][W

    PubMed Central

    Li, Xiaojuan; Wang, Xiaohua; Yang, Yong; Li, Ruili; He, Qihua; Fang, Xiaohong; Luu, Doan-Trung; Maurel, Christophe; Lin, Jinxing

    2011-01-01

    PIP2;1 is an integral membrane protein that facilitates water transport across plasma membranes. To address the dynamics of Arabidopsis thaliana PIP2;1 at the single-molecule level as well as their role in PIP2;1 regulation, we tracked green fluorescent protein–PIP2;1 molecules by variable-angle evanescent wave microscopy and fluorescence correlation spectroscopy (FCS). Single-particle tracking analysis revealed that PIP2;1 presented four diffusion modes with large dispersion of diffusion coefficients, suggesting that partitioning and dynamics of PIP2;1 are heterogeneous and, more importantly, that PIP2;1 can move into or out of membrane microdomains. In response to salt stress, the diffusion coefficients and percentage of restricted diffusion increased, implying that PIP2;1 internalization was enhanced. This was further supported by the decrease in PIP2;1 density on plasma membranes by FCS. We additionally demonstrated that PIP2;1 internalization involves a combination of two pathways: a tyrphostin A23-sensitive clathrin-dependent pathway and a methyl-β-cyclodextrin–sensitive, membrane raft–associated pathway. The latter was efficiently stimulated under NaCl conditions. Taken together, our findings demonstrate that PIP2;1 molecules are heterogeneously distributed on the plasma membrane and that clathrin and membrane raft pathways cooperate to mediate the subcellular trafficking of PIP2;1, suggesting that the dynamic partitioning and recycling pathways might be involved in the multiple modes of regulating water permeability. PMID:22010034

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

    PubMed

    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

  19. 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

  20. 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

  1. 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

  2. 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. PMID:22386862

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

    PubMed

    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

  4. Aquaporins in Plants.

    PubMed

    Maurel, Christophe; Boursiac, Yann; Luu, Doan-Trung; Santoni, Véronique; Shahzad, Zaigham; Verdoucq, Lionel

    2015-10-01

    Aquaporins are membrane channels that facilitate the transport of water and small neutral molecules across biological membranes of most living organisms. In plants, aquaporins occur as multiple isoforms reflecting a high diversity of cellular localizations, transport selectivity, and regulation properties. Plant aquaporins are localized in the plasma membrane, endoplasmic reticulum, vacuoles, plastids and, in some species, in membrane compartments interacting with symbiotic organisms. Plant aquaporins can transport various physiological substrates in addition to water. Of particular relevance for plants is the transport of dissolved gases such as carbon dioxide and ammonia or metalloids such as boron and silicon. Structure-function studies are developed to address the molecular and cellular mechanisms of plant aquaporin gating and subcellular trafficking. Phosphorylation plays a central role in these two processes. These mechanisms allow aquaporin regulation in response to signaling intermediates such as cytosolic pH and calcium, and reactive oxygen species. Combined genetic and physiological approaches are now integrating this knowledge, showing that aquaporins play key roles in hydraulic regulation in roots and leaves, during drought but also in response to stimuli as diverse as flooding, nutrient availability, temperature, or light. A general hydraulic control of plant tissue expansion by aquaporins is emerging, and their role in key developmental processes (seed germination, emergence of lateral roots) has been established. Plants with genetically altered aquaporin functions are now tested for their ability to improve plant tolerance to stresses. In conclusion, research on aquaporins delineates ever expanding fields in plant integrative biology thereby establishing their crucial role in plants. PMID:26336033

  5. Aquaporins in Clinical Medicine

    PubMed Central

    Verkman, A.S.

    2012-01-01

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

  6. 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. PMID:25204262

  7. 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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  8. Toward understanding the selective anticancer capacity of cold atmospheric plasma--a model based on aquaporins (Review).

    PubMed

    Yan, Dayun; Talbot, Annie; Nourmohammadi, Niki; Sherman, Jonathan H; Cheng, Xiaoqian; Keidar, Michael

    2015-01-01

    Selectively treating tumor cells is the ongoing challenge of modern cancer therapy. Recently, cold atmospheric plasma (CAP), a near room-temperature ionized gas, has been demonstrated to exhibit selective anticancer behavior. However, the mechanism governing such selectivity is still largely unknown. In this review, the authors first summarize the progress that has been made applying CAP as a selective tool for cancer treatment. Then, the key role of aquaporins in the H2O2 transmembrane diffusion is discussed. Finally, a novel model, based on the expression of aquaporins, is proposed to explain why cancer cells respond to CAP treatment with a greater rise in reactive oxygen species than homologous normal cells. Cancer cells tend to express more aquaporins on their cytoplasmic membranes, which may cause the H2O2 uptake speed in cancer cells to be faster than in normal cells. As a result, CAP treatment kills cancer cells more easily than normal cells. Our preliminary observations indicated that glioblastoma cells consumed H2O2 much faster than did astrocytes in either the CAP-treated or H2O2-rich media, which supported the selective model based on aquaporins. PMID:26700469

  9. Structural insights into eukaryotic aquaporin regulation.

    PubMed

    Törnroth-Horsefield, Susanna; Hedfalk, Kristina; Fischer, Gerhard; Lindkvist-Petersson, Karin; Neutze, Richard

    2010-06-18

    Aquaporin-mediated water transport across cellular membranes is an ancient, ubiquitous mechanism within cell biology. This family of integral membrane proteins includes both water selective pores (aquaporins) and transport facilitators of other small molecules such as glycerol and urea (aquaglyceroporins). Eukaryotic aquaporins are frequently regulated post-translationally by gating, whereby the rate of flux through the channel is controlled, or by trafficking, whereby aquaporins are shuttled from intracellular storage sites to the plasma membrane. A number of high-resolution X-ray structures of eukaryotic aquaporins have recently been reported and the new structural insights into gating and trafficking that emerged from these studies are described. Basic structural themes reoccur, illustrating how the problem of regulation in diverse biological contexts builds upon a limited set of possible solutions. PMID:20416297

  10. 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

  11. 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

    2013-02-01

    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. PMID:23311686

  12. Increased aquaporin 1 and 5 membrane expression in the lens epithelium of cataract patients.

    PubMed

    Barandika, Olatz; Ezquerra-Inchausti, Maitane; Anasagasti, Ander; Vallejo-Illarramendi, Ainara; Llarena, Irantzu; Bascaran, Lucia; Alberdi, Txomin; De Benedetti, Giacomo; Mendicute, Javier; Ruiz-Ederra, Javier

    2016-10-01

    In this work we have analyzed the expression levels of the main aquaporins (AQPs) expressed in human lens epithelial cells (HLECs) using 112 samples from patients treated with cataract surgery and 36 samples from individuals treated with refractive surgery, with transparent lenses as controls. Aquaporin-1 (AQP1) is the main AQP, representing 64.1% of total AQPs in HLECs, with aquaporin-5 (AQP5) representing 35.9% in controls. A similar proportion of each AQP in cataract was found. Although no differences were found at the mRNA level compared to controls, a significant 1.65-fold increase (p=0.001) in AQP1protein expression was observed in HLECs from cataract patients, with the highest differences being found for nuclear cataracts (2.1-fold increase; p<0.001). A similar trend was found for AQP5 (1.47-fold increase), although the difference was not significant (p=0.161). Moreover we have shown increased membrane AQP5 protein expression in HLECs of patients with cataracts. No association of AQP1 or AQP5 expression levels with age or sex was observed in either group. Our results suggest regulation of AQP1 and AQP5 at the post-translational level and support previous observations on the implication of AQP1 and 5 in maintenance of lens transparency in animal models. Our results likely reflect a compensatory response of the crystalline lens to delay cataract formation by increasing the water removal rate. PMID:27497833

  13. Structure and Stability of the Spinach Aquaporin SoPIP2;1 in Detergent Micelles and Lipid Membranes

    PubMed Central

    Plasencia, Inés; Survery, Sabeen; Ibragimova, Sania; Hansen, Jesper S.; Kjellbom, Per; Helix-Nielsen, Claus; Johanson, Urban; Mouritsen, Ole G.

    2011-01-01

    Background SoPIP2;1 constitutes one of the major integral proteins in spinach leaf plasma membranes and belongs to the aquaporin family. SoPIP2;1 is a highly permeable and selective water channel that has been successfully overexpressed and purified with high yields. In order to optimize reconstitution of the purified protein into biomimetic systems, we have here for the first time characterized the structural stability of SoPIP2;1. Methodology/Principal Finding We have characterized the protein structural stability after purification and after reconstitution into detergent micelles and proteoliposomes using circular dichroism and fluorescence spectroscopy techniques. The structure of SoPIP2;1 was analyzed either with the protein solubilized with octyl-β-D-glucopyranoside (OG) or reconstituted into lipid membranes formed by E. coli lipids, diphytanoylphosphatidylcholine (DPhPC), or reconstituted into lipid membranes formed from mixtures of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPE), 1-palmitoyl-2oleoyl-phosphatidylethanolamine (POPE), 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS), and ergosterol. Generally, SoPIP2;1 secondary structure was found to be predominantly α-helical in accordance with crystallographic data. The protein has a high thermal structural stability in detergent solutions, with an irreversible thermal unfolding occurring at a melting temperature of 58°C. Incorporation of the protein into lipid membranes increases the structural stability as evidenced by an increased melting temperature of up to 70°C. Conclusion/Significance The results of this study provide insights into SoPIP2;1 stability in various host membranes and suggest suitable choices of detergent and lipid composition for reconstitution of SoPIP2;1 into biomimetic membranes for biotechnological applications. PMID:21339815

  14. 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.

  15. Gas-tight triblock-copolymer membranes are converted to CO2 permeable by insertion of plant aquaporins

    PubMed Central

    Uehlein, Norbert; Otto, Beate; Eilingsfeld, Adrian; Itel, Fabian; Meier, Wolfgang; Kaldenhoff, Ralf

    2012-01-01

    We demonstrate that membranes consisting of certain triblock-copolymers were tight for CO2. Using a novel approach, we provide evidence for aquaporin facilitated CO2 diffusion. Plant aquaporins obtained from heterologous expression were inserted into triblock copolymer membranes. These were employed to separate a chamber with a solution maintaining high CO2 concentrations from one with depleted CO2 concentrations. CO2 diffusion was detected by measuring the pH change resulting from membrane CO2 diffusion from one chamber to the other. An up to 21 fold increase in diffusion rate was determined. Besides the supply of this proof of principle, we could provide additional arguments in favour of protein facilitated CO2 diffusion to the vivid on-going debate about the principles of membrane gas diffusion in living cells. PMID:22844579

  16. Layer-by-layer assembly of aquaporin Z-incorporated biomimetic membranes for water purification.

    PubMed

    Wang, Miaoqi; Wang, Zhining; Wang, Xida; Wang, Shuzheng; Ding, Wande; Gao, Congjie

    2015-03-17

    We fabricated a biomimetic nanofiltration (NF) membrane by immobilizing an Aquaporin Z (AqpZ)-incorporated supported lipid bilayer (SLB) on a layer-by-layer (LbL) complex polyelectrolyte membrane to achieve excellent permeability and salt rejection with a high stability. The polyelectrolyte membranes were prepared by LbL assembly of poly(ethylenimine) (PEI) with positive charges and poly(sodium 4-styrenesulfonate) (PSS) with negative charges alternately on a porous hydrolyzed polyacrylonitrile (H-PAN) substrate. AqpZ-incorporated 1,2-dioleloyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dioleoyl-3-trimethylammo-nium-propane (chloride salt) (DOTAP) vesicles with positive charges were deposited on the H-PAN/PEI/PSS polyelectrolytes membrane surface. The resulting biomimetic membrane exhibited a high flux of 22 L·m(-2)·h(-1) (LMH), excellent MgCl2 rejection of ∼97% and NaCl rejection of ∼75% under an operation pressure of 0.4 MPa. Due to the attractive electrostatic interaction between SLB and the polyelectrolyte membrane, the biomimetic membrane showed satisfactory stability and durability as well as stable NF flux and rejection for at least 36 h. In addition, the AqpZ-containing biomimetic membrane was immersed in a 0.24 mM (critical micellar concentration, CMC) Triton X-100 solution for 5 min. The flux and rejection were slightly influenced by the Triton X-100 treatment. The current investigation demonstrated that the AqpZ-incorporated biomimetic membranes fabricated by the LbL method led to excellent separation performances and robust structures that withstand a high operation pressure for a relatively long time. PMID:25730158

  17. Aquaporin-10 Represents an Alternative Pathway for Glycerol Efflux from Human Adipocytes

    PubMed Central

    Laforenza, Umberto; Scaffino, Manuela F.; Gastaldi, Giulia

    2013-01-01

    Background Glycerol outflow from adipocytes has been considered for a decade to be mediated by aquaporin-7, an aquaglyceroporin highly expressed in the adipose tissue. Its involvement in glycerol metabolism has been widely studied also in humans. Recent studies in different aquaporin-7 KO mice models pose two different questions 1) the exact localization of aquaporin-7 in human white adipose tissue; 2) the existence of other aquaglyceroporins that work with aquaporin-7 to guarantee glycerol efflux and thus a normal adiposity in humans. To this purpose we investigated the expression, the localization and the functioning of aquaglyceroporin-10 in subcutaneous white adipose tissue, in isolated and cultured differentiated adipocytes. Methodology/Principal Findings Aquaporin-7 and -10 were expressed in the white adipose tissue both at mRNA and at protein level. Immunofluorescence revealed aquaporin-7 and -10 labelling in the human adipose tissue both to the plasma membrane and to a thin rim of cytoplasm of adipocytes. Aquaporin-7, but not aquaporin-10, colocalized with the endothelial marker CD34. Human cultured differentiated adipocytes showed an aquaporin-7 and -10 labelling mainly in the cytoplasm and in the lipid droplets with insulin reinforcing the lipid droplets staining and isoproterenol inducing its translocation to the plasma membrane compartment. Water and glycerol permeability measurements using adipocytes and adipose membrane vesicles confirmed the presence of functioning aquaglyceroporins. Aquaporin-10 silencing in human differentiated adipocytes resulted in a 50% decrease of glycerol and osmotic water permeability. Conclusions/Significance The results indicate that aquaporin-7, differently from mice, is present in both adipocyte and capillary plasma membranes of human adipose tissue. Aquaporin-10, on the contrary, is expressed exclusively in the adipocytes. The expression of two aquaglyceroporins in human adipose tissue is particularly important for the

  18. Fluxes of Water through Aquaporin 9 Weaken Membrane-Cytoskeleton Anchorage and Promote Formation of Membrane Protrusions

    PubMed Central

    Karlsson, Thommie; Bolshakova, Anastasia; Magalhães, Marco A. O.; Loitto, Vesa M.; Magnusson, Karl-Eric

    2013-01-01

    All modes of cell migration require rapid rearrangements of cell shape, allowing the cell to navigate within narrow spaces in an extracellular matrix. Thus, a highly flexible membrane and a dynamic cytoskeleton are crucial for rapid cell migration. Cytoskeleton dynamics and tension also play instrumental roles in the formation of different specialized cell membrane protrusions, viz. lamellipodia, filopodia, and membrane blebs. The flux of water through membrane-anchored water channels, known as aquaporins (AQPs) has recently been implicated in the regulation of cell motility, and here we provide novel evidence for the role of AQP9 in the development of various forms of membrane protrusion. Using multiple imaging techniques and cellular models we show that: (i) AQP9 induced and accumulated in filopodia, (ii) AQP9-associated filopodial extensions preceded actin polymerization, which was in turn crucial for their stability and dynamics, and (iii) minute, local reductions in osmolarity immediately initiated small dynamic bleb-like protrusions, the size of which correlated with the reduction in osmotic pressure. Based on this, we present a model for AQP9-induced membrane protrusion, where the interplay of water fluxes through AQP9 and actin dynamics regulate the cellular protrusive and motile activity of cells. PMID:23573219

  19. Water Permeation Across Biological Membranes: Mechanism and Dynamics of Aquaporin-1 and GlpF

    NASA Astrophysics Data System (ADS)

    de Groot, Bert L.; Grubmüller, Helmut

    2001-12-01

    ``Real time'' molecular dynamics simulations of water permeation through human aquaporin-1 (AQP1) and the bacterial glycerol facilitator GlpF are presented. We obtained time-resolved, atomic-resolution models of the permeation mechanism across these highly selective membrane channels. Both proteins act as two-stage filters: Conserved fingerprint [asparagine-proline-alanine (NPA)] motifs form a selectivity-determining region; a second (aromatic/arginine) region is proposed to function as a proton filter. Hydrophobic regions near the NPA motifs are rate-limiting water barriers. In AQP1, a fine-tuned water dipole rotation during passage is essential for water selectivity. In GlpF, a glycerol-mediated ``induced fit'' gating motion is proposed to generate selectivity for glycerol over water.

  20. 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

  1. PIP1 aquaporins: Intrinsic water channels or PIP2 aquaporin modulators?

    PubMed

    Yaneff, Agustín; Vitali, Victoria; Amodeo, Gabriela

    2015-11-30

    The highly conserved plant aquaporins, known as Plasma membrane Intrinsic Proteins (PIPs), are the main gateways for cell membrane water exchange. Years of research have described in detail the properties of the PIP2 subfamily. However, characterizing the PIP1 subfamily has been difficult due to the failure to localize to the plasma membrane. In addition, the discovery of the PIP1-PIP2 interaction suggested that PIP1 aquaporins could be regulated by a complex posttranslational mechanism that involves trafficking, heteromerization and fine-tuning of channel activity. This review not only considers the evidence and findings but also discusses the complexity of PIP aquaporins. To establish a new benchmark in PIP regulation, we propose to consider PIP1-PIP2 pairs as functional units for the purpose of future research into their physiological roles. PMID:26526614

  2. Drought Stress-Induced Rma1H1, a RING Membrane-Anchor E3 Ubiquitin Ligase Homolog, Regulates Aquaporin Levels via Ubiquitination in Transgenic Arabidopsis Plants[C][W

    PubMed Central

    Lee, Hyun Kyung; Cho, Seok Keun; Son, Ora; Xu, Zhengyi; Hwang, Inhwan; Kim, Woo Taek

    2009-01-01

    Ubiquitination is involved in a variety of biological processes, but the exact role of ubiquitination in abiotic responses is not clearly understood in higher plants. Here, we investigated Rma1H1, a hot pepper (Capsicum annuum) homolog of a human RING membrane-anchor 1 E3 ubiquitin (Ub) ligase. Bacterially expressed Rma1H1 displayed E3 Ub ligase activity in vitro. Rma1H1 was rapidly induced by various abiotic stresses, including dehydration, and its overexpression in transgenic Arabidopsis thaliana plants conferred strongly enhanced tolerance to drought stress. Colocalization experiments with marker proteins revealed that Rma1H1 resides in the endoplasmic reticulum (ER) membrane. Overexpression of Rma1H1 in Arabidopsis inhibited trafficking of an aquaporin isoform PIP2;1 from the ER to the plasma membrane and reduced PIP2;1 levels in protoplasts and transgenic plants. This Rma1H1-induced reduction of PIP2;1 was inhibited by MG132, an inhibitor of the 26S proteasome. Furthermore, Rma1H1 interacted with PIP2;1 in vitro and ubiquitinated it in vivo. Similar to Rma1H1, Rma1, an Arabidopsis homolog of Rma1H1, localized to the ER, and its overexpression reduced the PIP2;1 protein level and inhibited trafficking of PIP2;1 from the ER to the plasma membrane in protoplasts. In addition, reduced expression of Rma homologs resulted in the increased level of PIP2;1 in protoplasts. We propose that Rma1H1 and Rma1 play a critical role in the downregulation of plasma membrane aquaporin levels by inhibiting aquaporin trafficking to the plasma membrane and subsequent proteasomal degradation as a response to dehydration in transgenic Arabidopsis plants. PMID:19234086

  3. 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

  4. Analysis of the aquaporin gene family in cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aquaporins are intrinsic membrane proteins present across kingdoms. In plants, aquaporins play roles in intercellular and intracellular water movement in response to osmotic and hydraulic potentials resulting from changing environmental conditions. In higher plants, aquaporins consist of five subfam...

  5. The Role of Aquaporins and Membrane Damage in Chilling and Hydrogen Peroxide Induced Changes in the Hydraulic Conductance of Maize Roots12

    PubMed Central

    Aroca, Ricardo; Amodeo, Gabriela; Fernández-Illescas, Silvia; Herman, Eliot M.; Chaumont, François; Chrispeels, Maarten J.

    2005-01-01

    When chilling-sensitive plants are chilled, root hydraulic conductance (Lo) declines precipitously; Lo also declines in chilling-tolerant plants, but it subsequently recovers, whereas in chilling-sensitive plants it does not. As a result, the chilling-sensitive plants dry out and may die. Using a chilling-sensitive and a chilling-tolerant maize genotype we investigated the effect of chilling on Lo, and its relationship to osmotic water permeability of isolated root cortex protoplasts, aquaporin gene expression, aquaporin abundance, and aquaporin phosphorylation, hydrogen peroxide (H2O2) accumulation in the roots and electrolyte leakage from the roots. Because chilling can cause H2O2 accumulation we also determined the effects of a short H2O2 treatment of the roots and examined the same parameters. We conclude from these studies that the recovery of Lo during chilling in the chilling-tolerant genotype is made possible by avoiding or repairing membrane damage and by a greater abundance and/or activity of aquaporins. The same changes in aquaporins take place in the chilling-sensitive genotype, but we postulate that membrane damage prevents the Lo recovery. It appears that the aquaporin response is necessary but not sufficient to respond to chilling injury. The plant must also be able to avoid the oxidative damage that accompanies chilling. PMID:15591439

  6. 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.

  7. Challenges in plasma membrane phosphoproteomics

    PubMed Central

    Orsburn, Benjamin C; Stockwin, Luke H; Newton, Dianne L

    2011-01-01

    The response to extracellular stimuli often alters the phosphorylation state of plasma membrane-associated proteins. In this regard, generation of a comprehensive membrane phosphoproteome can significantly enhance signal transduction and drug mechanism studies. However, analysis of this subproteome is regarded as technically challenging, given the low abundance and insolubility of integral membrane proteins, combined with difficulties in isolating, ionizing and fragmenting phosphopeptides. In this article, we highlight recent advances in membrane and phosphoprotein enrichment techniques resulting in improved identification of these elusive peptides. We also describe the use of alternative fragmentation techniques, and assess their current and future value to the field of membrane phosphoproteomics. PMID:21819303

  8. Lysosomes and the plasma membrane

    PubMed Central

    Andrews, Norma W.

    2002-01-01

    Studies of the cell invasion mechanism of the parasite Trypanosoma cruzi led to a series of novel findings, which revealed a previously unsuspected ability of conventional lysosomes to fuse with the plasma membrane. This regulated exocytic process, previously regarded mostly as a specialization of certain cell types, was recently shown to play an important role in the mechanism by which cells reseal their plasma membrane after injury. PMID:12147679

  9. 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. PMID:26248320

  10. 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.

  11. Fragment Screening of Human Aquaporin 1.

    PubMed

    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

  12. 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

  13. Aquaporins and cell migration.

    PubMed

    Papadopoulos, M C; Saadoun, S; Verkman, A S

    2008-07-01

    Aquaporin (AQP) water channels are expressed primarily in cell plasma membranes. In this paper, we review recent evidence that AQPs facilitate cell migration. AQP-dependent cell migration has been found in a variety of cell types in vitro and in mice in vivo. AQP1 deletion reduces endothelial cell migration, limiting tumor angiogenesis and growth. AQP4 deletion slows the migration of reactive astrocytes, impairing glial scarring after brain stab injury. AQP1-expressing tumor cells have enhanced metastatic potential and local infiltration. Impaired cell migration has also been seen in AQP1-deficient proximal tubule epithelial cells, and AQP3-deficient corneal epithelial cells, enterocytes, and skin keratinocytes. The mechanisms by which AQPs enhance cell migration are under investigation. We propose that, as a consequence of actin polymerization/depolymerization and transmembrane ionic fluxes, the cytoplasm adjacent to the leading edge of migrating cells undergoes rapid changes in osmolality. AQPs could thus facilitate osmotic water flow across the plasma membrane in cell protrusions that form during migration. AQP-dependent cell migration has potentially broad implications in angiogenesis, tumor metastasis, wound healing, glial scarring, and other events requiring rapid, directed cell movement. AQP inhibitors may thus have therapeutic potential in modulating these events, such as slowing tumor growth and spread, and reducing glial scarring after injury to allow neuronal regeneration. PMID:17968585

  14. 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. PMID:24215931

  15. 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

  16. Detecting Aquaporin Function and Regulation

    PubMed Central

    Madeira, Ana; Moura, Teresa F.; Soveral, Graça

    2016-01-01

    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 diagnostics 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. PMID:26870725

  17. 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.

  18. Detecting Aquaporin Function and Regulation.

    PubMed

    Madeira, Ana; Moura, Teresa F; Soveral, Graça

    2016-01-01

    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 diagnostics 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. PMID:26870725

  19. Regulation of the perilymphatic-endolymphatic water shunt in the cochlea by membrane translocation of aquaporin-5.

    PubMed

    Eckhard, A; Dos Santos, A; Liu, W; Bassiouni, M; Arnold, H; Gleiser, C; Hirt, B; Harteneck, C; Müller, M; Rask-Andersen, H; Löwenheim, H

    2015-12-01

    Volume homeostasis of the cochlear endolymph depends on radial and longitudinal endolymph movements (LEMs). LEMs measured in vivo have been exclusively recognized under physiologically challenging conditions, such as experimentally induced alterations of perilymph osmolarity or endolymph volume. The regulatory mechanisms that adjust LEMs to the physiological requirements of endolymph volume homeostasis remain unknown. Here, we describe the formation of an aquaporin (AQP)-based "water shunt" during the postnatal development of the mouse cochlea and its regulation by different triggers. The final complementary expression pattern of AQP5 (apical membrane) and AQP4 (basolateral membrane) in outer sulcus cells (OSCs) of the cochlear apex is acquired at the onset of hearing function (postnatal day (p)8-p12). In vitro, hyperosmolar perfusion of the perilymphatic fluid spaces or the administration of the muscarinic agonist pilocarpine in cochlear explants (p14) induced the translocation of AQP5 channel proteins into the apical membranes of OSCs. AQP5 membrane translocation was blocked by the muscarinic antagonist atropine. The muscarinic M3 acetylcholine (ACh) receptor (M3R) was identified in murine OSCs via mRNA expression, immunolabeling, and in vitro binding studies using an M3R-specific fluorescent ligand. Finally, the water shunt elements AQP4, AQP5, and M3R were also demonstrated in OSCs of the human cochlea. The regulation of the AQP4/AQP5 water shunt in OSCs of the cochlear apex provides a molecular basis for regulated endolymphatic volume homeostasis. Moreover, its dysregulation or disruption may have pathophysiologic implications for clinical conditions related to endolymphatic hydrops, such as Ménière's disease. PMID:26208470

  20. 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. PMID:27028365

  1. 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

  2. Multifaceted roles of aquaporins as molecular conduits in plant responses to abiotic stresses.

    PubMed

    Srivastava, Ashish Kumar; Penna, Suprasanna; Nguyen, Dong Van; Tran, Lam-Son Phan

    2016-01-01

    Abiotic stress has become a challenge to food security due to occurrences of climate change and environmental degradation. Plants initiate molecular, cellular and physiological changes to respond and adapt to various types of abiotic stress. Understanding of plant response mechanisms will aid in strategies aimed at improving stress tolerance in crop plants. One of the most common and early symptoms associated with these stresses is the disturbance in plant-water homeostasis, which is regulated by a group of proteins called "aquaporins". Aquaporins constitute a small family of proteins which are classified further on the basis of their localization, such as plasma membrane intrinsic proteins, tonoplast intrinsic proteins, nodulin26-like intrinsic proteins (initially identified in symbiosomes of legumes but also found in the plasma membrane and endoplasmic reticulum), small basic intrinsic proteins localized in ER (endoplasmic reticulum) and X intrinsic proteins present in plasma membrane. Apart from water, aquaporins are also known to transport CO2, H2O2, urea, ammonia, silicic acid, arsenite and wide range of small uncharged solutes. Besides, aquaporins also function to modulate abiotic stress-induced signaling. Such kind of versatile functions has made aquaporins a suitable candidate for development of transgenic plants with increased tolerance toward different abiotic stress. Toward this endeavor, the present review describes the versatile functions of aquaporins in water uptake, nutrient balancing, long-distance signal transfer, nutrient/heavy metal acquisition and seed development. Various functional genomic studies showing the potential of specific aquaporin isoforms for enhancing plant abiotic stress tolerance are summarized and future research directions are given to design stress-tolerant crops. PMID:25430890

  3. 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). PMID:16749244

  4. Identification of new aquaporin genes and single nucleotide polymorphism in bread wheat.

    PubMed

    Pandey, B; Sharma, P; Pandey, D M; Sharma, I; Chatrath, R

    2013-01-01

    Major facilitators of water movement through plant cell membranes include aquaporin proteins. Wheat is among the largest and most important cereal crops worldwide; however, unlike other model plants such as rice, maize and Arabidopsis, little has been reported on wheat major intrinsic proteins (MIPs). This study presents a comprehensive computational identification of 349 new wheat expressed sequence tags (ESTs), encoding 13 wheat aquaporin genes. Identified aquaporins consist of 6 plasma membrane intrinsic proteins (PIP) and 1 TIP showing high sequence similarity with rice aquaporins. We also identified 4 NOD26-like intrinsic proteins (NIP) and 2 SIP members that showed more divergence. Further, expression analysis of the aquaporin genes using the available EST information in UniGene revealed their transcripts were differentially regulated in various stress- and tissue-specific libraries. Allele specific Polymerase chain reaction (PCR) primers based on single nucleotide polymorphism (SNP) were designed using PIP as the target gene and validated on a core set of Indian wheat genotypes. A 3D theoretical model of the wheat aquaporin protein was built by homology modeling and could prove to be useful in the further functional characterization of this protein. Collectively with expression and bioinformatics analysis, our results support the idea that the genes identified in this study signify an important genetic resource providing potential targets to modify the water use properties of wheat. PMID:24250219

  5. 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. PMID:21352231

  6. Aquaporin water channels in the nervous system

    PubMed Central

    Papadopoulos, Marios C.; Verkman, Alan S.

    2013-01-01

    The aquaporins (AQPs) are plasma membrane water-transporting proteins. AQP4 is the principal member of this protein family in the CNS, where it is expressed in astrocytes and is involved in water movement, cell migration and neuroexcitation. AQP1 is expressed in the choroid plexus, where it facilitates cerebrospinal fluid secretion, and in dorsal root ganglion neurons, where it tunes pain perception. The AQPs are potential drug targets for several neurological conditions. Astrocytoma cells strongly express AQP4, which may facilitate their infiltration into the brain, and the neuroinflammatory disease neuromyelitis optica is caused by AQP4-specific autoantibodies that produce complement-mediated astrocytic damage. PMID:23481483

  7. Gene cloning and expression of an aquaporin (AQP-h3BL) in the basolateral membrane of water-permeable epithelial cells in osmoregulatory organs of the tree frog.

    PubMed

    Akabane, Gen; Ogushi, Yuji; Hasegawa, Takahiro; Suzuki, Masakazu; Tanaka, Shigeyasu

    2007-06-01

    An aquaporin (Hyla AQP-h3BL), consisting of 292 amino acid residues, has been cloned from the urinary bladder of Hyla japonica. In a swelling assay using Xenopus oocytes, AQP-h3BL cRNA-injected oocytes developed a sevenfold and 2.8-fold higher permeability to water and glycerol, respectively, than the water-injected oocytes. This permeability was inhibited by HgCl2. Immunofluorescence revealed that AQP-h3BL is localized in the basolateral plasma membrane of both granular cells in the ventral pelvic and dorsal skins and the secretory cells in the mucous glands. Immunopositive cells were also observed in the basolateral membrane of principal cells in the collecting ducts and in a portion of the late distal tubules in the kidneys, as well as in the principal cells of the urinary bladder. Sequence homology suggests that AQP-h3BL is a homolog to mammalian AQP3. This conclusion is supported by the observed localization of AQP-h3BL to the basolateral membrane in water- and glycerol-permeable epithelial cells. In ventral pelvic skins and urinary bladders, water enters into the cytoplasm through the apical plasma membrane at sites where AQP-h2, sometimes in association with AQP-h3, responds to stimulation by vasotocin; the water exits throughout AQP-h3BL to extracellular spaces. In the mucous glands, on the other hand, water enters throughout this AQP-h3BL and exits through AQP-x5, which is in the apical membrane of secretory cells. Thus, water homeostasis in the frog body is regulated by AQP-h3BL expressed in the basolateral membrane in concert with arginine vasotocin (AVT)-dependent or AVT-independent AQP. PMID:17332153

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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. PMID:18312385

  13. 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

  14. 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

  15. Developmental pattern of aquaporin expression in barley (Hordeum vulgare L.) leaves

    PubMed Central

    Besse, Matthieu; Knipfer, Thorsten; Miller, Anthony J.; Verdeil, Jean-Luc; Jahn, Thomas P.; Fricke, Wieland

    2011-01-01

    Aquaporins are multifunctional membrane channels which belong to the family of major intrinsic proteins (MIPs) and are best known for their ability to facilitate the movement of water. In the present study, earlier results from microarray experiments were followed up. These experiments had suggested that, in barley (Hordeum vulgare L.), aquaporin family members are expressed in distinct patterns during leaf development. Real-time PCR and in situ hybridization were used to analyse the level and tissue-distribution of expression of candidate aquaporins, focusing on plasma membrane and tonoplast intrinsic proteins (PIPs, TIPs). Water channel function of seven aquaporins, whose transcripts were the most abundant and the most variable, was tested through expression in yeast and, in part, through expression in oocytes. All PIP1 and PIP2 subfamily members changed in expression during leaf development, with expression being much higher or lower in growing compared with mature tissue. The same applied to those TIPs which were expressed at detectable levels. Specific roles during leaf development are proposed for particular aquaporins. PMID:21737414

  16. Aquaporin 4 as a NH3 Channel.

    PubMed

    Assentoft, Mette; Kaptan, Shreyas; Schneider, Hans-Peter; Deitmer, Joachim W; de Groot, Bert L; MacAulay, Nanna

    2016-09-01

    Ammonia is a biologically potent molecule, and the regulation of ammonia levels in the mammalian body is, therefore, strictly controlled. The molecular paths of ammonia permeation across plasma membranes remain ill-defined, but the structural similarity of water and NH3 has pointed to the aquaporins as putative NH3-permeable pores. Accordingly, a range of aquaporins from mammals, plants, fungi, and protozoans demonstrates ammonia permeability. Aquaporin 4 (AQP4) is highly expressed at perivascular glia end-feet in the mammalian brain and may, with this prominent localization at the blood-brain-interface, participate in the exchange of ammonia, which is required to sustain the glutamate-glutamine cycle. Here we observe that AQP4-expressing Xenopus oocytes display a reflection coefficient <1 for NH4Cl at pH 8.0, at which pH an increased amount of the ammonia occurs in the form of NH3 Taken together with an NH4Cl-mediated intracellular alkalization (or lesser acidification) of AQP4-expressing oocytes, these data suggest that NH3 is able to permeate the pore of AQP4. Exposure to NH4Cl increased the membrane currents to a similar extent in uninjected oocytes and in oocytes expressing AQP4, indicating that the ionic NH4 (+) did not permeate AQP4. Molecular dynamics simulations revealed partial pore permeation events of NH3 but not of NH4 (+) and a reduced energy barrier for NH3 permeation through AQP4 compared with that of a cholesterol-containing lipid bilayer, suggesting AQP4 as a favored transmembrane route for NH3 Our data propose that AQP4 belongs to the growing list of NH3-permeable water channels. PMID:27435677

  17. 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

  18. Radiofrequency plasma polymerized perfluoroionomer membrane materials

    SciTech Connect

    Danilich, M.J.; Gervasio, D.F.; Marchant, R.E.

    1993-12-31

    Ion exchange membranes have received considerable attention in recent years. Applications of ion exchange membranes have included such electrochemical systems as water and organic electrolyzers, redox-flow batteries, and sensors. This work is a study of radiofrequency plasma polymerization of perfluorinated acid-containing monomers and a perfluorinated {open_quotes}backbone{close_quotes} comonomer as a method for synthesizing novel polyionomer film coatings for use as membranes on electrodes and biomedical sensors. The results indicate that, by altering the deposition conditions, some control can be exercised over the retention of acid functional groups by plasma polymers. Using AC impedance measurements, the ionic conductivity of these films was found to be two to four orders of magnitude higher than their aqueous environments. In addition, several of the acid-containing plasma polymerized films were hydrophilic, having an advancing water contact angle of less than fifteen degrees. The initial results of this study have demonstrated the feasibility of using acid-containing plasma polymers as crosslinked membrane materials suitable for use with electrochemical sensors and biosensors.

  19. 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

  20. Regulation of salmonid fish sperm motility by osmotic shock-induced water influx across the plasma membrane.

    PubMed

    Takei, Gen Leon; Mukai, Chinatsu; Okuno, Makoto

    2015-04-01

    The motility of salmonid fish sperm is initiated by a decrease in the extracellular K(+) concentration. However, our previous studies revealed that salmonid fish sperm motility could be initiated in the presence of an inhibitory concentration of K(+) by drastic osmotic shock induced by suspension in a hypertonic glycerol solution and subsequent dilution in a hypotonic solution (glycerol-treatment). In the present study, we examined if an osmotic shock-induced water influx is involved in the regulation of salmonid fish sperm motility. HgCl2, a common inhibitor of aquaporins (AQPs), decreased the duration of salmonid fish sperm motility. Dilution of sperm cells in a hypotonic solution increased the cellular volume, whereas HgCl2 inhibited such an increase in cellular volume. Furthermore, the expression of AQP 1a and 10 in rainbow trout testes was confirmed. In contrast, HgCl2 did not affect glycerol-treated sperm motility, indicating that AQPs are not involved in glycerol-treated sperm motility. We also explored the possibility of aquaporin-independent water influx in glycerol-treated sperm by assessing the sperm membrane permeability using propidium iodide. The plasma membrane of glycerol-treated sperm was considerably permeabilized. The cellular volume was decreased in a hypertonic glycerol solution and increased upon subsequent hypoosmotic shock, indicating an AQP-independent water flux across the plasma membrane upon glycerol-treatment. Taken together, these results showed that water influx across the plasma membrane via AQP is crucial for the maintenance of salmonid fish sperm motility under normal conditions, whereas water influx by osmotic shock-induced membrane permeation is critical for the initiation of glycerol-treated sperm motility. PMID:25522712

  1. CO2 Transport by PIP2 Aquaporins of Barley

    PubMed Central

    Mori, Izumi C.; Rhee, Jiye; Shibasaka, Mineo; Sasano, Shizuka; Kaneko, Toshiyuki; Horie, Tomoaki; Katsuhara, Maki

    2014-01-01

    CO2 permeability of plasma membrane intrinsic protein 2 (PIP2) aquaporins of Hordeum vulgare L. was investigated. Five PIP2 members were heterologously expressed in Xenopus laevis oocytes. CO2 permeability was determined by decrease of cytosolic pH in CO2-enriched buffer using a hydrogen ion-selective microelectrode. HvPIP2;1, HvPIP2;2, HvPIP2;3 and HvPIP2;5 facilitated CO2 transport across the oocyte cell membrane. However, HvPIP2;4 that is highly homologous to HvPIP2;3 did not. The isoleucine residue at position 254 of HvPIP2;3 was conserved in PIP2 aquaporins of barley, except HvPIP2;4, which possesses methionine instead. CO2 permeability was lost by the substitution of the Ile254 of HvPIP2;3 by methionine, while water permeability was not affected. These results suggest that PIP2 aquaporins are permeable to CO2. and the conserved isoleucine at the end of the E-loop is crucial for CO2 selectivity. PMID:24406630

  2. 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. PMID:17114274

  3. Rupture of plasma membrane under tension.

    PubMed

    Tan, Samuel Chun Wei; Yang, Tianyi; Gong, Yingxue; Liao, Kin

    2011-04-29

    We present a study on the rupture behavior of single NIH 3T3 mouse fibroblasts under tension using micropipette aspiration. Membrane rupture was characterized by breaking and formation of an enclosed membrane linked to a tether at the cell apex. Three different rupture modes, namely: single break, initial multiple breaks, and continuous multiple breaks, were observed under similar loading condition. The measured mean tensile strengths of plasma membrane were 3.83 ± 1.94 and 3.98 ± 1.54mN/m for control cells and cells labeled with TubulinTracker, respectively. The tensile strength data was described by Weibull distribution. For the control cells, the Weibull modulus and characteristic strength were 1.86 and 4.40 mN/m, respectively; for cells labeled with TubulinTracker, the Weibull modulus and characteristic strength were 2.68 and 4.48 mN/m, respectively. Based on the experimental data, the estimated average transmembrane proteins-lipid cleavage strength was 2.64 ± 0.64 mN/m. From the random sampling of volume ratio of transmembrane proteins in cell membrane, we concluded that the Weibull characteristic of plasma membrane strength was likely to be originated from the variation in transmembrane proteins-lipid interactions. PMID:21288526

  4. 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.

  5. Membrane raft association is a determinant of plasma membrane localization

    PubMed Central

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

    2014-01-01

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

  6. Plasma Membrane Transporters in Modern Liver Pharmacology

    PubMed Central

    Marin, Jose J. G.

    2012-01-01

    The liver plays a crucial role in the detoxification of drugs used in the treatment of many diseases. The liver itself is the target for drugs aimed to modify its function or to treat infections and tumours affecting this organ. Both detoxification and pharmacological processes occurring in the liver require the uptake of the drug by hepatic cells and, in some cases, the elimination into bile. These steps have been classified as detoxification phase 0 and phase III, respectively. Since most drugs cannot cross the plasma membrane by simple diffusion, the involvement of transporters is mandatory. Several members of the superfamilies of solute carriers (SLC) and ATP-binding cassette (ABC) proteins, with a minor participation of other families of transporters, account for the uptake and efflux, respectively, of endobiotic and xenobiotic compounds across the basolateral and apical membranes of hepatocytes and cholangiocytes. These transporters are also involved in the sensitivity and refractoriness to the pharmacological treatment of liver tumours. An additional interesting aspect of the role of plasma membrane transporters in liver pharmacology regards the promiscuity of many of these carriers, which accounts for a variety of drug-drug, endogenous substances-drug and food components-drug interactions with clinical relevance. PMID:24278693

  7. 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. PMID:27215210

  8. From genome to function: the Arabidopsis aquaporins

    PubMed Central

    Quigley, Francoise; Rosenberg, Joshua M; Shachar-Hill, Yair; Bohnert, Hans J

    2002-01-01

    Background In the post-genomic era newly sequenced genomes can be used to deduce organismal functions from our knowledge of other systems. Here we apply this approach to analyzing the aquaporin gene family in Arabidopsis thaliana. The aquaporins are intrinsic membrane proteins that have been characterized as facilitators of water flux. Originally termed major intrinsic proteins (MIPs), they are now also known as water channels, glycerol facilitators and aqua-glyceroporins, yet recent data suggest that they facilitate the movement of other low-molecular-weight metabolites as well. Results The Arabidopsis genome contains 38 sequences with homology to aquaporin in four subfamilies, termed PIP, TIP, NIP and SIP. We have analyzed aquaporin family structure and expression using the A. thaliana genome sequence, and introduce a new NMR approach for the purpose of analyzing water movement in plant roots in vivo. Conclusions Our preliminary data indicate a strongly transcellular component for the flux of water in roots. PMID:11806824

  9. Regulation of Plasma Membrane Recycling by CFTR

    NASA Astrophysics Data System (ADS)

    Bradbury, Neil A.; Jilling, Tamas; Berta, Gabor; Sorscher, Eric J.; Bridges, Robert J.; Kirk, Kevin L.

    1992-04-01

    The gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) is defective in patients with cystic fibrosis. Although the protein product of the CFTR gene has been proposed to function as a chloride ion channel, certain aspects of its function remain unclear. The role of CFTR in the adenosine 3',5'-monophosphate (cAMP)-dependent regulation of plasma membrane recycling was examined. Adenosine 3',5'-monophosphate is known to regulate endocytosis and exocytosis in chloride-secreting epithelial cells that express CFTR. However, mutant epithelial cells derived from a patient with cystic fibrosis exhibited no cAMP-dependent regulation of endocytosis and exocytosis until they were transfected with complementary DNA encoding wild-type CFTR. Thus, CFTR is critical for cAMP-dependent regulation of membrane recycling in epithelial tissues, and this function of CFTR could explain in part the pleiotropic nature of cystic fibrosis.

  10. Electron microscopy methods for studying plasma membranes.

    PubMed

    Beckett, Alison J; Prior, Ian A

    2015-01-01

    Electron microscopy allows direct visualization of the underlying organization of cell surface components on a nano-scale. Immuno-gold labelling of isolated plasma membranes generates point patterns that enable mapping of protein and lipid distributions. 2D spatial statistics reveals the extent to which these distributions are clustered or dispersed and allows the extent of co-localization between different cell surface components to be precisely determined. This approach has been successfully applied to the study of signalling network organization and the consequences of physiological changes in modulating cell surface function. PMID:25331134

  11. Binding contribution between synaptic vesicle membrane and plasma membrane proteins in neurons: an AFM study.

    PubMed

    Sritharan, K C; Quinn, A S; Taatjes, D J; Jena, B P

    1998-01-01

    The final step in the exocytotic process is the docking and fusion of membrane-bound secretory vesicles at the cell plasma membrane. This docking and fusion is brought about by several participating vesicle membrane, plasma membrane and soluble cytosolic proteins. A clear understanding of the interactions between these participating proteins giving rise to vesicle docking and fusion is essential. In this study, the binding force profiles between synaptic vesicle membrane and plasma membrane proteins have been examined for the first time using the atomic force microscope. Binding force contributions of a synaptic vesicle membrane protein VAMP1, and the plasma membrane proteins SNAP-25 and syntaxin, are also implicated from these studies. Our study suggests that these three proteins are the major, if not the only contributors to the interactive binding force that exist between the two membranes. PMID:10452835

  12. Aquaporins and unloading of phloem-imported water in coats of developing bean seeds.

    PubMed

    Zhou, Yuchan; Setz, Nathan; Niemietz, Christa; Qu, Hongxia; Offler, Christina E; Tyerman, Stephen D; Patrick, John W

    2007-12-01

    Nutrients are imported into developing legume seeds by mass flow through the phloem, and reach developing embryos following secretion from their symplasmically isolated coats. To sustain homeostasis of seed coat water relations, phloem-delivered nutrients and water must exit seed coats at rates commensurate with those of import through the phloem. In this context, coats of developing French bean seeds were screened for expression of aquaporin genes resulting in cloning PvPIP1;1, PvPIP2;2 and PvPIP2;3. These genes were differentially expressed in all vegetative organs, but exhibited their strongest expression in seed coats. In seed coats, expression was localized to cells of the nutrient-unloading pathway. Transport properties of the PvPIPs were characterized by expression in Xenopus oocytes. Only PvPIP2;3 showed significant water channel activity (Pos = 150-200 microm s(-1)) even when the plasma membrane intrinsic proteins (PIPs) were co-expressed in various combinations. Permeability increases to glycerol, methylamine and urea were not detected in oocytes expressing PvPIPs. Transport active aquaporins in native plasma membranes of seed coats were demonstrated by measuring rates of osmotic shrinkage of membrane vesicles in the presence and absence of mercuric chloride and silver nitrate. The functional significance of aquaporins in nutrient and water transport in developing seeds is discussed. PMID:17927694

  13. What are aquaporins for?

    PubMed

    Hill, A E; Shachar-Hill, B; Shachar-Hill, Y

    2004-01-01

    The prime function of aquaporins (AQPs) is generally believed to be that of increasing water flow rates across membranes by raising their osmotic or hydraulic permeability. In addition, this applies to other small solutes of physiological importance. Notable applications of this 'simple permeability hypothesis' (SPH) have been epithelial fluid transport in animals, water exchanges associated with transpiration, growth and stress in plants, and osmoregulation in microbes. We first analyze the need for such increased permeabilities and conclude that in a range of situations at the cellular, subcellular and tissue levels the SPH cannot satisfactorily account for the presence of AQPs. The analysis includes an examination of the effects of the genetic elimination or reduction of AQPs (knockouts, antisense transgenics and null mutants). These either have no effect, or a partial effect that is difficult to explain, and we argue that they do not support the hypothesis beyond showing that AQPs are involved in the process under examination. We assume that since AQPs are ubiquitous, they must have an important function and suggest that this is the detection of osmotic and turgor pressure gradients. A mechanistic model is proposed--in terms of monomer structure and changes in the tetrameric configuration of AQPs in the membrane--for how AQPs might function as sensors. Sensors then signal within the cell to control diverse processes, probably as part of feedback loops. Finally, we examine how AQPs as sensors may serve animal, plant and microbial cells and show that this sensor hypothesis can provide an explanation of many basic processes in which AQPs are already implicated. Aquaporins are molecules in search of a function; osmotic and turgor sensors are functions in search of a molecule. PMID:15014915

  14. 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

  15. Selective photosensitizer delivery into plasma membrane for effective photodynamic therapy.

    PubMed

    Kim, Jiyoung; Santos, Olavo Amorim; Park, Ji-Ho

    2014-10-10

    Subcellular localization of photosensitizers (PSs) determines the therapeutic efficacy in the photodynamic therapy. However, among the subcellular compartments, there has been little effort to deliver the PSs selectively into the plasma membrane and examine the phototherapeutic efficacy of membrane-localized PSs. Here, we developed a liposomal delivery system to localize the hydrophobic PSs selectively into the plasma membrane. The membrane fusogenic liposomes (MFLs), the membrane of which is engineered to fuse with the plasma membrane, was prepared for the membrane localization of PSs. The phototherapeutic efficacy of cells treated with ZnPc-loaded MFLs was superior over that of cells treated with ZnPc-loaded non-fusogenic liposomes, which is the conventional liposomal formulation that delivers the PSs into the intracellular compartments via endocytosis. The membrane localization of ZnPc molecules led to rapid membrane disruption upon irradiation and subsequent necrosis-like cell death. The membrane-localized generation of reactive oxygen species in the cells treated with ZnPc-loaded MFLs was likely to account for the effective disruption of plasma membrane. Thus, this work provides a novel delivery method to localize the PSs selectively into the plasma membrane with the enhanced phototherapeutic efficacy. PMID:24892975

  16. 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

  17. Order of lipid phases in model and plasma membranes

    PubMed Central

    Kaiser, Hermann-Josef; Lingwood, Daniel; Levental, Ilya; Sampaio, Julio L.; Kalvodova, Lucie; Rajendran, Lawrence; Simons, Kai

    2009-01-01

    Lipid rafts are nanoscopic assemblies of sphingolipids, cholesterol, and specific membrane proteins that contribute to lateral heterogeneity in eukaryotic membranes. Separation of artificial membranes into liquid-ordered (Lo) and liquid-disordered phases is regarded as a common model for this compartmentalization. However, tight lipid packing in Lo phases seems to conflict with efficient partitioning of raft-associated transmembrane (TM) proteins. To assess membrane order as a component of raft organization, we performed fluorescence spectroscopy and microscopy with the membrane probes Laurdan and C-laurdan. First, we assessed lipid packing in model membranes of various compositions and found cholesterol and acyl chain dependence of membrane order. Then we probed cell membranes by using two novel systems that exhibit inducible phase separation: giant plasma membrane vesicles [Baumgart et al. (2007) Proc Natl Acad Sci USA 104:3165–3170] and plasma membrane spheres. Notably, only the latter support selective inclusion of raft TM proteins with the ganglioside GM1 into one phase. We measured comparable small differences in order between the separated phases of both biomembranes. Lateral packing in the ordered phase of giant plasma membrane vesicles resembled the Lo domain of model membranes, whereas the GM1 phase in plasma membrane spheres exhibited considerably lower order, consistent with different partitioning of lipid and TM protein markers. Thus, lipid-mediated coalescence of the GM1 raft domain seems to be distinct from the formation of a Lo phase, suggesting additional interactions between proteins and lipids to be effective. PMID:19805351

  18. 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

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

    PubMed

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

    2015-10-01

    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

  20. A major integral protein of the plant plasma membrane binds flavin.

    PubMed

    Lorenz, Astrid; Kaldenhoff, Ralf; Hertel, Rainer

    2003-05-01

    Abundant flavin binding sites have been found in membranes of plants and fungi. With flavin mononucleotide-agarose affinity columns, riboflavin-binding activity from microsomes of Cucurbita pepoL. hypocotyls was purified and identified as a specific PIP1-homologous protein of the aquaporin family. Sequences such as gi|2149955 in Phaseolus vulgaris, PIP1b of Arabidopsis thaliana, and NtAQP1 of tobacco are closely related. The identification as a riboflavin-binding protein was confirmed by binding tests with an extract of Escherichia coli cells expressing the tobacco NtAQP1 as well as leaves of transgenic tobacco plants that overexpress NtAQP1 or were inhibited in PIP1 expression by antisense constructs. When binding was assayed in the presence of dithionite, the reduced flavin formed a relatively stable association with the protein. Upon dilution under oxidizing conditions, the adduct was resolved, and free flavin reappeared with a half time of about 30 min. Such an association can also be induced photochemically, with oxidized flavin by blue light at 450 nm, in the presence of an electron donor. Several criteria, localization in the plasma membrane, high abundance, affinity to roseoflavin, and photochemistry, argue for a role of the riboflavin-binding protein PIP1 as a photoreceptor. PMID:12768338

  1. Expression and functional analysis of the rice plasma-membrane intrinsic protein gene family.

    PubMed

    Guo, Lei; Wang, Zi Yi; Lin, Hong; Cui, Wei Er; Chen, Jun; Liu, Meihua; Chen, Zhang Liang; Qu, Li Jia; Gu, Hongya

    2006-03-01

    Plasma membrane intrinsic proteins (PIPs) are a subfamily of aquaporins that enable fast and controlled translocation of water across the membrane. In this study, we systematically identified and cloned ten PIP genes from rice. Based on the similarity of the amino acid sequences they encoded, these rice PIP genes were classified into two groups and designated as OsPIP1-1 to OsPIP1-3 and OsPIP2-1 to OsPIP2-7 following the nomenclature of PIP genes in maize. Quantitative RT-PCR analysis identified three root-specific and one leaf-specific OsPIP genes. Furthermore, the expression profile of each OsPIP gene in response to salt, drought and ABA treatment was examined in detail. Analysis on transgenic plants over-expressing of either OsPIP1 (OsPIP1-1) or OsPIP2 (OsPIP2-2) in wild-type Arabidopsis, showed enhanced tolerance to salt (100 mM of NaCl) and drought (200 mM of mannitol), but not to salt treatment of higher concentration (150 mM of NaCl). Taken together, these data suggest a distinct role of each OsPIP gene in response to different stresses, and should add a new layer to the understanding of the physiological function of rice PIP genes. PMID:16541126

  2. Easy measurement of diffusion coefficients of EGFP-tagged plasma membrane proteins using k-Space Image Correlation Spectroscopy.

    PubMed

    Arnspang, Eva C; Koffman, Jennifer S; Marlar, Saw; Wiseman, Paul W; Nejsum, Lene N

    2014-01-01

    Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new insights to plasma membrane protein function and regulation. Recently, k-Space Image Correlation Spectroscopy (kICS)(1) was developed to enable routine measurements of diffusion coefficients directly from images of fluorescently tagged plasma membrane proteins, that avoided systematic biases introduced by probe photophysics. Although the theoretical basis for the analysis is complex, the method can be implemented by nonexperts using a freely available code to measure diffusion coefficients of proteins. kICS calculates a time correlation function from a fluorescence microscopy image stack after Fourier transformation of each image to reciprocal (k-) space. Subsequently, circular averaging, natural logarithm transform and linear fits to the correlation function yields the diffusion coefficient. This paper provides a step-by-step guide to the image analysis and measurement of diffusion coefficients via kICS. First, a high frame rate image sequence of a fluorescently labeled plasma membrane protein is acquired using a fluorescence microscope. Then, a region of interest (ROI) avoiding intracellular organelles, moving vesicles or protruding membrane regions is selected. The ROI stack is imported into a freely available code and several defined parameters (see Method section) are set for kICS analysis. The program then generates a "slope of slopes" plot from the k-space time correlation functions, and the diffusion coefficient is calculated from the slope of the plot. Below is a step-by-step kICS procedure to measure the diffusion coefficient of a membrane protein using the renal water channel aquaporin-3 tagged with EGFP as a canonical example. PMID:24893770

  3. POST-GOLGI SUPRAMOLECULAR ASSEMBLY OF AQUAPORIN-4 IN ORTHOGONAL ARRAYS

    PubMed Central

    Rossi, Andrea; Baumgart, Florian; van Hoek, Alfred N.; Verkman, Alan S.

    2012-01-01

    The supramolecular assembly of aquaporin-4 (AQP4) in orthogonal arrays of particles (OAPs) involves N-terminus interactions of the M23-AQP4 isoform. We found AQP4 OAPs in cell plasma membranes but not in endoplasmic reticulum (ER) or Golgi, as shown by: (i) native gel electrophoresis of brain and AQP4-transfected cells; (ii) photobleaching recovery of GFP-AQP4 chimeras in live cells; and (iii) freeze-fracture electron microscopy (FFEM). We found that AQP4 OAP formation in plasma membranes but not Golgi was not related to AQP4 density, pH, membrane lipid composition, C-terminal PDZ-domain interactions or α-syntrophin expression. Remarkably, however, fusion of AQP4-containing Golgi vesicles with (AQP4-free) plasma membrane vesicles produced OAPs, suggesting the involvement of plasma membrane factor(s) in AQP4 OAP formation. In investigating additional possible determinants of OAP assembly we discovered membrane curvature-dependent OAP assembly, in which OAPs were disrupted by extrusion of plasma membrane vesicles to ~110 nm diameter, but not to ~220 nm diameter. We conclude that AQP4 supramolecular assembly in OAPs is a post-Golgi phenomenon involving plasma membrane-specific factor(s). Post-Golgi and membrane curvature-dependent OAP assembly may be important for vesicle transport of AQP4 in the secretory pathway and AQP4-facilitated astrocyte migration, and suggests a novel therapeutic approach for neuromyelitis optica (NMO). PMID:21981006

  4. Relative Abundance of Integral Plasma Membrane Proteins in Arabidopsis Leaf and Root Tissue Determined by Metabolic Labeling and Mass Spectrometry

    PubMed Central

    Bernfur, Katja; Larsson, Olaf; Larsson, Christer; Gustavsson, Niklas

    2013-01-01

    Metabolic labeling of proteins with a stable isotope (15N) in intact Arabidopsis plants was used for accurate determination by mass spectrometry of differences in protein abundance between plasma membranes isolated from leaves and roots. In total, 703 proteins were identified, of which 188 were predicted to be integral membrane proteins. Major classes were transporters, receptors, proteins involved in membrane trafficking and cell wall-related proteins. Forty-one of the integral proteins, including nine of the 13 isoforms of the PIP (plasma membrane intrinsic protein) aquaporin subfamily, could be identified by peptides unique to these proteins, which made it possible to determine their relative abundance in leaf and root tissue. In addition, peptides shared between isoforms gave information on the proportions of these isoforms. A comparison between our data for protein levels and corresponding data for mRNA levels in the widely used database Genevestigator showed an agreement for only about two thirds of the proteins. By contrast, localization data available in the literature for 21 of the 41 proteins show a much better agreement with our data, in particular data based on immunostaining of proteins and GUS-staining of promoter activity. Thus, although mRNA levels may provide a useful approximation for protein levels, detection and quantification of isoform-specific peptides by proteomics should generate the most reliable data for the proteome. PMID:23990937

  5. Syntaxin specificity of aquaporins in the inner medullary collecting duct.

    PubMed

    Mistry, Abinash C; Mallick, Rickta; Klein, Janet D; Weimbs, Thomas; Sands, Jeff M; Fröhlich, Otto

    2009-08-01

    Proper targeting of the aquaporin-2 (AQP2) water channel to the collecting duct apical plasma membrane is critical for the urine concentrating mechanism and body water homeostasis. However, the trafficking mechanisms that recruit AQP2 to the plasma membrane are still unclear. Snapin is emerging as an important mediator in the initial interaction of trafficked proteins with target soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (t-SNARE) proteins, and this interaction is functionally important for AQP2 regulation. We show that in AQP2-Madin-Darby canine kidney cells subjected to adenoviral-mediated expression of both snapin and syntaxins, the association of AQP2 with both syntaxin-3 and syntaxin-4 is highly enhanced by the presence of snapin. In pull-down studies, snapin detected AQP2, syntaxin-3, syntaxin-4, and SNAP23 from the inner medullary collecting duct. AQP2 transport activity, as probed by AQP2's urea permeability, was greatly enhanced in oocytes that were coinjected with cRNAs of SNARE components (snapin+syntaxin-3+SNAP23) over those injected with AQP2 cRNA alone. It was not enhanced when syntaxin-3 was replaced by syntaxin-4 (snapin+syntaxin-4+SNAP23). On the other hand, the latter combination significantly enhanced the transport activity of the related AQP3 water channel while the presence of syntaxin-3 did not. This AQP-syntaxin interaction agrees with the polarity of these proteins' expression in the inner medullary collecting duct epithelium. Thus our findings suggest a selectivity of interactions between different aquaporin and syntaxin isoforms, and thus in the regulation of AQP2 and AQP3 activities in the plasma membrane. Snapin plays an important role as a linker between the water channel and the t-SNARE complex, leading to the fusion event, and the pairing with specific t-SNAREs is essential for the specificity of membrane recognition and fusion. PMID:19515809

  6. The movement of water and cryoprotectants across the plasma membrane of mammalian oocytes and embryos and its relevance to vitrification

    PubMed Central

    EDASHIGE, Keisuke

    2016-01-01

    The permeability of the plasma membrane to water and cryoprotectants is one of the most important factors for determining suitable conditions for vitrification of mammalian oocytes and embryos. In mouse oocytes and early stage embryos, water and cryoprotectants move slowly, principally by simple diffusion. In contrast, in morulae (and probably blastocysts), water, glycerol, and ethylene glycerol move rapidly, principally by facilitated diffusion via aquaporin 3, and DMSO moves rapidly via channels other than aquaporin 3. However, propylene glycol moves principally by simple diffusion. In cows and pigs, similar results were obtained. However, in bovine morulae, DMSO moves principally by simple diffusion. In pigs, permeability to water, glycerol, and ethylene glycol increases not at the morula stage but at the blastocyst stage, and increases further at the expanded blastocyst stage. Therefore, in general, the permeability of mammalian oocytes and early stage embryos to water and cryoprotectants is low. Then, at later stages, the permeability to water and some cryoprotectants markedly increases and occurs by facilitated diffusion via channels, although there are some species-specific differences. PMID:27193425

  7. The movement of water and cryoprotectants across the plasma membrane of mammalian oocytes and embryos and its relevance to vitrification.

    PubMed

    Edashige, Keisuke

    2016-08-25

    The permeability of the plasma membrane to water and cryoprotectants is one of the most important factors for determining suitable conditions for vitrification of mammalian oocytes and embryos. In mouse oocytes and early stage embryos, water and cryoprotectants move slowly, principally by simple diffusion. In contrast, in morulae (and probably blastocysts), water, glycerol, and ethylene glycerol move rapidly, principally by facilitated diffusion via aquaporin 3, and DMSO moves rapidly via channels other than aquaporin 3. However, propylene glycol moves principally by simple diffusion. In cows and pigs, similar results were obtained. However, in bovine morulae, DMSO moves principally by simple diffusion. In pigs, permeability to water, glycerol, and ethylene glycol increases not at the morula stage but at the blastocyst stage, and increases further at the expanded blastocyst stage. Therefore, in general, the permeability of mammalian oocytes and early stage embryos to water and cryoprotectants is low. Then, at later stages, the permeability to water and some cryoprotectants markedly increases and occurs by facilitated diffusion via channels, although there are some species-specific differences. PMID:27193425

  8. Dynamics of photoinduced cell plasma membrane injury.

    PubMed Central

    Thorpe, W P; Toner, M; Ezzell, R M; Tompkins, R G; Yarmush, M L

    1995-01-01

    We have developed a video microscopy system designed for real-time measurement of single cell damage during photolysis under well defined physicochemical and photophysical conditions. Melanoma cells cultured in vitro were treated with the photosensitizer (PS), tin chlorin e6 (SnCe6) or immunoconjugate (SnCe6 conjugated to a anti-ICAM monoclonal antibody), and illuminated with a 10 mW He/Ne laser at a 630 nm wavelength. Cell membrane integrity was assessed using the vital dye calcein-AM. In experiments in which the laser power density and PS concentration were varied, it was determined that the time lag before cell rupture was inversely proportional to the estimated singlet oxygen flux to the cell surface. Microscopic examination of the lytic event indicated that photo-induced lysis was caused by a point rupture of the plasma membrane. The on-line nature of this microscopy system offers an opportunity to monitor the dynamics of the cell damage process and to gain insights into the mechanism governing photolytic cell injury processes. Images FIGURE 2 FIGURE 3 FIGURE 6 FIGURE 7 PMID:7612864

  9. Channelopathies linked to plasma membrane phosphoinositides

    PubMed Central

    Logothetis, Diomedes E.; Petrou, Vasileios I.; Adney, Scott K.; Mahajan, Rahul

    2014-01-01

    The plasma membrane phosphoinositide phosphatidylinositol 4,5-bisphosphate (PIP2) controls the activity of most ion channels tested thus far through direct electrostatic interactions. Mutations in channel proteins that change their apparent affinity to PIP2 can lead to channelopathies. Given the fundamental role that membrane phosphoinositides play in regulating channel activity, it is surprising that only a small number of channelopathies have been linked to phosphoinositides. This review proposes that for channels whose activity is PIP2-dependent and for which mutations can lead to channelopathies, the possibility that the mutations alter channel-PIP2 interactions ought to be tested. Similarly, diseases that are linked to disorders of the phosphoinositide pathway result in altered PIP2 levels. In such cases, it is proposed that the possibility for a concomitant dysregulation of channel activity also ought to be tested. The ever-growing list of ion channels whose activity depends on interactions with PIP2 promises to provide a mechanism by which defects on either the channel protein or the phosphoinositide levels can lead to disease. PMID:20396900

  10. Membrane Compartment Occupied by Can1 (MCC) and Eisosome Subdomains of the Fungal Plasma Membrane

    PubMed Central

    Douglas, Lois M.; Wang, Hong X.; Li, Lifang; Konopka, James B.

    2011-01-01

    Studies on the budding yeast Saccharomyces cerevisiae have revealed that fungal plasma membranes are organized into different subdomains. One new domain termed MCC/eisosomes consists of stable punctate patches that are distinct from lipid rafts. The MCC/eisosome domains correspond to furrows in the plasma membrane that are about 300 nm long and 50 nm deep. The MCC portion includes integral membrane proteins, such as the tetraspanners Sur7 and Nce102. The adjacent eisosome includes proteins that are peripherally associated with the membrane, including the BAR domains proteins Pil1 and Lsp1 that are thought to promote membrane curvature. Genetic analysis of the MCC/eisosome components indicates these domains broadly affect overall plasma membrane organization. The mechanisms regulating the formation of MCC/eisosomes in model organisms will be reviewed as well as the role of these plasma membrane domains in fungal pathogenesis and response to antifungal drugs. PMID:22368779

  11. 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

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

    PubMed

    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

  13. Plasma Membrane Water Permeability of Cultured Cells and Epithelia Measured by Light Microscopy with Spatial Filtering

    PubMed Central

    Farinas, Javier; Kneen, Malea; Moore, Megan; Verkman, A.S.

    1997-01-01

    A method was developed to measure the osmotic water permeability (Pf) of plasma membranes in cell layers and applied to cells and epithelia expressing molecular water channels. It was found that the integrated intensity of monochromatic light in a phase contrast or dark field microscope was dependent on relative cell volume. For cells of different size and shape (Sf9, MDCK, CHO, A549, tracheal epithelia, BHK), increased cell volume was associated with decreased signal intensity; generally the signal decreased 10–20% for a twofold increase in cell volume. A theory relating signal intensity to relative cell volume was developed based on spatial filtering and changes in optical path length associated with cell volume changes. Theory predictions were confirmed by signal measurements of cell layers bathed in solutions of various osmolarities and refractive indices. The excellent signal-to-noise ratio of the transmitted light detection permitted measurement of cell volume changes of <1%. The method was applied to characterize transfected cells and tissues that natively express water channels. Pf in control Chinese hamster ovary cells was low (0.0012 cm/s at 23°C) and increased more than fourfold upon stable transfection with aquaporins 1, 2, 4, or 5. Pf in apical and basolateral membranes in polarized epithelial cells grown on porous supports was measured. Pfbl and Pfap were 0.0011 and 0.0024 cm/s (MDCK cells), and 0.0039 and 0.0052 cm/s (human tracheal cells) at 23°C. In intact toad urinary bladder, basolateral Pf was 0.036 cm/s and apical membrane Pf after vasopressin stimulation was 0.025 cm/s at 23°C. The results establish light microscopy with spatial filtering as a technically simple and quantitative method to measure water permeability in cell layers and provide the first measurement of the apical and basolateral membrane permeabilities of several important epithelial cell types. PMID:9276754

  14. 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

  15. 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. PMID:26786101

  16. 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

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

    PubMed

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

    1995-06-01

    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

  18. 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

  19. 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. PMID:19096770

  20. A Minireview on Vasopressin-regulated Aquaporin-2 in Kidney Collecting Duct Cells

    PubMed Central

    Park, Eui-Jung

    2015-01-01

    The kidney collecting duct is an important renal tubular segment for the regulation of body water and salt homeostasis. Water reabsorption in the collecting duct cells is regulated by arginine vasopressin (AVP) via the vasopressin V2-receptor (V2R). AVP increases the osmotic water permeability of the collecting duct cells through aquaporin-2 (AQP2) and aquaporin-3 (AQP3). AVP induces the apical targeting of AQP2 and transcription of AQP2 gene in the kidney collecting duct principal cells. The signaling transduction pathways resulting in the AQP2 trafficking to the apical plasma membrane of the collecting duct principal cells, include AQP2 phosphorylation, RhoA phosphorylation, actin depolymerization and calcium mobilization, and the changes of AQP2 protein abundance in water balance disorders have been extensively studied. These studies elucidate the underlying cellular and molecular mechanisms of body water homeostasis and provide the basis for the treatment of body water balance disorders. PMID:26240594

  1. A Minireview on Vasopressin-regulated Aquaporin-2 in Kidney Collecting Duct Cells.

    PubMed

    Park, Eui-Jung; Kwon, Tae-Hwan

    2015-06-01

    The kidney collecting duct is an important renal tubular segment for the regulation of body water and salt homeostasis. Water reabsorption in the collecting duct cells is regulated by arginine vasopressin (AVP) via the vasopressin V2-receptor (V2R). AVP increases the osmotic water permeability of the collecting duct cells through aquaporin-2 (AQP2) and aquaporin-3 (AQP3). AVP induces the apical targeting of AQP2 and transcription of AQP2 gene in the kidney collecting duct principal cells. The signaling transduction pathways resulting in the AQP2 trafficking to the apical plasma membrane of the collecting duct principal cells, include AQP2 phosphorylation, RhoA phosphorylation, actin depolymerization and calcium mobilization, and the changes of AQP2 protein abundance in water balance disorders have been extensively studied. These studies elucidate the underlying cellular and molecular mechanisms of body water homeostasis and provide the basis for the treatment of body water balance disorders. PMID:26240594

  2. No primexine and plasma membrane undulation is essential for primexine deposition and plasma membrane undulation during microsporogenesis in Arabidopsis.

    PubMed

    Chang, Hai-Shuang; Zhang, Cheng; Chang, Yu-Hua; Zhu, Jun; Xu, Xiao-Feng; Shi, Zhi-Hao; Zhang, Xiao-Lei; Xu, Ling; Huang, Hai; Zhang, Sen; Yang, Zhong-Nan

    2012-01-01

    Primexine deposition and plasma membrane undulation are the initial steps of pollen wall formation. However, little is known about the genes involved in this important biological process. Here, we report a novel gene, NO PRIMEXINE AND PLASMA MEMBRANE UNDULATION (NPU), which functions in the early stage of pollen wall development in Arabidopsis (Arabidopsis thaliana). Loss of NPU function causes male sterility due to a defect in callose synthesis and sporopollenin deposition, resulting in disrupted pollen in npu mutants. Transmission electronic microscopy observation demonstrated that primexine deposition and plasma membrane undulation are completely absent in the npu mutants. NPU encodes a membrane protein with two transmembrane domains and one intracellular domain. In situ hybridization analysis revealed that NPU is strongly expressed in microspores and the tapetum during the tetrad stage. All these results together indicate that NPU plays a vital role in primexine deposition and plasma membrane undulation during early pollen wall development. PMID:22100644

  3. Aquaporins--new players in cancer biology.

    PubMed

    Verkman, A S; Hara-Chikuma, Mariko; Papadopoulos, Marios C

    2008-05-01

    The aquaporins (AQPs) are small, integral-membrane proteins that selectively transport water across cell plasma membranes. A subset of AQPs, the aquaglyceroporins, also transport glycerol. AQPs are strongly expressed in tumor cells of different origins, particularly aggressive tumors. Recent discoveries of AQP involvement in cell migration and proliferation suggest that AQPs play key roles in tumor biology. AQP1 is ubiquitously expressed in tumor vascular endothelium, and AQP1-null mice show defective tumor angiogenesis resulting from impaired endothelial cell migration. AQP-expressing cancer cells show enhanced migration in vitro and greater local tumor invasion, tumor cell extravasation, and metastases in vivo. AQP-dependent cell migration may involve AQP-facilitated water influx into lamellipodia at the front edge of migrating cells. The aquaglyceroporin AQP3, which is found in normal epidermis and becomes upregulated in basal cell carcinoma, facilitates cell proliferation in different cell types. Remarkably, AQP3-null mice are resistant to skin tumorigenesis by a mechanism that may involve reduced tumor cell glycerol metabolism and ATP generation. Together, the data suggest that AQP expression in tumor cells and tumor vessels facilitates tumor growth and spread, suggesting AQP inhibition as a novel antitumor therapy. PMID:18311471

  4. Roles of aquaporin-3 in the epidermis.

    PubMed

    Hara-Chikuma, Mariko; Verkman, A S

    2008-09-01

    Aquaporin-3 (AQP3) is a membrane transporter of water and glycerol expressed in plasma membranes in the basal layer keratinocytes of epidermis in normal skin. AQP3 expression in human skin is increased in response to skin stress in diseases such as atopic eczema, to various agents such as retinoic acid, and in skin carcinomas. AQP3-knockout mice have reduced stratum corneum water content and elasticity compared with wild-type mice, as well as impaired wound healing and epidermal biosynthesis. Reduced AQP3-dependent glycerol transport in AQP3-deficient epidermis appears to be responsible for these phenotype findings, as evidenced by reduced glycerol content in epidermis and stratum corneum in AQP3-knockout mice, and correction of the phenotype abnormalities by glycerol replacement. Recent data implicate AQP3 as an important determinant in epidermal proliferation and skin tumorigenesis, in which AQP3-knockout mice are resistant to tumor formation by a mechanism that may involve reduced cell glycerol content and ATP energy for biosynthesis. AQP3 is thus a key player in epidermal biology and a potential target for drug development. PMID:18548108

  5. Functional analysis of the aquaporin gene family in Caenorhabditis elegans.

    PubMed

    Huang, Chunyi George; Lamitina, Todd; Agre, Peter; Strange, Kevin

    2007-05-01

    Aquaporin channels facilitate the transport of water, glycerol, and other small solutes across cell membranes. The physiological roles of many aquaporins remain unclear. To better understand aquaporin function, we characterized the aquaporin gene family in the nematode Caenorhabditis elegans. Eight canonical aquaporin-encoding genes (aqp) are present in the worm genome. Expression of aqp-2, aqp-3, aqp-4, aqp-6, or aqp-7 in Xenopus oocytes increased water permeability five- to sevenfold. Glycerol permeability was increased three to sevenfold by expression of aqp-1, aqp-3, or aqp-7. Green fluorescent protein transcriptional and translational reporters demonstrated that aqp genes are expressed in numerous C. elegans cell types, including the intestine, excretory cell, and hypodermis, which play important roles in whole animal osmoregulation. To define the role of C. elegans aquaporins in osmotic homeostasis, we isolated deletion alleles for four aqp genes, aqp-2, aqp-3, aqp-4, and aqp-8, which are expressed in osmoregulatory tissues and mediate water transport. Single, double, triple, and quadruple aqp mutant animals exhibited normal survival, development, growth, fertility, and movement under normal and hypertonic culture conditions. aqp-2;aqp-3;aqp-4;aqp-8 quadruple mutants exhibited a slight defect in recovery from hypotonic stress but survived hypotonic stress as well as wild-type animals. These results suggest that C. elegans aquaporins are not essential for whole animal osmoregulation and/or that deletion of aquaporin genes activates mechanisms that compensate for loss of water channel function. PMID:17229810

  6. Characterization of α-Crystallin-Plasma Membrane Binding*

    PubMed Central

    Cobb, Brian A.; Petrash, J. Mark

    2010-01-01

    α-Crystallin, a large lenticular protein complex made up of two related subunits (αA- and αB-crystallin), is known to associate increasingly with fiber cell plasma membranes with age and/or the onset of cataract. To understand better the binding mechanism, we developed a sensitive membrane binding assay using lens plasma membranes and recombinant human αA- and αB-crystallins conjugated to a small fluorescent tag (Alexa350®). Both αA and αB homopolymer complexes, as well as a reconstituted 3:1 heteromeric complex, bind to lens membranes in a specific, saturable, and partially irreversible manner that is sensitive to both time and temperature. The amount of α-crystallin that binds to the membrane increases under acidic pH conditions and upon removal of exposed intrinsic membrane protein domains but is not affected at high ionic strength, suggesting that α-crystallin binds to the fiber cell plasma membranes mainly through hydrophobic interactions. The binding capacity and affinity for the reconstituted 3:1 heteromeric complex were measured to be 3.45 ± 0.11 ng/μg of membrane and 4.57 ± 0.50 × 10−4 μg−1 of membrane, respectively. The present membrane binding data support the hypothesis that the physical properties of a mixed α-crystallin complex may hold particular relevance for the function of α-crystallin within the lens. PMID:10692476

  7. 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.

  8. 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.

  9. 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

  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. Glycan Moieties as Bait to Fish Plasma Membrane Proteins.

    PubMed

    Fang, Fei; Zhao, Qun; Sui, Zhigang; Liang, Yu; Jiang, Hao; Yang, Kaiguang; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

    2016-05-17

    Plasma membrane proteome analysis is of significance for screening candidate biomarkers and drug targets. However, due to their low abundance and lack of specific groups that can enable their capture, the plasma membrane proteins (PMPs) are under-represented. On the basis of the fact that PMPs are embedded in or anchored to the phospholipid bilayer of the plasma membrane and the glycan moieties of proteins and lipids located on the plasma membrane are exposed outside of the cell surface, we proposed a strategy to capture PMPs, termed as glycan moieties-directed PMPs enrichment (GMDPE). With the glycan moieties exposed outside of the cells as bait to ensure the selectivity and the phospholipid bilayer as raft to provide the sensitivity, we applied this strategy into the plasma membrane proteome analysis of HeLa cells, and in total, 772 PMPs were identified, increased by 4.5 times compared to those identified by the reported cell surface biotinylation method. Notably, among them, 86 CD antigens and 16 ion channel proteins were confidently identified. All these results demonstrated that our proposed approach has great potential in the large scale plasma membrane proteome profiling. PMID:27088673

  12. 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. PMID:27339748

  13. Aquaporin-mediated long-distance polyphosphate translocation directed towards the host in arbuscular mycorrhizal symbiosis: application of virus-induced gene silencing.

    PubMed

    Kikuchi, Yusuke; Hijikata, Nowaki; Ohtomo, Ryo; Handa, Yoshihiro; Kawaguchi, Masayoshi; Saito, Katsuharu; Masuta, Chikara; Ezawa, Tatsuhiro

    2016-09-01

    Arbuscular mycorrhizal fungi translocate polyphosphate through hyphae over a long distance to deliver to the host. More than three decades ago, suppression of host transpiration was found to decelerate phosphate delivery of the fungal symbiont, leading us to hypothesize that transpiration provides a primary driving force for polyphosphate translocation, probably via creating hyphal water flow in which fungal aquaporin(s) may be involved. The impact of transpiration suppression on polyphosphate translocation through hyphae of Rhizophagus clarus was evaluated. An aquaporin gene expressed in intraradical mycelia was characterized and knocked down by virus-induced gene silencing to investigate the involvement of the gene in polyphosphate translocation. Rhizophagus clarus aquaporin 3 (RcAQP3) that was most highly expressed in intraradical mycelia encodes an aquaglyceroporin responsible for water transport across the plasma membrane. Knockdown of RcAQP3 as well as the suppression of host transpiration decelerated polyphosphate translocation in proportion to the levels of knockdown and suppression, respectively. These results provide the first insight into the mechanism underlying long-distance polyphosphate translocation in mycorrhizal associations at the molecular level, in which host transpiration and the fungal aquaporin play key roles. A hypothetical model of the translocation is proposed for further elucidation of the mechanism. PMID:27136716

  14. 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

  15. Homeostasis of plasma membrane viscosity in fluctuating temperatures.

    PubMed

    Martinière, Alexandre; Shvedunova, Maria; Thomson, Adrian J W; Evans, Nicola H; Penfield, Steven; Runions, John; McWatters, Harriet G

    2011-10-01

    Temperature has a direct effect at the cellular level on an organism. For instance, in the case of biomembranes, cooling causes lipids to lose entropy and pack closely together. Reducing temperature should, in the absence of other factors, increase the viscosity of a lipid membrane. We have investigated the effect of temperature variation on plasma membrane (PM) viscosity. We used dispersion tracking of photoactivated green fluorescent protein (GFP) and fluorescence recovery after photobleaching in wild-type and desaturase mutant Arabidopsis thaliana plants along with membrane lipid saturation analysis to monitor the effect of temperature and membrane lipid composition on PM viscosity. Plasma membrane viscosity in A. thaliana is negatively correlated with ambient temperature only under constant-temperature conditions. In the more natural environment of temperature cycles, plants actively manage PM viscosity to counteract the direct effects of temperature. Plasma membrane viscosity is regulated by altering the proportion of desaturated fatty acids. In cold conditions, cell membranes accumulate desaturated fatty acids, which decreases membrane viscosity and vice versa. Moreover, we show that control of fatty acid desaturase 2 (FAD2)-dependent lipid desaturation is essential for this homeostasis of membrane viscosity. Finally, a lack of FAD2 function results in aberrant temperature responses. PMID:21762166

  16. Limited and selective transfer of plasma membrane glycoproteins to membrane of secondary lysosomes

    SciTech Connect

    Haylett, T.; Thilo, L.

    1986-10-01

    Radioactive galactose, covalently bound to cell surface glycoconjugates on mouse macrophage cells, P388D/sub 1/, was used as a membrane marker to study the composition, and the kinetics of exchange, of plasma membrane-derived constituents in the membrane of secondary lysosomes. Secondary lysosomes were separated from endosomes and plasma membrane by self-forming Percoll density gradients. Horseradish peroxidase, taken up by fluid-phase pinocytosis, served as a vesicle contents marker to monitor transfer of endosomal contents into secondary lysosomes. Concurrently, the fraction of plasma membrane-derived label of secondary lysosomes increased by first order kinetics from <0.1% to a steady-state level of approx.2.5% of the total label. As analyzed by NaDodSO/sub 4/ PAGE, labeled molecules of M/sub r/ 160-190 kD were depleted and of the M/sub r/ 100-120 kD were enriched in lysosome membrane compared with the relative composition of label on the cell surface. No corresponding selectivity was observed for the degradation of label, with all M/sub r/ classes being affected to the same relative extent. The results indicate that endocytosis-derived transfer of plasma membrane constitutents to secondary lysosomes is a limited and selective process, and that only approx.1% of internalized membrane is recycled via a membrane pool of secondary lysosomes.

  17. Plasma jet accelerator optimization with supple membrane model

    NASA Astrophysics Data System (ADS)

    Galkin, S. A.; Bogatu, I. N.; Kim, J. S.

    2006-10-01

    High density (>=3x10^17cm-3) and high Mach number (M>10) plasma jets have important applications such as plasma rotation, refueling and disruption mitigation in tokamaks. The most deleterious blow-by instability occurs in coaxial plasma accelerators; hence electrode shape optimization is required to accelerate plasmas to ˜200 km/s [1]. A full 3D particle simulation takes a huge computational time. We have developed a membrane model to provide a good starting point and further physical insight for a full 3D optimization. Our model approximates the axisymmetrical plasma by a thin supple conducting membrane with a distributed mass, located between the electrodes, and connects them to model dynamics of the blow-by instability and to conduct the optimization. The supple membrane is allowed to slip along the conductors freely or with some friction as affected by Lorenz force, generated by magnetic field inside the chamber and current on membrane. The total mass and the density distribution represent the initial plasma. The density is redistributed adiabatically during the acceleration. An external electrical circuit with capacitance, inductance and resistivity is a part of the model. The membrane model simulation results will be compared to the 2D fluid MACH2 results and then will be used to guide a full 3D optimization by the LSP code. 1. http://hyperv.com/projects/pic/

  18. 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

  19. Nuclear Receptor Regulation of Aquaporin-2 in the Kidney.

    PubMed

    Zhang, Xiao-Yan; Wang, Bing; Guan, You-Fei

    2016-01-01

    Aquaporin-2 (AQP2) is a vasopressin-regulated water channel responsible for regulating water reabsorption through the apical plasma membrane of the principal cells of renal collecting ducts. It has been found that dysregulation and dysfunction of AQP2 cause many disorders related to water balance in people and animals, including polyuria and dilutional hyponatremia. Classically, AQP2 mRNA and protein expression and its membrane translocation are regulated by systemic vasopressin involving short-term regulation of AQP2 trafficking to and from the apical plasma membrane and long-term regulation of the total amount of the AQP2 protein in the cell. Recently, increasing evidence has demonstrated that collecting duct AQP2 expression and membrane translocation are also under the control of many other local factors, especially nuclear receptors. Here, we briefly review the progress of studies in this area and discuss the role of nuclear receptors in the regulation of water reabsorption via affecting AQP2 expression and function. PMID:27409611

  20. Nuclear Receptor Regulation of Aquaporin-2 in the Kidney

    PubMed Central

    Zhang, Xiao-Yan; Wang, Bing; Guan, You-Fei

    2016-01-01

    Aquaporin-2 (AQP2) is a vasopressin-regulated water channel responsible for regulating water reabsorption through the apical plasma membrane of the principal cells of renal collecting ducts. It has been found that dysregulation and dysfunction of AQP2 cause many disorders related to water balance in people and animals, including polyuria and dilutional hyponatremia. Classically, AQP2 mRNA and protein expression and its membrane translocation are regulated by systemic vasopressin involving short-term regulation of AQP2 trafficking to and from the apical plasma membrane and long-term regulation of the total amount of the AQP2 protein in the cell. Recently, increasing evidence has demonstrated that collecting duct AQP2 expression and membrane translocation are also under the control of many other local factors, especially nuclear receptors. Here, we briefly review the progress of studies in this area and discuss the role of nuclear receptors in the regulation of water reabsorption via affecting AQP2 expression and function. PMID:27409611

  1. Surface modification of nanoporous alumina membranes by plasma polymerization

    NASA Astrophysics Data System (ADS)

    Losic, Dusan; Cole, Martin A.; Dollmann, Björn; Vasilev, Krasimir; Griesser, Hans J.

    2008-06-01

    The deposition of plasma polymer coatings onto porous alumina (PA) membranes was investigated with the aim of adjusting the surface chemistry and the pore size of the membranes. PA membranes from commercial sources with a range of pore diameters (20, 100 and 200 nm) were used and modified by plasma polymerization using n-heptylamine (HA) monomer, which resulted in a chemically reactive polymer surface with amino groups. Heptylamine plasma polymer (HAPP) layers with a thickness less than the pore diameter do not span the pores but reduce their diameter. Accordingly, by adjusting the deposition time and thus the thickness of the plasma polymer coating, it is feasible to produce any desired pore diameter. The structural and chemical properties of modified membranes were studied by scanning electron microscopy (SEM), atomic force microscopy (AFM) and x-ray electron spectroscopy (XPS). The resultant PA membranes with specific surface chemistry and controlled pore size are applicable for molecular separation, cell culture, bioreactors, biosensing, drug delivery, and engineering complex composite membranes.

  2. Adipocyte cell size enlargement involves plasma membrane area increase.

    PubMed

    Chowdhury, H H; Zorec, R

    2012-07-01

    The adipocyte enlargement is associated with an increase in the cytoplasmic lipid content, but how the plasma membrane area follows this increase is poorly understood. We monitored single-cell membrane surface area fluctuations, which mirror the dynamics of exocytosis and endocytosis. We employed the patch-clamp technique to measure membrane capacitance (C(m)), a parameter linearly related to the plasma membrane area. Specifically, we studied whether insulin affects membrane area dynamics in adipocytes. A five-minute cell exposure to insulin increased resting C(m) by 12 ± 4%; in controls the change in C(m) was not different from zero. We measured cell diameter of isolated rat adipocytes microscopically. Twenty-four hour exposure of cells to insulin resulted in a significant increase in cell diameter by 5.1 ± 0.6%. We conclude that insulin induces membrane area increase, which may in chronic hyperinsulinemia promote the enlargement of plasma membrane area, acting in concert with other insulin-mediated metabolic effects on adipocytes. PMID:22540353

  3. Modification of polyethylene terephthalate track membrane properties by ammonia plasma

    NASA Astrophysics Data System (ADS)

    Kravets, Lyubov; Dmitriev, Serguei; Dinescu, George; Lazea, Andrada; Raiciu, Eric

    2004-09-01

    The properties of polyethylene terephthalate track membranes (PET TM) exposed to ammonia are investigated. The influence of the conditions of plasma treatment on the basic characteristics of the membranes, namely pore size and shape, wettability, water permeability, is studied. PET TM of the thickness of 10 μ m with the effective pore diameter of 0.215 μ m (pore density 2\\cdot 10^8 cm-2) were under study. The plasma treatment was performed on a plasma-chemical installation realizing a RF-discharge on the frequency 13.56 MHz. The process was conducted in a dynamic mode. Before delivering vapours of the plasma forming gas, the chamber was beforehand vacuumed down to residual pressure of 10-2 Torr. One side of the membranes was subjected to plasma. The discharge parameters (gas pressure in the vacuum chamber, discharge power) and the duration of plasma action were varied. It has been figured out that when treating the membranes in plasma of the explored gas there are two competing processes: etching of a polymeric matrix and deposition of a polymeric layer on their surface. It has been shown that at a short time of plasma action and low values of the discharge parameters, an etching process is mainly observed. Decrease in the thickness of the membranes and increase in the effective pore diameter testifies it. A result of the gas-discharge etching is also a hydrophilization of the TM surface stipulated by formation of polar function groups in the points of breaking chemical bonds. Here the value of the water contact angle of surface decreases down to 45-50 degrees in some cases. It has been shown that at a longer action of the plasma and increase of the discharge parameters, as accumulation in the chamber of etch products takes place, a process of deposition of a polymeric film becomes dominating, and it is proved by increasing the width of the membranes and changing their color. The value of the water contact angle of surface in this case is grown and, depending

  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. PMID:22088195

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. Expression and Prognostic Value of Aquaporin 1, 3 in Cervical Carcinoma in Women of Uygur Ethnicity from Xinjiang, China

    PubMed Central

    Chen, Rui; Shi, Yonghua; Amiduo, Reshalaity; Tuokan, Talaf; Suzuk, Lalai

    2014-01-01

    Background Overexpression of several aquaporins has been reported in different types of human cancer but the role of aquaporins in carcinogenesis has not yet been clearly defined. There is few report concerning role of aquaporins in human cervical carcinogenesis so far. Here, we determined the expression and prognostic value of aquaporin 1, 3 in cervical carcinoma in Chinese women of Uygur ethnicity. Methods and Results Real-time PCR analyses demonstrated aquaporin 1, 3 mRNA were differentially expressed in cervical carcinoma, CIN 2-3 and mild cervicitis. Immunofluorescent and immunohistochemical analyses demonstrated aquaporin 1 was predominantly localized to stromal endothelial cells in cervical lesions. Aquaporin 3 was localized to the membrane of normal squamous epithelium, CIN and carcinoma cells. Aquaporin 1 and 3 were upregulated in cervical cancer compared to mild cervicitis and CIN2-3 (P<0.05); Tumor expression of aquaporin 1, 3 significantly increased in advanced stage disease, and patients with deeper tumor infiltration, lymph node metastases or larger tumor volume (P<0.05). Multivariate analysis demonstrated that aquaporin 1, 3 were not independent prognostic factors in cervical carcinoma. Conclusion Aquaporins may participate in the initiation and progression of cervical carcinoma by promoting tumor growth, invasion or lymph node metastasis. Further study is required to determine whether aquaporins have potential as prognostic factors in cervical cancer. PMID:24918928

  10. Crystal structure of the plasma membrane proton pump.

    PubMed

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

    2007-12-13

    A prerequisite for life is the ability to maintain electrochemical imbalances across biomembranes. In all eukaryotes the plasma membrane potential and secondary transport systems are energized by the activity of P-type ATPase membrane proteins: H+-ATPase (the proton pump) in plants and fungi, and Na+,K+-ATPase (the sodium-potassium pump) in animals. The name P-type derives from the fact that these proteins exploit a phosphorylated reaction cycle intermediate of ATP hydrolysis. The plasma membrane proton pumps belong to the type III P-type ATPase subfamily, whereas Na+,K+-ATPase and Ca2+-ATPase are type II. Electron microscopy has revealed the overall shape of proton pumps, however, an atomic structure has been lacking. Here we present the first structure of a P-type proton pump determined by X-ray crystallography. Ten transmembrane helices and three cytoplasmic domains define the functional unit of ATP-coupled proton transport across the plasma membrane, and the structure is locked in a functional state not previously observed in P-type ATPases. The transmembrane domain reveals a large cavity, which is likely to be filled with water, located near the middle of the membrane plane where it is lined by conserved hydrophilic and charged residues. Proton transport against a high membrane potential is readily explained by this structural arrangement. PMID:18075595

  11. Aquaporins in Sensory and Pain Transmission

    PubMed Central

    Borsani, Elisa

    2010-01-01

    Recent data suggest a possible involvement of Aquaporins (AQPs) in pain transmission. AQPs are small membrane channel proteins involved in osmoregulation and, to date, AQP1, AQP2, AQP3, AQP4, AQP5, AQP8 and AQP9 have been found in the nervous system. Nevertheless only AQP1, AQP2 and AQP4 seem to be involved in nociception. In this review, direct and indirect evidences of the role of AQPs in pain processing will be reported. PMID:21119883

  12. Sub-genomic level sequence analysis of the aquaporin multi-gene family in cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aquaporins function mainly as water transport channel proteins that facilitate water movement across intracellular and intercellular membranes in most living organisms. Plant aquaporins belong to a multi-gene family and are commonly categorized into 5 subfamilies according to sequence similarity. Re...

  13. 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.

  14. 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.

  15. Functional Implications of Plasma Membrane Condensation for T Cell Activation

    PubMed Central

    Quinn, Carmel M.; Engelhardt, Karin; Williamson, David; Grewal, Thomas; Jessup, Wendy; Harder, Thomas; Gaus, Katharina

    2008-01-01

    The T lymphocyte plasma membrane condenses at the site of activation but the functional significance of this receptor-mediated membrane reorganization is not yet known. Here we demonstrate that membrane condensation at the T cell activation sites can be inhibited by incorporation of the oxysterol 7-ketocholesterol (7KC), which is known to prevent the formation of raft-like liquid-ordered domains in model membranes. We enriched T cells with 7KC, or cholesterol as control, to assess the importance of membrane condensation for T cell activation. Upon 7KC treatment, T cell antigen receptor (TCR) triggered calcium fluxes and early tyrosine phosphorylation events appear unaltered. However, signaling complexes form less efficiently on the cell surface, fewer phosphorylated signaling proteins are retained in the plasma membrane and actin restructuring at activation sites is impaired in 7KC-enriched cells resulting in compromised downstream activation responses. Our data emphasizes lipids as an important medium for the organization at T cell activation sites and strongly indicates that membrane condensation is an important element of the T cell activation process. PMID:18509459

  16. 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. PMID:27018128

  17. An Endosome-to-Plasma Membrane Pathway Involved in Trafficking of a Mutant Plasma Membrane ATPase in Yeast

    PubMed Central

    Luo, Wen-jie; Chang, Amy

    2000-01-01

    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°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. PMID:10679016

  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. 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

  20. Exclusive photorelease of signalling lipids at the plasma membrane.

    PubMed

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

    2015-01-01

    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

  1. On the puzzling distribution of cholesterol in the plasma membrane.

    PubMed

    Giang, H; Schick, M

    2016-09-01

    The distribution of cholesterol between the two leaves of the plasma membrane in mammalian cells presents a conundrum; given cholesterol's known affinity for sphingomyelin, which resides predominantly in the exoplasmic leaf, why is it that experiment finds a majority of the cholesterol in the cytoplasmic leaf? This article reviews a recently proposed solution to this puzzle. PMID:26724709

  2. Granuphilin exclusively mediates functional granule docking to the plasma membrane

    PubMed Central

    Mizuno, Kouichi; Fujita, Takuji; Gomi, Hiroshi; Izumi, Tetsuro

    2016-01-01

    In regulated exocytosis, it is generally assumed that vesicles must stably “dock” at the plasma membrane before they are primed to become fusion-competent. However, recent biophysical analyses in living cells that visualize fluorescent secretory granules have revealed that exocytic behaviors are not necessarily uniform: some granules beneath the plasma membrane are resistant to Ca2+ -triggered release, while others are accelerated to fuse without a pause for stable docking. These findings suggest that stable docking is unnecessary, and can even be inhibitory or nonfunctional, for fusion. Consistently, pancreatic β cells deficient in the Rab27 effector, granuphilin, lack insulin granules directly attached to the plasma membrane in electron micrographs but nevertheless exhibit augmented exocytosis. Here we directly compare the exocytic behaviors between granuphilin-positive and -negative insulin granules. Although granuphilin makes granules immobile and fusion-reluctant beneath the plasma membrane, those granuphilin-positive, docked granules release a portion of granuphilin upon fusion, and fuse at a frequency and time course similar to those of granuphilin-negative undocked granules. Furthermore, granuphilin forms a 180-nm cluster at the site of each docked granule, along with granuphilin-interacting Rab27a and Munc18-1 clusters. These findings indicate that granuphilin is an exclusive component of the functional and fusion-inhibitory docking machinery of secretory granules. PMID:27032672

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  4. A TRiP to the plasma membrane

    PubMed Central

    Ghosh, Debapriya; Voets, Thomas

    2015-01-01

    TRP ion channels are ubiquitously present in the mammalian body and take part in numerous key physiological functions, including temperature sensing, taste perception, osmo-regulation, cardiac function, renal function, development, and glucose homeostasis. The mechanisms whereby TRP channels are transported to the plasma membrane, where most of them exert their physiological actions, remains a poorly understood aspect of TRP channel biology.

  5. 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. PMID:24251774

  6. Yeast Ist2 Recruits the Endoplasmic Reticulum to the Plasma Membrane and Creates a Ribosome-Free Membrane Microcompartment

    PubMed Central

    Lorenz, Holger; Schwappach, Blanche; Seedorf, Matthias

    2012-01-01

    The endoplasmic reticulum (ER) forms contacts with the plasma membrane. These contacts are known to function in non-vesicular lipid transport and signaling. Ist2 resides in specific domains of the ER in Saccharomyces cerevisiae where it binds phosphoinositide lipids at the cytosolic face of the plasma membrane. Here, we report that Ist2 recruits domains of the yeast ER to the plasma membrane. Ist2 determines the amount of cortical ER present and the distance between the ER and the plasma membrane. Deletion of IST2 resulted in an increased distance between ER and plasma membrane and allowed access of ribosomes to the space between the two membranes. Cells that overexpress Ist2 showed an association of the nucleus with the plasma membrane. The morphology of the ER and yeast growth were sensitive to the abundance of Ist2. Moreover, Ist2-dependent effects on cytosolic pH and genetic interactions link Ist2 to the activity of the H+ pump Pma1 in the plasma membrane during cellular adaptation to the growth phase of the culture. Consistently we found a partial colocalization of Ist2-containing cortical ER and Pma1-containing domains of the plasma membrane. Hence Ist2 may be critically positioned in domains that couple functions of the ER and the plasma membrane. PMID:22808051

  7. Control of Plasma Membrane Permeability by ABC Transporters.

    PubMed

    Khakhina, Svetlana; Johnson, Soraya S; Manoharlal, Raman; Russo, Sarah B; Blugeon, Corinne; Lemoine, Sophie; Sunshine, Anna B; Dunham, Maitreya J; Cowart, L Ashley; Devaux, Frédéric; Moye-Rowley, W Scott

    2015-05-01

    ATP-binding cassette transporters Pdr5 and Yor1 from Saccharomyces cerevisiae control the asymmetric distribution of phospholipids across the plasma membrane as well as serving as ATP-dependent drug efflux pumps. Mutant strains lacking these transporter proteins were found to exhibit very different resistance phenotypes to two inhibitors of sphingolipid biosynthesis that act either late (aureobasidin A [AbA]) or early (myriocin [Myr]) in the pathway leading to production of these important plasma membrane lipids. These pdr5Δ yor1 strains were highly AbA resistant but extremely sensitive to Myr. We provide evidence that these phenotypic changes are likely due to modulation of the plasma membrane flippase complexes, Dnf1/Lem3 and Dnf2/Lem3. Flippases act to move phospholipids from the outer to the inner leaflet of the plasma membrane. Genetic analyses indicate that lem3Δ mutant strains are highly AbA sensitive and Myr resistant. These phenotypes are fully epistatic to those seen in pdr5Δ yor1 strains. Direct analysis of AbA-induced signaling demonstrated that loss of Pdr5 and Yor1 inhibited the AbA-triggered phosphorylation of the AGC kinase Ypk1 and its substrate Orm1. Microarray experiments found that a pdr5Δ yor1 strain induced a Pdr1-dependent induction of the entire Pdr regulon. Our data support the view that Pdr5/Yor1 negatively regulate flippase function and activity of the nuclear Pdr1 transcription factor. Together, these data argue that the interaction of the ABC transporters Pdr5 and Yor1 with the Lem3-dependent flippases regulates permeability of AbA via control of plasma membrane protein function as seen for the high-affinity tryptophan permease Tat2. PMID:25724885

  8. [Aquaporins--a new element in the regulation of body water homeostasis].

    PubMed

    Ciechanowicz, Andrzej; Krzyształowska, Maja; Bińcjczak-Kuleta, Agnieszka

    2009-08-01

    Aquaporins represent an ubiquitous class of integral membrane proteins that are serving in the passage of water across cell membrane. A subset of aquaporins may additionally facilitate transmembrane permeation of small neutral solutes such as glicerol (aquaglyceroporins). The widespread occurrence and an unique function of aquaporins give rise to the high degree of their intra- and interspecies homology, especially in their regions forming the internal wall of channel. The review presents current knowledge about the role of water channels in regulation of body homeostasis basing on results of experiments with mice lacking various aquaporins genes and studies on humans with inherited or acquired abnormalities in their function as well as about potential perspectives of pharmacological regulation of aquaporin activity. PMID:19856883

  9. Effect of clofibrate on the enzyme activity of rat liver plasma membranes.

    PubMed

    Renaud, G; Foliot, A; Marais, J; Infante, R

    1980-03-15

    The activity of 3 plasma membranes marker enzymes (5'-nucleotidase, Mg++-ATPase and alkaline phosphodiesterase-I) was determined in plasma membranes isolated from liver of control and of clofibrate-treated rats. A complete indentity of plasma membranes enzyme activity in the 2 groups of experimental animals was observed for the 3 enzymes studied. PMID:6102923

  10. Imaging plasma membrane deformations with pTIRFM.

    PubMed

    Passmore, Daniel R; Rao, Tejeshwar C; Peleman, Andrew R; Anantharam, Arun

    2014-01-01

    To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress. Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or diD. DiD intercalates in the membrane plane with a "fixed" orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561 nm laser (p-pol, s-pol). A 488 nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation. PMID:24747638

  11. Proteome Analysis of the Plasma Membrane of Mycobacterium Tuberculosis

    PubMed Central

    Arora, Shalini; Kosalai, K.; Namane, Abdelkader; Pym, Alex S.; Cole, Stewart T.

    2002-01-01

    The plasma membrane of Mycobacterium tuberculosis is likely to contain proteins that could serve as novel drug targets, diagnostic probes or even components of a vaccine against tuberculosis. With this in mind, we have undertaken proteome analysis of the membrane of M. tuberculosis H37Rv. Isolated membrane vesicles were extracted with either a detergent (Triton X114) or an alkaline buffer (carbonate) following two of the protocols recommended for membrane protein enrichment. Proteins were resolved by 2D-GE using immobilized pH gradient (IPG) strips, and identified by peptide mass mapping utilizing the M. tuberculosis genome database. The two extraction procedures yielded patterns with minimal overlap. Only two proteins, both HSPs, showed a common presence. MALDI–MS analysis of 61 spots led to the identification of 32 proteins, 17 of which were new to the M. tuberculosis proteome database. We classified 19 of the identified proteins as ‘membrane-associated’; 14 of these were further classified as ‘membrane-bound’, three of which were lipoproteins. The remaining proteins included four heat-shock proteins and several enzymes involved in energy or lipid metabolism. Extraction with Triton X114 was found to be more effective than carbonate for detecting ‘putative’ M. tuberculosis membrane proteins. The protocol was also found to be suitable for comparing BCG and M. tuberculosis membranes, identifying ESAT-6 as being expressed selectively in M. tuberculosis. While this study demonstrates for the first time some of the membrane proteins of M. tuberculosis, it also underscores the problems associated with proteomic analysis of a complex membrane such as that of a mycobacterium. PMID:18629250

  12. Fractionation of liver plasma membranes prepared by zonal centrifugation

    PubMed Central

    Evans, W. H.

    1970-01-01

    1. Plasma membranes were isolated from crude nuclear sediments from mouse and rat liver by a rate-dependent centrifugation through a sucrose density gradient contained in the `A' type zonal rotor. 2. The membranes were further purified by isopycnic centrifugation, and characterized enzymically, chemically and morphologically. 3. When the plasma-membrane fraction of sucrose density 1.17g/cm3 was dispersed in a tight-fitting homogenizer, two subfractions of densities 1.12 and 1.18 were obtained by isopycnic centrifugation. 4. The light subfraction contained 5′-nucleotidase, nucleoside diphosphatase, leucine naphthylamidase and Mg2+-stimulated adenosine triphosphatase activities at higher specific activities than unfractionated membranes. The heavy subfraction was deficient in the above enzymes but contained higher Na++K+-stimulated adenosine triphosphatase activity. 5. The light subfraction contained twice as much phospholipid and cholesterol, and three times as much N-acetylneuraminic acid relative to unit protein weight as the heavy subfraction. Polyacrylamide-gel electrophoresis indicated differences in protein composition. 6. Electron microscopy showed the light subfraction to be vesicular. The heavy subfraction contained membrane strips with junctional complexes in addition to vesicles. ImagesPLATE 2PLATE 3PLATE 1 PMID:4315049

  13. Isolation of plasma membranes from cultured glioma cells and application to evaluation of membrane sphingomyelin turnover

    SciTech Connect

    Cook, H.W.; Palmer, F.B.; Byers, D.M.; Spence, M.W.

    1988-11-01

    A rapid and reliable method for the isolation of plasma membranes and microsomes of high purity and yield from cultured glioma cells is described. The procedure involves disruption by N2 cavitation, preliminary separation by centrifugation in Tricine buffer, and final separation on a gradient formed from 40% Percoll at pH 9.3. Enzyme and chemical markers indicated greater than 60% yield with six- to eightfold enrichment for plasma membranes and greater than 25% yield with three- to fourfold enrichment for a microsomal fraction consisting mainly of endoplasmic reticulum. The final fractions were obtained with high reproducibility in less than 1 h from the time of cell harvesting. Application of this procedure to human fibroblasts in culture is assessed. The isolation procedure was applied to investigations of synthesis and turnover of sphingomyelin and phosphatidylcholine in plasma membranes of glioma cells following incubation for 4-24 h with (methyl-/sup 3/H)choline. These studies indicated that radioactivity from phosphatidylcholine synthesized in microsomes from exogenous choline may serve as a precursor of the head-group of sphingomyelin accumulating in the plasma membrane.

  14. An aquaporin protein is associated with drought stress tolerance.

    PubMed

    Li, Jun; Ban, Liping; Wen, Hongyu; Wang, Zan; Dzyubenko, Nikolay; Chapurin, Vladimir; Gao, Hongwen; Wang, Xuemin

    2015-04-01

    Water channel proteins known as aquaporins (AQPs) regulate the movement of water and other small molecules across plant vacuolar and plasma membranes; they are associated with plant tolerance of biotic and abiotic stresses. In this study, a PIP type AQPs gene, designated as GoPIP1, was cloned from Galega orientalis, a high value leguminous forage crop. The GoPIP1 gene consists of an 870 bp open reading frame encoding a protein of 289 amino acids, and belongs to the PIP1 subgroup of the PIP subfamily. The transcript level of GoPIP1 was higher in the root of G. orientalis than in the leaf and stem. The level of GoPIP1 transcript increased significantly when treated with 200 mM NaCl or 20% polyethylene glycol (PEG) 6000. Transient expression of GoPIP1 in onion epidermal cells revealed that the GoPIP1 protein was localized to the plasma membrane. Over-expression of GoPIP1 increased the rosette/root ratio and increased sensitivity to drought in transgenic Arabidopsis plants. However, GoPIP1 over-expression in Arabidopsis had no significant effect under saline condition. The present data provides a gene resource that contributes to furthering our understanding of water channel protein and their application in plant stress tolerance. PMID:25701792

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  16. The three-dimensional structure of aquaporin-1

    NASA Astrophysics Data System (ADS)

    Walz, Thomas; Hirai, Teruhisa; Murata, Kazuyoshi; Heymann, J. Bernard; Mitsuoka, Kaoru; Fujiyoshi, Yoshinori; Smith, Barbara L.; Agre, Peter; Engel, Andreas

    1997-06-01

    The entry and exit of water from cells is a fundamental process of life. Recognition of the high water permeability of red blood cells led to the proposal that specialized water pores exist in the plasma membrane. Expression in Xenopus oocytes and functional studies of an erythrocyte integral membrane protein of relative molecular mass 28,000, identified it as the mercury-sensitive water channel, aquaporin-1 (AQP1). Many related proteins, all belonging to the major intrinsic protein (MIP) family, are found throughout nature. AQP1 is a homotetramer containing four independent aqueous channels. When reconstituted into lipid bilayers, the protein forms two-dimensional lattices with a unit cell containing two tetramers in opposite orientation. Here we present the three-dimensional structure of AQP1 determined at 6Å resolution by cryo-electron microscopy. Each AQP1 monomer has six tilted, bilayer-spanning α-helices which form a right-handed bundle surrounding a central density. These results, together with functional studies, provide a model that identifies the aqueous pore in the AQP1 molecule and indicates the organization of the tetrameric complex in the membrane.

  17. The three-dimensional structure of aquaporin-1.

    PubMed

    Walz, T; Hirai, T; Murata, K; Heymann, J B; Mitsuoka, K; Fujiyoshi, Y; Smith, B L; Agre, P; Engel, A

    1997-06-01

    The entry and exit of water from cells is a fundamental process of life. Recognition of the high water permeability of red blood cells led to the proposal that specialized water pores exist in the plasma membrane. Expression in Xenopus oocytes and functional studies of an erythrocyte integral membrane protein of relative molecular mass 28,000, identified it as the mercury-sensitive water channel, aquaporin-1 (AQP1). Many related proteins, all belonging to the major intrinsic protein (MIP) family, are found throughout nature. AQP1 is a homotetramer containing four independent aqueous channels. When reconstituted into lipid bilayers, the protein forms two-dimensional lattices with a unit cell containing two tetramers in opposite orientation. Here we present the three-dimensional structure of AQP1 determined at 6A resolution by cryo-electron microscopy. Each AQP1 monomer has six tilted, bilayer-spanning alpha-helices which form a right-handed bundle surrounding a central density. These results, together with functional studies, provide a model that identifies the aqueous pore in the AQP1 molecule and indicates the organization of the tetrameric complex in the membrane. PMID:9177353

  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. In Situ Quantification of Protein Binding to the Plasma Membrane

    PubMed Central

    Smith, Elizabeth M.; Hennen, Jared; Chen, Yan; Mueller, Joachim D.

    2015-01-01

    This study presents a fluorescence-based assay that allows for direct measurement of protein binding to the plasma membrane inside living cells. An axial scan through the cell generates a fluorescence intensity profile that is analyzed to determine the membrane-bound and cytoplasmic concentrations of a peripheral membrane protein labeled by the enhanced green fluorescent protein (EGFP). The membrane binding curve is constructed by mapping those concentrations for a population of cells with a wide range of protein expression levels, and a fit of the binding curve determines the number of binding sites and the dissociation coefficient. We experimentally verified the technique, using myosin-1C-EGFP as a model system and fit its binding curve. Furthermore, we studied the protein-lipid interactions of the membrane binding domains from lactadherin and phospholipase C-δ1 to evaluate the feasibility of using competition binding experiments to identify specific lipid-protein interactions in living cells. Finally, we applied the technique to determine the lipid specificity, the number of binding sites, and the dissociation coefficient of membrane binding for the Gag matrix domain of human T-lymphotropic virus type 1, which provides insight into early assembly steps of the retrovirus. PMID:26039166

  20. b-Type Cytochromes in Higher Plant Plasma Membranes 1

    PubMed Central

    Asard, Han; Venken, Mireille; Caubergs, Roland; Reijnders, Willem; Oltmann, Fred L.; De Greef, Jan A.

    1989-01-01

    The composition and characteristics of b-type cytochromes from higher plant plasma membranes, purified using aqueous two-phase partitioning, were investigated. At least three different cytochromes were identified by their wavelength maxima and redox midpoint potentials (E0′). Cytochrome b-560.7 (E0′ from + 110 to + 160 millivolts) was present in zucchini (Cucurbita pepo) hypocotyls and bean (Phaseolus vulgaris L.) hooks, although in different concentrations. The main component in cauliflower (Brassica oleracea L.) inflorescences (cytochrome b-558.8) is probably functionally similar to this cytochrome. The plasma membrane generally contains two to three cytochrome species. However, the occurrence and concentrations were species dependent. The high potential cytochrome can be reduced by ascorbate but not NADH, and may be involved in blue light perception. PMID:16666854

  1. Neobiosynthesis of glycosphingolipids by plasma membrane-associated glycosyltransferases.

    PubMed

    Crespo, Pilar M; Demichelis, Vanina Torres; Daniotti, José L

    2010-09-17

    Gangliosides, complex glycosphingolipids containing sialic acids, are synthesized in the endoplasmic reticulum and in the Golgi complex. These neobiosynthesized gangliosides move via vesicular transport to the plasma membrane, becoming components of the external leaflet. Gangliosides can undergo endocytosis followed by recycling to the cell surface or sorting to the Golgi complex or lysosomes for remodeling and catabolism. Recently, glycosphingolipid catabolic enzymes (glycohydrolases) have been found to be associated with the plasma membrane, where they display activity on the membrane components. In this work, we demonstrated that ecto-ganglioside glycosyltransferases may catalyze ganglioside synthesis outside the Golgi compartment, particularly at the cell surface. Specifically, we report the first direct evidence of expression and activity of CMP-NeuAc:GM3 sialyltransferase (Sial-T2) at the cell surface of epithelial and melanoma cells, with membrane-integrated ecto-Sial-T2 being able to sialylate endogenously synthesized GM3 ganglioside as well as exogenously incorporated substrate. Interestingly, we also showed that ecto-Sial-T2 was able to synthesize GD3 ganglioside at the cell surface using the endogenously synthesized cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) available at the extracellular milieu. In addition, the expression of UDP-GalNAc:LacCer/GM3/GD3 N-acetylgalactosaminyltransferase (GalNAc-T) was also detected at the cell surface of epithelial cells, whose catalytic activity was only observed after feeding the cells with exogenous GM3 substrate. Thus, the relative interplay between the plasma membrane-associated glycosyltransferase and glycohydrolase activities, even when acting on a common substrate, emerges as a potential level of regulation of the local glycosphingolipid composition in response to different external and internal stimuli. PMID:20639193

  2. 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. PMID:20099798

  3. Detecting protein association at the T cell plasma membrane.

    PubMed

    Baumgart, Florian; Schütz, Gerhard J

    2015-04-01

    At the moment, many models on T cell signaling rely on results obtained via rather indirect methodologies, which makes direct comparison and conclusions to the in vivo situation difficult. Recently, a variety of new imaging methods were developed, which have the potential to directly shed light onto the mysteries of protein association at the T cell membrane. While the new modalities are extremely promising, for a broad readership it may be difficult to judge the results, since technological shortcomings are not always obvious. In this review article, we put key questions on the mechanism of protein interactions in the T cell plasma membrane into relation with techniques that allow to address such questions. We discuss applicability of the techniques, their strengths and weaknesses. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling. PMID:25300585

  4. 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

  5. Transformable DNA nanocarriers for plasma membrane targeted delivery of cytokine.

    PubMed

    Sun, Wujin; Ji, Wenyan; Hu, Quanyin; Yu, Jicheng; Wang, Chao; Qian, Chenggen; Hochu, Gabrielle; Gu, Zhen

    2016-07-01

    Direct delivery of cytokines using nanocarriers holds great promise for cancer therapy. However, the nanometric scale of the vehicles made them susceptible to size-dependent endocytosis, reducing the plasma membrane-associated apoptosis signaling. Herein, we report a tumor microenvironment-responsive and transformable nanocarrier for cell membrane targeted delivery of cytokine. This formulation is comprised of a phospholipase A2 (PLA2) degradable liposome as a shell, and complementary DNA nanostructures (designated as nanoclews) decorated with cytokines as the cores. Utilizing the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as a model cytokine, we demonstrate that the TRAIL loaded DNA nanoclews are capable of transforming into nanofibers after PLA2 activation. The nanofibers with micro-scaled lengths efficiently present the loaded TRAIL to death receptors on the cancer cell membrane and amplified the apoptotic signaling with reduced TRAIL internalization. PMID:27131597

  6. 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

  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. Aquaporin-2 Regulation in Health and Disease

    PubMed Central

    Radin, M. Judith; Yu, Ming-Jiun; Stoedkilde, Lene; Miller, R. Lance; Hoffert, Jason D.; Frokiaer, Jorgen; Pisitkun, Trairak; Knepper, Mark A.

    2012-01-01

    Aquaporin-2 (AQP2), the vasopressin-regulated water channel of the renal collecting duct, is dysregulated in numerous water balance disorders in humans and animals including those associated with polyuria (e.g. urinary tract obstruction, hypokalemia, inflammation, and lithium toxicity) and dilutional hyponatremia (e.g. SIADH). Normal regulation of AQP2 by vasopressin involves two independent regulatory mechanisms: 1) short-term regulation of AQP2 trafficking to and from the apical plasma membrane, and 2) long term regulation of the total abundance of the AQP2 protein in the cells. Most water balance disorders are the result of dysregulation of processes that regulate the total abundance of AQP2 in collecting duct cells. In general, the level of AQP2 in a collecting duct cell is determined by a balance between production via translation of AQP2 mRNA and removal via degradation and/or secretion into the urine in exosomes. AQP2 abundance increases in response to vasopressin chiefly due to increased translation subsequent to increases in AQP2 mRNA. Vasopressin-mediated regulation AQP2 gene transcription is poorly understood, although several transcription factor binding elements in the 5’ flanking region of the AQP2 gene have been identified and candidate transcription factors corresponding to these element have been discovered in proteomics studies. Here we review progress in this area and discuss elements of vasopressin signaling in the collecting duct that may impinge on regulation of AQP2 in health and in the context of examples of polyuric diseases. PMID:23130944

  9. A brief survey of aquaporins and their implications for renal physiology.

    PubMed

    Gade, Wayne; Robinson, Brooke

    2006-01-01

    Aquaporins (AQPs) are an important family of proteins that efficiently channel water through the cell membranes. Although water can diffuse across biological membranes at measurable rates, physiologists had long predicted the existence of channels to facilitate rapid reabsorption of water by renal tubular cells. With AQPs present, water can "gush" through the membrane at the extraordinary rate of three billion water molecules per second per aquaporin channel. In their absence, water only trickles across the hydrophobic lipid bilayers of cell membranes. Aquaporins have fascinated researchers over the last decade, culminating in the 2003 Nobel Prize for Chemistry given to their discoverer, Dr. Peter Agre. During the 1990s, scientists identified and characterized members of the mammalian aquaporin family, now designated as AQP0 through AQP10. AQPs are also found in many plant and bacterial species. However, their relevance to the clinical laboratory is only recently emerging. Dr. Agre's Nobel symposium address provides an excellent mini-review of aquaporins in medicine. 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 AQP2 system controlled by anti-diuretic hormone (ADH). Virtually all mammalian cells incorporate aquaporins into their cell membranes, and many cells produce multiple aquaporins, each with a specific function. It is therefore not surprising that malfunctions have important clinical conditions. The present article discusses the implications of aquaporins for renal physiology, while the accompanying article is focused on the clinical aspects of aquaporins. PMID:16749243

  10. 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. PMID:25461277

  11. Astrocyte Aquaporin Dynamics in Health and Disease

    PubMed Central

    Potokar, Maja; Jorgačevski, Jernej; Zorec, Robert

    2016-01-01

    The family of aquaporins (AQPs), membrane water channels, consists of diverse types of proteins that are mainly permeable to water; some are also permeable to small solutes, such as glycerol and urea. They have been identified in a wide range of organisms, from microbes to vertebrates and plants, and are expressed in various tissues. Here, we focus on AQP types and their isoforms in astrocytes, a major glial cell type in the central nervous system (CNS). Astrocytes have anatomical contact with the microvasculature, pia, and neurons. Of the many roles that astrocytes have in the CNS, they are key in maintaining water homeostasis. The processes involved in this regulation have been investigated intensively, in particular regulation of the permeability and expression patterns of different AQP types in astrocytes. Three aquaporin types have been described in astrocytes: aquaporins AQP1 and AQP4 and aquaglyceroporin AQP9. The aim here is to review their isoforms, subcellular localization, permeability regulation, and expression patterns in the CNS. In the human CNS, AQP4 is expressed in normal physiological and pathological conditions, but astrocytic expression of AQP1 and AQP9 is mainly associated with a pathological state. PMID:27420057

  12. Astrocyte Aquaporin Dynamics in Health and Disease.

    PubMed

    Potokar, Maja; Jorgačevski, Jernej; Zorec, Robert

    2016-01-01

    The family of aquaporins (AQPs), membrane water channels, consists of diverse types of proteins that are mainly permeable to water; some are also permeable to small solutes, such as glycerol and urea. They have been identified in a wide range of organisms, from microbes to vertebrates and plants, and are expressed in various tissues. Here, we focus on AQP types and their isoforms in astrocytes, a major glial cell type in the central nervous system (CNS). Astrocytes have anatomical contact with the microvasculature, pia, and neurons. Of the many roles that astrocytes have in the CNS, they are key in maintaining water homeostasis. The processes involved in this regulation have been investigated intensively, in particular regulation of the permeability and expression patterns of different AQP types in astrocytes. Three aquaporin types have been described in astrocytes: aquaporins AQP1 and AQP4 and aquaglyceroporin AQP9. The aim here is to review their isoforms, subcellular localization, permeability regulation, and expression patterns in the CNS. In the human CNS, AQP4 is expressed in normal physiological and pathological conditions, but astrocytic expression of AQP1 and AQP9 is mainly associated with a pathological state. PMID:27420057

  13. Experimental Evaluation of Proposed Small-Molecule Inhibitors of Water Channel Aquaporin-1.

    PubMed

    Esteva-Font, Cristina; Jin, Byung-Ju; Lee, Sujin; Phuan, Puay-Wah; Anderson, Marc O; Verkman, A S

    2016-06-01

    The aquaporin-1 (AQP1) water channel is a potentially important drug target, as AQP1 inhibition is predicted to have therapeutic action in edema, tumor growth, glaucoma, and other conditions. Here, we measured the AQP1 inhibition efficacy of 12 putative small-molecule AQP1 inhibitors reported in six recent studies, and one AQP1 activator. Osmotic water permeability was measured by stopped-flow light scattering in human and rat erythrocytes that natively express AQP1, in hemoglobin-free membrane vesicles from rat and human erythrocytes, and in plasma membrane vesicles isolated from AQP1-transfected Chinese hamster ovary cell cultures. As a positive control, 0.3 mM HgCl2 inhibited AQP1 water permeability by >95%. We found that none of the tested compounds at 50 µM significantly inhibited or increased AQP1 water permeability in these assays. Identification of AQP1 inhibitors remains an important priority. PMID:26993802

  14. Dysferlinopathy Fibroblasts Are Defective in Plasma Membrane Repair

    PubMed Central

    Matsuda, Chie; Kiyosue, Kazuyuki; Nishino, Ichizo; Goto, Yuichi; Hayashi, Yukiko K.

    2015-01-01

    Background: Dysferlin is a sarcolemmal protein that is defective in Miyoshi myopathy and limb-girdle muscular dystrophy type 2B, and is involved in sarcolemmal repair. Primary cultured myoblasts and myotubes established from patient muscle biopsies have been widely utilized to explore the molecular mechanism of dysferlinopathy. Objectives: The purpose of this study was to explore the possible utility of dermal fibroblasts from dysferlin-deficient patients and SJL mice as a tool for studying dysferlinopathy. Methods: Dysferlin protein expression in fibroblasts from dysferlin-deficient patients and SJL mice was analyzed by immunoblotting and immunocytochemistry. The membrane wound-repair assay was performed on the fibroblasts using a confocal microscope equipped with a UV-laser. The membrane blebbing assay using hypotonic shock, in which normal membrane blebbing is detected only in the presence of dysferlin, was also performed using human and mouse fibroblasts. Results: Mis-sense mutated dysferlin was expressed at a very low level in fibroblasts from a dysferlinopathy patient, and lower expression level of truncated dysferlin was observed in SJL mouse fibroblast. Fibroblasts from patients with dysferlinopathy and SJL mice showed attenuated membrane repair and did not form membrane blebs in response to hypoosmotic shock. Proteosomal inhibitior increased mis-sense mutated or truncated dysferlin levels, and restored membrane blebbing, however, proteosomal inhibition failed to improve levels of dysferlin with non-sense or frame-shift mutation. Conclusion: Fibroblasts from dysferlinopathy patients and SJL mice showed attenuated plasma membrane repair, and could be a tool for studying dysferlinopathy. PMID:26579332

  15. 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. PMID:27016447

  16. Structural context shapes the aquaporin selectivity filter

    PubMed Central

    Savage, David F.; O’Connell, Joseph D.; Miercke, Larry J. W.; Finer-Moore, Janet; Stroud, Robert M.

    2010-01-01

    Aquaporins are transmembrane channels that facilitate the permeation of water and small, uncharged amphipathic molecules across cellular membranes. One distinct aquaporin subfamily contains pure water channels, whereas a second subfamily contains channels that conduct small alditols such as glycerol, in addition to water. Distinction between these substrates is central to aquaporin function, though the contributions of protein structural motifs required for selectivity are not yet fully characterized. To address this question, we sequentially engineered three signature amino acids of the glycerol-conducting subfamily into the Escherichia coli water channel aquaporin Z (AqpZ). Functional analysis of these mutant channels showed a decrease in water permeability but not the expected increase in glycerol conduction. Using X-ray crystallography, we determined the atomic resolution structures of the mutant channels. The structures revealed a channel surprisingly similar in size to the wild-type AqpZ pore. Comparison with measured rates of transport showed that, as the size of the selectivity filter region of the channel approaches that of water, channel hydrophilicity dominated water conduction energetics. In contrast, the major determinant of selectivity for larger amphipathic molecules such as glycerol was channel cross-section size. Finally, we find that, although the selectivity filter region is indeed central to substrate transport, other structural elements that do not directly interact with the substrates, such as the loop connecting helices M6 and M7, and the C loop between helices C4 and C5, play an essential role in facilitating selectivity. PMID:20855585

  17. 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

  18. 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

  19. Lysosomal involvement in cellular turnover of plasma membrane sphingomyelin.

    PubMed

    Sutrina, S L; Chen, W W

    1984-04-18

    At least two isoenzymes of sphingomyelinase (sphingomyelin cholinephosphohydrolase, EC 3.1.4.12), including lysosomal acid sphingomyelinase and nonlysosomal magnesium-dependent neutral sphingomyelinase, catalyse the degradation of sphingomyelin in cultured human skin fibroblasts. A genetically determined disorder of sphingomyelin metabolism, type A Niemann-Pick disease, is characterized by a deficiency of lysosomal acid sphingomyelinase. To investigate the involvement of lysosomes in the degradation of cellular membrane sphingomyelin, we have undertaken studies to compare the turnover of plasma membrane sphingomyelin in fibroblasts from a patient with type A Niemann-Pick disease, which completely lack acid sphingomyelinase activity but retain nonlysosomal neutral sphingomyelinase activity, with turnover in fibroblasts from normal individuals. Plasma membrane sphingomyelin was labeled by incubating cells at low temperature with phosphatidylcholine vesicles containing radioactive sphingomyelin. A fluorescent analog of sphingomyelin, N-4-nitrobenzo-2-oxa-1,3-diazoleaminocaproyl sphingosylphosphorylcholine (NBD-sphingomyelin) is seen to be readily transferred at low temperature from phosphatidylcholine liposomes to the plasma membranes of cultured human fibroblasts. Moreover, when kinetic studies were done in parallel, a constant ratio of [14C]oleoylsphingosylphosphorylcholine ( [14C]sphingomyelin) to NBD-sphingomyelin was taken up at low temperature by the fibroblast cells, suggesting that [14C]sphingomyelin undergoes a similar transfer. The comparison of sphingomyelin turnover at 37 degrees C in normal fibroblasts compared to Niemann-Pick diseased fibroblasts shows that a rapid turnover of plasma membrane-associated sphingomyelin within the first 30 min appears to be similar in both normal and Niemann-Pick diseased cells. This rapid turnover appears to be primarily due to rapid removal of the [14C]sphingomyelin from the cell surface into the incubation medium. During

  20. 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

  1. A specific gastrin receptor on plasma membranes of antral smooth muscle.

    PubMed

    Baur, S; Bacon, V C

    1976-12-20

    Plasma membranes with a 17 fold enrichment in 5'-nucleotidase over homogenate were prepared from antral smooth muscle. A specific gastrin receptor on the plasma membranes has been demonstrated. By Scatchard analysis receptor has a Kaff of 2x10(9)M(-1) and a binding capacity of 5x10(-14) moles/mg of membrane protein. PMID:15625862

  2. Lateral transport of Smoothened from the plasma membrane to the membrane of the cilium

    PubMed Central

    Milenkovic, Ljiljana

    2009-01-01

    The function of primary cilia depends critically on the localization of specific proteins in the ciliary membrane. A major challenge in the field is to understand protein trafficking to cilia. The Hedgehog (Hh) pathway protein Smoothened (Smo), a 7-pass transmembrane protein, moves to cilia when a ligand is received. Using microscopy-based pulse-chase analysis, we find that Smo moves through a lateral transport pathway from the plasma membrane to the ciliary membrane. Lateral movement, either via diffusion or active transport, is quite distinct from currently studied pathways of ciliary protein transport in mammals, which emphasize directed trafficking of Golgi-derived vesicles to the base of the cilium. We anticipate that this alternative route will be used by other signaling proteins that function at cilia. The path taken by Smo may allow novel strategies for modulation of Hh signaling in cancer and regeneration. PMID:19948480

  3. 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.

  4. Reducing Plasma Membrane Sphingomyelin Increases Insulin Sensitivity ▿

    PubMed Central

    Li, Zhiqiang; Zhang, Hongqi; Liu, Jing; Liang, Chien-Ping; Li, Yan; Li, Yue; Teitelman, Gladys; Beyer, Thomas; Bui, Hai H.; Peake, David A.; Zhang, Youyan; Sanders, Phillip E.; Kuo, Ming-Shang; Park, Tae-Sik; Cao, Guoqing; Jiang, Xian-Cheng

    2011-01-01

    It has been shown that inhibition of de novo sphingolipid synthesis increases insulin sensitivity. For further exploration of the mechanism involved, we utilized two models: heterozygous serine palmitoyltransferase (SPT) subunit 2 (Sptlc2) gene knockout mice and sphingomyelin synthase 2 (Sms2) gene knockout mice. SPT is the key enzyme in sphingolipid biosynthesis, and Sptlc2 is one of its subunits. Homozygous Sptlc2-deficient mice are embryonic lethal. However, heterozygous Sptlc2-deficient mice that were viable and without major developmental defects demonstrated decreased ceramide and sphingomyelin levels in the cell plasma membranes, as well as heightened sensitivity to insulin. Moreover, these mutant mice were protected from high-fat diet-induced obesity and insulin resistance. SMS is the last enzyme for sphingomyelin biosynthesis, and SMS2 is one of its isoforms. Sms2 deficiency increased cell membrane ceramide but decreased SM levels. Sms2 deficiency also increased insulin sensitivity and ameliorated high-fat diet-induced obesity. We have concluded that Sptlc2 heterozygous deficiency- or Sms2 deficiency-mediated reduction of SM in the plasma membranes leads to an improvement in tissue and whole-body insulin sensitivity. PMID:21844222

  5. 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.

  6. Reconstitution of a plasma-membrane H(+)-ATPase into bilayer lipid membrane.

    PubMed

    Ziegler, W; Slayman, C L; Cartwright, C P

    1993-10-01

    The plasma membrane H(+)-ATPase of Neurospora has been reconstituted into planar lipid bilayer membranes by means of the vesicle-fusion technique described by Finkelstein and his collaborators (Zimmerberg et al., 1980; Cohen et al., 1980, 1984; Akabas et al., 1984). Enzyme was first transferred from isolated plasma membrane fragments into asolectin vesicles by a detergent-dialysis procedure (Perlin et al., 1984). After H(+)-pumping activity had been checked by quenching of acridine orange fluorescence, the vesicles were fused into performed bilayers. Critical features of the fusion process include (i) attachment of the vesicles to the bilayer in the presence of divalent cations (Mg++), and (ii) rapid osmotic swelling, which was enhanced by prior sonication or freeze-thawing of the vesicles, and/or by inclusions of physiologic channels. Enough proton pumps could be thus incorporated into bilayers to achieve ATP-driven, vanadate-sensitive currents of 0.04-0.4 pA. Aqueous solutions of low ionic strength were used to suppress conductance fluctuations due to the channels, and when that precaution was taken, we could demonstrate the proton pump the work against membrane potentials of at least 50 mV. PMID:8181690

  7. On the definition, nomenclature and classification of water channel proteins (aquaporins and relatives).

    PubMed

    Benga, Gheorghe

    2012-01-01

    A water channel protein (WCP) or a water channel can be defined as a transmembrane protein that has a specific three-dimensional structure with a pore that provides a pathway for water permeation across biological membranes. The pore is formed by two highly conserved regions in the amino acid sequence, called NPA boxes (or motifs) with three amino acid residues (asparagine-proline-alanine, NPA) and several surrounding amino acids. The NPA boxes have been called the "signature" sequence of WCPs. WCPs are a family of proteins belonging to the Membrane Intrinsic Proteins (MIPs) superfamily. In addition, in the MIP superfamily (with more than 1000 members) there are also proteins with no channel activity. The WCP family include three subfamilies: aquaporins, aquaglyceroporins and S-aquaporins. (1) The aquaporins (AQPs) are water selective or specific water channels, also named by various authors as "orthodox", "ordinary", "conventional", "classical", "pure", "normal", or "sensu strictu" aquaporins); (2) The aquaglyceroporins are permeable to water, but also to other small uncharged molecules, in particular glycerol; this family includes the glycerol facilitators, abbreviated as GlpFs, from glycerol permease facilitators. The "signature" sequence for aquaglyceroporins is the aspartic acid residue (D) in the second NPA box. (3) The third subfamily of WCPs have little conserved amino acid sequences around the NPA boxes, unclassifiable to the first two subfamilies. I recommend to use always for this subfamily the name S-aquaporins. They are also named "superaquaporins", "aquaporins with unusual (or deviated) NPA boxes", "subcellular aquaporins", or "sip-like aquaporins". I also recommend to use always the spelling aquaporin (not aquaporine), and, for various AQPs, the abbreviation AQP followed immediately by the number, (e.g. AQP1), with no space or--which might create confusions with "minus". PMID:22542572

  8. 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. PMID:26338867

  9. 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.

  10. A water-specific aquaporin involved in aphid osmoregulation.

    PubMed

    Shakesby, A J; Wallace, I S; Isaacs, H V; Pritchard, J; Roberts, D M; Douglas, A E

    2009-01-01

    The osmotic pressure of plant phloem sap is generally higher than that of insect body fluids. Water cycling from the distal to proximal regions of the gut is believed to contribute to the osmoregulation of aphids and other phloem-feeding insects, with the high flux of water mediated by a membrane-associated aquaporin. A putative aquaporin referred to as ApAQP1 was identified by RT-PCR of RNA isolated from the guts of pea aphids Acyrthosiphon pisum. The ApAQP1 protein has a predicted molecular mass 28.94kDa. Molecular modeling suggests that ApAQP1 has the general aquaporin topology and possesses the conserved pore properties of water-specific aquaporins. When expressed in Xenopus oocytes, ApAQP1 showed the hallmarks of aquaporin-mediated water transport, including an 18-fold increase in the osmotic water permeability of the oolemma, a reduced activation energy, and inhibition of elevated water transport activity by Hg ions. The ApAQP1 transcript was localised to the stomach and distal intestine, and RNAi-mediated knockdown of its expression resulted in elevated osmotic pressure of the haemolymph. Taken together, these data suggest that ApAQP1 contributes to the molecular basis of water cycling in the aphid gut. PMID:18983920

  11. 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

  12. Changes in Air CO₂ Concentration Differentially Alter Transcript Levels of NtAQP1 and NtPIP2;1 Aquaporin Genes in Tobacco Leaves.

    PubMed

    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

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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. PMID:25659484

  19. Subproteomics: identification of plasma membrane proteins from the yeast Saccharomyces cerevisiae.

    PubMed

    Navarre, Catherine; Degand, Hervé; Bennett, Keiryn L; Crawford, Janne S; Mørtz, Ejvind; Boutry, Marc

    2002-12-01

    As a consequence of their poor solubility during isoelectric focusing, integral membrane proteins are generally absent from two-dimensional gel proteome maps. In order to analyze the yeast plasma membrane proteome, a plasma membrane purification protocol was optimized in order to reduce contaminating membranes and cytosolic proteins. Specifically, the new fractionation scheme largely depleted the plasma membrane fraction of cytosolic proteins by deoxycholate stripping and ribosomal proteins by sucrose gradient flotation. The plasma membrane complement was resolved by two-dimensional electrophoresis using the cationic detergent cetyl trimethyl ammonium bromide in the first, and sodium dodecyl sulfate in the second dimension, and fifty spots were identified by matrix-assisted laser desorption/ionization-time of flight mass spectometry. In spite of the presence of still contaminating ribosomal proteins, major proteins corresponded to known plasma membrane residents, the ABC transporters Pdr5p and Snq2p, the P-type H(+)-ATPase Pma1p, the glucose transporter Hxt7p, the seven transmembrane-span Mrh1p, the low affinity Fe(++) transporter Fet4p, the twelve-span Ptr2p, and the plasma membrane anchored casein kinase Yck2p. The four transmembrane-span proteins Sur7p and Nce102p were also present in the isolated plasma membranes, as well as the unknown protein Ygr266wp that probably contains a single transmembrane span. Thus, combining subcellular fractionation with adapted two-dimensional electrophoresis resulted in the identification of intrinsic plasma membrane proteins. PMID:12469340

  20. 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

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

    PubMed

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

    2015-06-01

    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 Ca(2+) signaling. PMID:25908859

  2. 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. PMID:27226538

  3. 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

  4. 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. PMID:20037196

  5. 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. PMID:26721276

  6. Polyamine Binding to Plasma Membrane Vesicles Isolated from Zucchini Hypocotyls.

    PubMed Central

    Tassoni, A.; Antognoni, F.; Bagni, N.

    1996-01-01

    The general features of [14C]spermidine binding to plasmalemma vesicles isolated from zucchini (Cucurbita pepo L.) etiolated hypocotyls are reported in the present paper. The specific interaction of the polyamine with the plasma membranes was reversible and thermolabile, since it decreased by about 50% in the assay performed at 40[deg]C compared to that carried out on ice. On the contrary, nonspecific binding was unaffected by temperature. Specific spermidine binding showed a pH dependence with a maximum at pH 8.0 and it reached saturation between 0.75 and 1 mM external spermidine concentration. The value of the dissociation constant calculated from Scatchard analysis was 4.4 x 10-5 M. Specific spermidine interaction appeared to be sensitive to detergents and was markedly reduced by the presence of divalent cations, such as Mg2+ and Ca2+, whereas it was stimulated by monovalent cations. Polyamine binding sites were highly sensitive to pronase treatment. Competition experiments, performed using a series of compounds structurally related to spermidine, may provide some indication of the characteristics of spermidine binding sites. The results presented here suggest that specific spermidine binding occurs mainly with the protein component of the plasma membrane. PMID:12226221

  7. Supramolecular architecture of endoplasmic reticulum-plasma membrane contact sites.

    PubMed

    Fernández-Busnadiego, Rubén

    2016-04-15

    The endoplasmic reticulum (ER) forms membrane contact sites (MCS) with most other cellular organelles and the plasma membrane (PM). These ER-PM MCS, where the membranes of the ER and PM are closely apposed, were discovered in the early days of electron microscopy (EM), but only recently are we starting to understand their functional and structural diversity. ER-PM MCS are nowadays known to mediate excitation-contraction coupling (ECC) in striated muscle cells and to play crucial roles in Ca(2+)and lipid homoeostasis in all metazoan cells. A common feature across ER-PM MCS specialized in different functions is the preponderance of cooperative phenomena that result in the formation of large supramolecular assemblies. Therefore, characterizing the supramolecular architecture of ER-PM MCS is critical to understand their mechanisms of function. Cryo-electron tomography (cryo-ET) is a powerful EM technique uniquely positioned to address this issue, as it allows 3D imaging of fully hydrated, unstained cellular structures at molecular resolution. In this review I summarize our current structural knowledge on the molecular organization of ER-PM MCS and its functional implications, with special emphasis on the emerging contributions of cryo-ET. PMID:27068966

  8. 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. PMID:23547882

  9. Loop A is critical for the functional interaction of two Beta vulgaris PIP aquaporins.

    PubMed

    Jozefkowicz, Cintia; Rosi, Pablo; Sigaut, Lorena; Soto, Gabriela; Pietrasanta, Lía Isabel; Amodeo, Gabriela; Alleva, Karina

    2013-01-01

    Research done in the last years strongly support the hypothesis that PIP aquaporin can form heterooligomeric assemblies, specially combining PIP2 monomers with PIP1 monomers. Nevertheless, the structural elements involved in the ruling of homo versus heterooligomeric organization are not completely elucidated. In this work we unveil some features of monomer-monomer interaction in Beta vulgaris PIP aquaporins. Our results show that while BvPIP2;2 is able to interact with BvPIP1;1, BvPIP2;1 shows no functional interaction. The lack of functional interaction between BvPIP2;1 and BvPIP1;1 was further corroborated by dose-response curves of water permeability due to aquaporin activity exposed to different acidic conditions. We also found that BvPIP2;1 is unable to translocate BvPIP1;1-ECFP from an intracellular position to the plasma membrane when co-expressed, as BvPIP2;2 does. Moreover we postulate that the first extracellular loop (loop A) of BvPIP2;1, could be relevant for the functional interaction with BvPIP1;1. Thus, we investigate BvPIP2;1 loop A at an atomic level by Molecular Dynamics Simulation (MDS) and by direct mutagenesis. We found that, within the tetramer, each loop A presents a dissimilar behavior. Besides, BvPIP2;1 loop A mutants restore functional interaction with BvPIP1;1. This work is a contribution to unravel how PIP2 and PIP1 interact to form functional heterooligomeric assemblies. We postulate that BvPIP2;1 loop A is relevant for the lack of functional interaction with BvPIP1;1 and that the monomer composition of PIP assemblies determines their functional properties. PMID:23483963

  10. Loop A Is Critical for the Functional Interaction of Two Beta vulgaris PIP Aquaporins

    PubMed Central

    Jozefkowicz, Cintia; Rosi, Pablo; Sigaut, Lorena; Soto, Gabriela; Pietrasanta, Lía Isabel; Amodeo, Gabriela; Alleva, Karina

    2013-01-01

    Research done in the last years strongly support the hypothesis that PIP aquaporin can form heterooligomeric assemblies, specially combining PIP2 monomers with PIP1 monomers. Nevertheless, the structural elements involved in the ruling of homo versus heterooligomeric organization are not completely elucidated. In this work we unveil some features of monomer-monomer interaction in Beta vulgaris PIP aquaporins. Our results show that while BvPIP2;2 is able to interact with BvPIP1;1, BvPIP2;1 shows no functional interaction. The lack of functional interaction between BvPIP2;1 and BvPIP1;1 was further corroborated by dose-response curves of water permeability due to aquaporin activity exposed to different acidic conditions. We also found that BvPIP2;1 is unable to translocate BvPIP1;1-ECFP from an intracellular position to the plasma membrane when co-expressed, as BvPIP2;2 does. Moreover we postulate that the first extracellular loop (loop A) of BvPIP2;1, could be relevant for the functional interaction with BvPIP1;1. Thus, we investigate BvPIP2;1 loop A at an atomic level by Molecular Dynamics Simulation (MDS) and by direct mutagenesis. We found that, within the tetramer, each loop A presents a dissimilar behavior. Besides, BvPIP2;1 loop A mutants restore functional interaction with BvPIP1;1. This work is a contribution to unravel how PIP2 and PIP1 interact to form functional heterooligomeric assemblies. We postulate that BvPIP2;1 loop A is relevant for the lack of functional interaction with BvPIP1;1 and that the monomer composition of PIP assemblies determines their functional properties. PMID:23483963

  11. 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

  12. Phosphorylation regulates the water channel activity of the seed-specific aquaporin alpha-TIP.

    PubMed

    Maurel, C; Kado, R T; Guern, J; Chrispeels, M J

    1995-07-01

    The vacuolar membrane protein alpha-TIP is a seed-specific protein of the Major Intrinsic Protein family. Expression of alpha-TIP in Xenopus oocytes conferred a 4- to 8-fold increase in the osmotic water permeability (Pf) of the oocyte plasma membrane, showing that alpha-TIP forms water channels and is thus a new aquaporin. alpha-TIP has three putative phosphorylation sites on the cytoplasmic side of the membrane (Ser7, Ser23 and Ser99), one of which (Ser7) has been shown to be phosphorylated. We present several lines of evidence that the activity of this aquaporin is regulated by phosphorylation. First, mutation of the putative phosphorylation sites in alpha-TIP (Ser7Ala, Ser23Ala and Ser99Ala) reduced the apparent water transport activity of alpha-TIP in oocytes, suggesting that phosphorylation of alpha-TIP occurs in the oocytes and participates in the control of water channel activity. Second, exposure of oocytes to the cAMP agonists 8-bromoadenosine 3',5'-cyclic monophosphate, forskolin and 3-isobutyl-1-methylxanthine, which stimulate endogenous protein kinase A (PKA), increased the water transport activity of alpha-TIP by 80-100% after 60 min. That the protein can be phosphorylated by PKA was demonstrated by phosphorylating alpha-TIP in isolated oocyte membranes with the bovine PKA catalytic subunit. Third, the integrity of the three sites at positions 7, 23 and 99 was necessary for the cAMP-dependent increase in the Pf of oocytes expressing alpha-TIP, as well as for in vitro phosphorylation of alpha-TIP. These findings demonstrate that the alpha-TIP water channel can be modulated via phosphorylation of Ser7, Ser23 and Ser99.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7542585

  13. Assembly and Comparison of Plasma Membrane SNARE Acceptor Complexes.

    PubMed

    Kreutzberger, Alex J B; Liang, Binyong; Kiessling, Volker; Tamm, Lukas K

    2016-05-24

    Neuronal exocytotic membrane fusion occurs on a fast timescale and is dependent on interactions between the vesicle SNARE synaptobrevin-2 and the plasma membrane SNAREs syntaxin-1a and SNAP-25 with a 1:1:1 stoichiometry. Reproducing fast fusion rates as observed in cells by reconstitution in vitro has been hindered by the spontaneous assembly of a 2:1 syntaxin-1a:SNAP-25 complex on target membranes that kinetically alters the binding of synaptobrevin-2. Previously, an artificial SNARE acceptor complex consisting of 1:1:1 syntaxin-1a(residues 183-288):SNAP-25:syb(residues 49-96) was found to greatly accelerate the rates of lipid mixing of reconstituted target and vesicle SNARE proteoliposomes. Here we present two (to our knowledge) new procedures to assemble membrane-bound 1:1 SNARE acceptor complexes that produce fast and efficient fusion without the need of the syb(49-96) peptide. In the first procedure, syntaxin-1a is purified in a strictly monomeric form and subsequently assembled with SNAP-25 in detergent with the correct 1:1 stoichiometry. In the second procedure, monomeric syntaxin-1a and dodecylated (d-)SNAP-25 are separately reconstituted into proteoliposomes and subsequently assembled in the plane of merged target lipid bilayers. Examining single particle fusion between synaptobrevin-2 proteoliposomes and planar-supported bilayers containing the two different SNARE acceptor complexes revealed similar fast rates of fusion. Changing the stoichiometry of syntaxin-1a and d-SNAP-25 in the target bilayer had significant effects on docking, but little effect on the rates of synaptobrevin-2 proteoliposome fusion. PMID:27178662

  14. The initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes

    PubMed Central

    Zak, Elena; Norling, Birgitta; Maitra, Radhashree; Huang, Fang; Andersson, Bertil; Pakrasi, Himadri B.

    2001-01-01

    During oxygenic photosynthesis in cyanobacteria and chloroplasts of plants and eukaryotic algae, conversion of light energy to biologically useful chemical energy occurs in the specialized thylakoid membranes. Light-induced charge separation at the reaction centers of photosystems I and II, two multisubunit pigment-protein complexes in the thylakoid membranes, energetically drive sequential photosynthetic electron transfer reactions in this membrane system. In general, in the prokaryotic cyanobacterial cells, the thylakoid membrane is distinctly different from the plasma membrane. We have recently developed a two-dimensional separation procedure to purify thylakoid and plasma membranes from the genetically widely studied cyanobacterium Synechocystis sp. PCC 6803. Immunoblotting analysis demonstrated that the purified plasma membrane contained a number of protein components closely associated with the reaction centers of both photosystems. Moreover, these proteins were assembled in the plasma membrane as chlorophyll-containing multiprotein complexes, as evidenced from nondenaturing green gel and low-temperature fluorescence spectroscopy data. Furthermore, electron paramagnetic resonance spectroscopic analysis showed that in the partially assembled photosystem I core complex in the plasma membrane, the P700 reaction center was capable of undergoing light-induced charge separation. Based on these data, we propose that the plasma membrane, and not the thylakoid membrane, is the site for a number of the early steps of biogenesis of the photosynthetic reaction center complexes in these cyanobacterial cells. PMID:11687660

  15. The Plasma Membrane Ca2+ ATPase and the Plasma Membrane Sodium Calcium Exchanger Cooperate in the Regulation of Cell Calcium

    PubMed Central

    Brini, Marisa; Carafoli, Ernesto

    2011-01-01

    Calcium is an ambivalent signal: it is essential for the correct functioning of cell life, but may also become dangerous to it. The plasma membrane Ca2+ ATPase (PMCA) and the plasma membrane Na+/Ca2+ exchanger (NCX) are the two mechanisms responsible for Ca2+ extrusion. The NCX has low Ca2+ affinity but high capacity for Ca2+ transport, whereas the PMCA has a high Ca2+ affinity but low transport capacity for it. Thus, traditionally, the PMCA pump has been attributed a housekeeping role in maintaining cytosolic Ca2+, and the NCX the dynamic role of counteracting large cytosolic Ca2+ variations (especially in excitable cells). This view of the roles of the two Ca2+ extrusion systems has been recently revised, as the specific functional properties of the numerous PMCA isoforms and splicing variants suggests that they may have evolved to cover both the basal Ca2+ regulation (in the 100 nM range) and the Ca2+ transients generated by cell stimulation (in the μM range). PMID:21421919

  16. 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. PMID:26369275

  17. Aquaporin tetramer composition modifies the function of tobacco aquaporins.

    PubMed

    Otto, Beate; Uehlein, Norbert; Sdorra, Sven; Fischer, Matthias; Ayaz, Muhammad; Belastegui-Macadam, Xana; Heckwolf, Marlies; Lachnit, Magdalena; Pede, Nadine; Priem, Nadine; Reinhard, André; Siegfart, Sven; Urban, Michael; Kaldenhoff, Ralf

    2010-10-01

    Heterologous expression in yeast cells revealed that NtAQP1, a member of the so-called PIP1 aquaporin subfamily, did not display increased water transport activity in comparison with controls. Instead, an increased CO(2)-triggered intracellular acidification was observed. NtPIP2;1, which belongs to the PIP2 subfamily of plant aquaporins, behaved as a true aquaporin but lacked a CO(2)-related function. Results from split YFP experiments, protein chromatography, and gel electrophoresis indicated that the proteins form heterotetramers when coexpressed in yeast. Tetramer composition had effects on transport activity as demonstrated by analysis of artificial heterotetramers with a defined proportion of NtAQP1 to NtPIP2;1. A single NtPIP2;1 aquaporin in a tetramer was sufficient to significantly increase the water permeability of the respective yeast cells. With regard to CO(2)-triggered intracellular acidification, a cooperative effect was observed, where maximum rates were measured when the tetramer consisted of NtAQP1 aquaporins only. The results confirm the model of an aquaporin monomer as a functional unit for water transport and suggest that, for CO(2)-related transport processes, a structure built up by the tetramer is the basis of this function. PMID:20657033

  18. Aquaporin Tetramer Composition Modifies the Function of Tobacco Aquaporins*

    PubMed Central

    Otto, Beate; Uehlein, Norbert; Sdorra, Sven; Fischer, Matthias; Ayaz, Muhammad; Belastegui-Macadam, Xana; Heckwolf, Marlies; Lachnit, Magdalena; Pede, Nadine; Priem, Nadine; Reinhard, André; Siegfart, Sven; Urban, Michael; Kaldenhoff, Ralf

    2010-01-01

    Heterologous expression in yeast cells revealed that NtAQP1, a member of the so-called PIP1 aquaporin subfamily, did not display increased water transport activity in comparison with controls. Instead, an increased CO2-triggered intracellular acidification was observed. NtPIP2;1, which belongs to the PIP2 subfamily of plant aquaporins, behaved as a true aquaporin but lacked a CO2-related function. Results from split YFP experiments, protein chromatography, and gel electrophoresis indicated that the proteins form heterotetramers when coexpressed in yeast. Tetramer composition had effects on transport activity as demonstrated by analysis of artificial heterotetramers with a defined proportion of NtAQP1 to NtPIP2;1. A single NtPIP2;1 aquaporin in a tetramer was sufficient to significantly increase the water permeability of the respective yeast cells. With regard to CO2-triggered intracellular acidification, a cooperative effect was observed, where maximum rates were measured when the tetramer consisted of NtAQP1 aquaporins only. The results confirm the model of an aquaporin monomer as a functional unit for water transport and suggest that, for CO2-related transport processes, a structure built up by the tetramer is the basis of this function. PMID:20657033

  19. Expression and Localization of Aquaporins in Benign Prostate Hyperplasia and Prostate Cancer

    PubMed Central

    Hwang, Insang; Hwang, Eu-Chang; Song, Seung Hee; Lee, Hyun-Suk; Kim, Sun-Ouck; Kang, Taek-Won; Kwon, Dongdeuk; Park, Kwangsung

    2012-01-01

    The aquaporin (AQP) families of water channels are intrinsic membrane proteins that facilitate selective water and small solute movement across the plasma membrane. The purposes of this study were to determine the expression and localization of AQPs in benign prostatic hyperplasia and prostate cancer. Prostatic tissue was collected from patients with benign prostatic hyperplasia or prostate cancer by transurethral resection of the prostate. The expression and cellular localization of the AQPs were determined in the human prostate by Western blot and immunohistochemistry. AQP1, 3, and 9 were expressed in the human prostate. Western blot analysis revealed bands at 28-36 kDa for the AQP1, 3, and 9 proteins. Of these proteins, AQP3 and 9 were expressed in the epithelium. Immunolabeling showed that AQP1 was mainly expressed in the capillaries and venules of the prostate, AQP9 was expressed in the cytoplasm of the epithelium, and AQP3 was mainly associated with the plasma membrane of the prostatic epithelium. Only AQP3 expression was localized in the cell membrane, and expressed AQP3 was translocated to the cytoplasm in prostate cancer. The epithelium in the human prostate expresses AQP3 and 9 proteins, and the capillaries and venules of the prostate express AQP1. Characterizing or modifying the expression of AQP3 may lead to an understanding of the role of the AQPs in human prostatic disease. PMID:23323224

  20. Aquaporin family genes exhibit developmentally-regulated and host-dependent transcription patterns in the sea louse Caligus rogercresseyi.

    PubMed

    Farlora, Rodolfo; Valenzuela-Muñoz, Valentina; Chávez-Mardones, Jacqueline; Gallardo-Escárate, Cristian

    2016-07-01

    Aquaporins are small integral membrane proteins that function as pore channels for the transport of water and other small solutes across the cell membrane. Considering the important roles of these proteins in several biological processes, including host-parasite interactions, there has been increased research on aquaporin proteins recently. The present study expands on the knowledge of aquaporin family genes in parasitic copepods, examining diversity and expression during the ontogeny of the sea louse Caligus rogercresseyi. Furthermore, aquaporin expression was evaluated during the early infestation of Atlantic (Salmo salar) and Coho salmon (Oncorhynchus kisutch). Deep transcriptome sequencing data revealed eight full length and two partial open reading frames belonging to the aquaporin protein family. Clustering analyses with identified Caligidae sequences revealed three major clades of aquaglyceroporins (Cr-Glp), classical aquaporin channels (Cr-Bib and Cr-PripL), and unorthodox aquaporins (Cr-Aqp12-like). In silico analysis revealed differential expression of aquaporin genes between developmental stages and between sexes. Male-biased expression of Cr-Glp1_v1 and female-biased expression of Cr-Bib were further confirmed in adults by RT-qPCR. Additionally, gene expressions were measured for seven aquaporins during the early infestation stage. The majority of aquaporin genes showed significant differential transcription expressions between sea lice parasitizing different hosts, with Atlantic salmon sea lice exhibiting overall reduced expression as compared to Coho salmon. The observed differences in the regulation of aquaporin genes may reveal osmoregulatory adaptations associated with nutrient ingestion and metabolite waste export, exposing complex host-parasite relationships in C. rogercresseyi. PMID:27016299

  1. Isolation and expression of two aquaporin-encoding genes from the marine phanerogam Posidonia oceanica.

    PubMed

    Maestrini, Pierluigi; Giordani, Tommaso; Lunardi, Andrea; Cavallini, Andrea; Natali, Lucia

    2004-12-01

    Seagrasses such as Posidonia oceanica (L.) Delile are marine phanerogams, widespread in various seas, where they form large prairies representing dynamic substrates exceeding the area of the sediment surface several times over and allowing settlement of epiphyte organisms. Studying mechanisms involved in water transport in marine plants, we isolated two aquaporin-encoding genes, PoPIP1;1 and PoTIP1;1, showing high similarity to plasma membrane- and tonoplast-intrinsic protein-encoding genes, respectively. PoPIP1;1 is unique in the genome of P. oceanica, while PoTIP1;1 belongs to an aquaporin subfamily of at least four members. PoPIP1;1 and PoTIP1;1 encode functional proteins, as indicated by expression experiments in Xenopus oocytes. Both genes are constitutively expressed in the leaves, with higher levels of transcripts in young than in differentiated leaf tissues. Variations of salt concentration in aquarium determined different PoPIP1;1 and PoTIP1;1 transcript accumulation, indicating the existence of adaptation mechanisms related to gene expression also in marine plants, i.e. adapted to very high salt concentrations. Hyposalinity induced lower levels of PIP1 transcripts, while hypersalinity determined more PIP1 transcripts than normal salinity. TIP1 transcripts increased in response to both hypo- and hypersalinity after 2 days of treatment and went back to control levels after 5 d. PMID:15653802

  2. Rapid shoot-to-root signalling regulates root hydraulic conductance via aquaporins.

    PubMed

    Vandeleur, Rebecca K; Sullivan, Wendy; Athman, Asmini; Jordans, Charlotte; Gilliham, Matthew; Kaiser, Brent N; Tyerman, Stephen D

    2014-02-01

    We investigated how root hydraulic conductance (normalized to root dry weight, Lo ) is regulated by the shoot. Shoot topping (about 30% reduction in leaf area) reduced Lo of grapevine (Vitis vinifera L.), soybean (Glycine max L.) and maize (Zea mays L.) by 50 to 60%. More detailed investigations with soybean and grapevine showed that the reduction in Lo was not correlated with the reduction in leaf area, and shading or cutting single leaves had a similar effect. Percentage reduction in Lo was largest when initial Lo was high in soybean. Inhibition of Lo by weak acid (low pH) was smaller after shoot damage or leaf shading. The half time of reduction in Lo was approximately 5 min after total shoot decapitation. These characteristics indicate involvement of aquaporins. We excluded phloem-borne signals and auxin-mediated signals. Xylem-mediated hydraulic signals are possible since turgor rapidly decreased within root cortex cells after shoot topping. There was a significant reduction in the expression of several aquaporins in the plasma membrane intrinsic protein (PIP) family of both grapevine and soybean. In soybean, there was a five- to 10-fold reduction in GmPIP1;6 expression over 0.5-1 h which was sustained over the period of reduced Lo . PMID:23926961

  3. Actin dynamics provides membrane tension to merge fusing vesicles into the plasma membrane.

    PubMed

    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

  4. A PIP1 Aquaporin Contributes to Hydrostatic Pressure-Induced Water Transport in Both the Root and Rosette of Arabidopsis1[C][W

    PubMed Central

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

    2010-01-01

    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 (Kros) was measured by a novel pressure chamber technique. Exposure of Arabidopsis plants to darkness increased Kros by up to 90%. Mercury and azide, two aquaporin inhibitors with distinct modes of action, were able to induce similar inhibition of Kros 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 Kros 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. PMID:20034965

  5. Complement-mediated production of plasma-membrane vesicles from rat fat-cells.

    PubMed

    Richardson, P J; Luzio, J P

    1980-03-15

    1. Rat isolated fat-cells were coated with rabbit anti-(rat erythrocyte) antibody and incubated with fresh guinea-pig serum for 25 min at 37 degrees C, which resulted in a more than 95% release of the cytosolic enzyme lactate dehydrogenase. 2. Under these conditions fragmentation of the plasma membrane was examined by following the plasma-membrane markers 5'-nucleotidase, adrenaline-sensitive adenylate cyclase and membrane-bound rabbit immunoglobulin G through a differential-centrifugation fractionation procedure. 3. Approx. 50% of the plasma-membrane markers remained associated with triacylglycerol. Of the remainder more than half was pelleted by centrifugation at 10 000 g for 30 min. 4. The 10 000 g supernatant was fractionated by centrifugation on a sucrose density gradient (15-50%, w/w). This procedure resulted in the production of two visible white bands on the density gradient. The bands consisted of vesicles derived from the plasma membrane, since they coincided with peaks of 5'-nucleotidase activity, contained membrane-bound immunoglobulin G and the denser one had adenylate cyclase activity. The phospholipid and protein contents of the vesicles were determined and compared with those in purified plasma membrane. 5. It is suggested that complement-mediated lysis of rat fat-cells caused the production of plasma-membrane vesicles that differ in composition from the whole plasma membrane. PMID:6249263

  6. 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. PMID:25087500

  7. The isolation and partial characterization of the plasma membrane from Trypanosoma brucei.

    PubMed

    Voorheis, H P; Gale, J S; Owen, M J; Edwards, W

    1979-04-15

    Whole sheets of plasma membrane, each with their attached flagellum, were purified from Trypanosoma brucei. The method devised for their isolation included a new technique of cell breakage that used a combination of osmotic stress followed by mechanical sheer and avoided the problem of extreme vesiculation as well as the trapping of organelles in cell 'ghosts'. The purified membranes all contained the pellicular microtubular array. The antigenic surface coat was completely released from the plasma membrane during the isolation procedure. The membranes had a very high cholesterol/phospholipid ratio (1.54). A large proportion (42%) of the cellular DNA was recovered in the plasma-membrane fraction unless a step involving deoxyribonuclease treatment, which decreased the DNA content to less than 13%, was included before secrose-density gradient centrifugation. This step also aided the separation of plasma membranes from other cellular components. The ouabain-sensitive Na+ + K+-stimulated adenosine triphosphatase and adenylate cyclase co-purified with the plasma membranes. Although 5'-nucleotidase was thought to be a plasma-membrane component, it was easily detached from the membrane. The purified membranes were essentially free of L-alanine-alpha-oxoglutarate aminotransferase, L-asparte-alpha-oxoglutarate aminotransferase, malate dehydrogenase, oligomycin-sensitive adenosine triphosphatase, glucose 6-phosphatase, Mg2+-stimulated p-nitrophenyl phosphatase and catalase. PMID:486094

  8. Detection of boar sperm plasma membrane protein using Rhodamine 640; implications for cryobiology and physiology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  9. Thymocyte plasma membrane: the location of specific glucocorticoid binding sites

    SciTech Connect

    Sergeev, P.V.; Kalinin, G.V.; Dukhanin, A.S.

    1987-01-01

    In modern molecular endocrinology it is now possible to determine the localization of receptors for biologically active substances with the aid of ligands, with high affinity for the receptor, immobilized on polymers. The purpose of this paper is to study the ability of hydrocortisone (HC), immobilized on polyvinylpyrrolidone (PVP-HC), to reduce binding of tritium-HC by thymocytes of adrenalectomized rats. It is determined that specific binding sites for HC on rat thymocytes are also accessible for PVP-HC, which, due to the fact that this immobilized version of HC does not penetrate into the cell, leads to the conclusion that the binding sites for HC itself are located in the plasma membrane.

  10. [Roles of Aquaporins in Brain Disorders].

    PubMed

    Yasui, Masato

    2015-06-01

    Aquaporin (AQP) is a water channel protein that is expressed in the cell membranes. AQPs are related to several kinds of human diseases such as cataract. In the mammalian central nervous system (CNS), AQP4 is specifically expressed in the astrocyte membranes lining the perivascular and periventricular structures. AQP4 plays a role in the development of brain edema associated with certain brain disorders. Neuromyelitis optica (NMO) is a demyelinating disorder, and patients with NMO develop autoimmune antibodies against AQP4 in their serum. Therefore, AQP4 is involved in NMO pathogenesis. A new concept referred to as "glymphatic pathway" has been recently proposed to explain the lymphatic system in the CNS. Dysfunction of the "glymphatic pathway" may cause several neurodegenerative diseases and mood disorders. Importantly, AQP4 may play a role in the "glymphatic pathway". Further investigation of AQP4 in CNS disorders is necessary, and a new drug against AQP4 is expected. PMID:26062588

  11. Abnormal aquaporin-3 protein expression in hyperproliferative skin disorders.

    PubMed

    Voss, Kristen E; Bollag, Roni J; Fussell, Nicole; By, Charya; Sheehan, Daniel J; Bollag, Wendy B

    2011-10-01

    Non-melanoma skin cancers (NMSCs) and psoriasis represent common hyperproliferative skin disorders, with approximately one million new NMSC diagnoses each year in the United States alone and a psoriasis prevalence of about 2% worldwide. We recently demonstrated that the glycerol channel, aquaporin-3 (AQP3) and the enzyme phospholipase D2 (PLD2) interact functionally in epidermal keratinocytes of the skin to inhibit their proliferation. However, others have suggested that AQP3 is pro-proliferative in keratinocytes and is upregulated in the NMSC, squamous cell carcinoma (SCC). To evaluate the AQP3/PLD2 signaling module in skin diseases, we determined their levels in SCC, basal cell carcinoma (BCC) and psoriasis as compared to normal epidermis. Skin biopsies with the appropriate diagnoses (10 normal, 5 SCC, 13 BCC and 10 plaque psoriasis samples) were obtained from the pathology archives and examined by immunohistochemistry using antibodies recognizing AQP3 and PLD2. In normal epidermis AQP3, an integral membrane protein, was localized mainly to the plasma membrane and PLD2 to the cell periphery, particularly in suprabasal layers. In BCC, AQP3 and PLD2 levels were reduced as compared to the normal-appearing overlying epidermis. In SCC, AQP3 staining was "patchy," with areas of reduced AQP3 immunoreactivity exhibiting positivity for Ki67, a marker of proliferation. PLD2 staining was unchanged in SCC. In psoriasis, AQP3 staining was usually observed in the cytoplasm rather than in the membrane. Also, in the majority of psoriatic samples, PLD2 showed weak immunoreactivity or aberrant localization. These results suggest that abnormalities in the AQP3/PLD2 signaling module correlate with hyperproliferation in psoriasis and the NMSCs. PMID:21400035

  12. Whole gene family expression and drought stress regulation of aquaporins.

    PubMed

    Alexandersson, Erik; Fraysse, Laure; Sjövall-Larsen, Sara; Gustavsson, Sofia; Fellert, Maria; Karlsson, Maria; Johanson, Urban; Kjellbom, Per

    2005-10-01

    Since many aquaporins (AQPs) act as water channels, they are thought to play an important role in plant water relations. It is therefore of interest to study the expression patterns of AQP isoforms in order to further elucidate their involvement in plant water transport. We have monitored the expression patterns of all 35 Arabidopsis AQPs in leaves, roots and flowers by cDNA microarrays, specially designed for AQPs, and by quantitative real-time reverse transcriptase PCR (Q-RT-PCR). This showed that many AQPs are pre-dominantly expressed in either root or flower organs, whereas no AQP isoform seem to be leaf specific. Looking at the AQP subfamilies, most plasma membrane intrinsic proteins (PIPs) and some tonoplast intrinsic proteins (TIPs) have a high level of expression, while NOD26-like proteins (NIPs) are present at a much lower level. In addition, we show that PIP transcripts are generally down-regulated upon gradual drought stress in leaves, with the exception of AtPIP1;4 and AtPIP2;5, which are up-regulated. AtPIP2;6 and AtSIP1;1 are constitutively expressed and not significantly affected by the drought stress. The transcriptional down-regulation of PIP genes upon drought stress could also be observed on the protein level. PMID:16235111

  13. Polyphenols as Modulators of Aquaporin Family in Health and Disease

    PubMed Central

    Fiorentini, Diana; Zambonin, Laura; Vieceli Dalla Sega, Francesco; Hrelia, Silvana

    2015-01-01

    Polyphenols are bioactive molecules widely distributed in fruits, vegetables, cereals, and beverages. Polyphenols in food sources are extensively studied for their role in the maintenance of human health and in the protection against development of chronic/degenerative diseases. Polyphenols act mainly as antioxidant molecules, protecting cell constituents against oxidative damage. The enormous number of polyphenolic compounds leads to huge different mechanisms of action not fully understood. Recently, some evidence is emerging about the role of polyphenols, such as curcumin, pinocembrin, resveratrol, and quercetin, in modulating the activity of some aquaporin (AQP) isoforms. AQPs are integral, small hydrophobic water channel proteins, extensively expressed in many organs and tissues, whose major function is to facilitate the transport of water or glycerol over cell plasma membranes. Here we summarize AQP physiological functions and report emerging evidence on the implication of these proteins in a number of pathophysiological processes. In particular, this review offers an overview about the role of AQPs in brain, eye, skin diseases, and metabolic syndrome, focusing on the ability of polyphenols to modulate AQP expression. This original analysis can contribute to elucidating some peculiar effects exerted by polyphenols and can lead to the development of an innovative potential preventive/therapeutic strategy. PMID:26346093

  14. STIM Proteins and the Endoplasmic Reticulum-Plasma Membrane Junctions

    PubMed Central

    Carrasco, Silvia; Meyer, Tobias

    2013-01-01

    Eukaryotic organelles can interact with each other through stable junctions where the two membranes are kept in close apposition. The junction that connects the endoplasmic reticulum to the plasma membrane (ER-PM junction) is unique in providing a direct communication link between the ER and the PM. In a recently discovered signaling process, STIM (stromal-interacting molecule) proteins sense a drop in ER Ca2+ levels and directly activate Orai PM Ca2+ channels across the junction space. In an inverse process, a voltage-gated PM Ca2+ channel can directly open ER ryanodine-receptor Ca2+ channels in striated-muscle cells. Although ER-PM junctions were first described 50 years ago, their broad importance in Ca2+ signaling, as well as in the regulation of cholesterol and phosphatidylinositol lipid transfer, has only recently been realized. Here, we discuss research from different fields to provide a broad perspective on the structures and unique roles of ER-PM junctions in controlling signaling and metabolic processes. PMID:21548779

  15. Hydrating skin by stimulating biosynthesis of aquaporins.

    PubMed

    Dumas, Marc; Sadick, Neil S; Noblesse, Emmanuelle; Juan, Milène; Lachmann-Weber, Nadège; Boury-Jamot, Mathieu; Sougrat, Rachid; Verbavatz, Jean Marc; Schnebert, Sylvianne; Bonté, Frédéric

    2007-06-01

    Aquaporins (AQPs) are proteins that facilitate the transport of water across cell membranes. AQP3 expression is related to the expressions of other epidermal proteins involved in water maintenance (ie, CD44, claudin-1, and filaggrin). The expressions of AQP3 water channels are strongly affected by age and chronic sun exposure, and a defective osmotic equilibrium could occur in the epidermis, which would account for the skin dryness observed in older people and skin areas most exposed to sunlight. We investigated active ingredients that are able to increase AQP3 levels in order to improve hydration in human skin keratinocytes. We selected an ethanolic/water (70/30 v/v) extract of Ajuga turkestanica, a plant from Central Asia, as the hydrating agent. After 17 days of treatment every 2 days with this extract (2.5 microg/mL) in vitro, AQP3 expression measured at the protein level in human reconstructed epidermis was significantly increased. Water transport through both aquaporins and aquaglyceroporins and glycerol transport through aquaglyceroporins alone are important to skin hydration. The distribution and the variability of aquaporins in human skin cells suggest that these channels may have important roles in skin physiology. AQPs appear to be key protein targets to improve the resistance and quality of the skin surface as well as to improve aging and sun exposure-induced dryness as shown by their roles in 1) hydrating the living layers of the epidermis where the keratinocyte differentiation takes place and 2) barrier formation and recovery. PMID:17691206

  16. Comparative Analysis of Techniques to Purify Plasma Membrane Proteins

    PubMed Central

    Weekes, Michael P.; Antrobus, Robin; Lill, Jennie R.; Duncan, Lidia M.; Hör, Simon; Lehner, Paul J.

    2010-01-01

    The aim of this project was to identify the best method for the enrichment of plasma membrane (PM) proteins for proteomics experiments. Following tryptic digestion and extended liquid chromatography-tandem mass spectrometry acquisitions, data were processed using MaxQuant and Gene Ontology (GO) terms used to determine protein subcellular localization. The following techniques were examined for the total number and percentage purity of PM proteins identified: (a) whole cell lysate (total number, 84–112; percentage purity, 9–13%); (b) crude membrane preparation (104–111; 17–20%); (c) biotinylation of surface proteins with N-hydroxysulfosuccinimydyl-S,S-biotin and streptavidin pulldown (78–115; 27–31%); (d) biotinylation of surface glycoproteins with biocytin hydrazide and streptavidin pulldown (41–54; 59–85%); or (e) biotinylation of surface glycoproteins with amino-oxy-biotin (which labels the sialylated fraction of PM glycoproteins) and streptavidin pulldown (120; 65%). A two- to threefold increase in the overall number of proteins identified was achieved by using stop and go extraction tip (StageTip)-based anion exchange (SAX) fractionation. Combining technique (e) with SAX fractionation increased the number of proteins identified to 281 (54%). Analysis of GO terms describing these proteins identified a large subset of proteins integral to the membrane with no subcellular assignment. These are likely to be of PM location and bring the total PM protein identifications to 364 (68%). This study suggests that selective biotinylation of the cell surface using amino-oxy-biotin in combination with SAX fractionation is a useful method for identification of sialylated PM proteins. PMID:20808639

  17. 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

  18. Members of rice plasma membrane intrinsic proteins subfamily are involved in arsenite permeability and tolerance in plants.

    PubMed

    Mosa, Kareem A; Kumar, Kundan; Chhikara, Sudesh; Mcdermott, Joseph; Liu, Zijuan; Musante, Craig; White, Jason C; Dhankher, Om Parkash

    2012-12-01

    Rice accumulates high level of arsenic (As) in its edible parts and thus plays an important role in the transfer of As into the food chain. However, the mechanisms of As uptake and its detoxification in rice are not well understood. Recently, members of the Nodulin 26-like intrinsic protein (NIP) subfamily of plant aquaporins were shown to transport arsenite in rice and Arabidopsis. Here we report that members of the rice plasma membrane intrinsic protein (PIP) subfamily are also involved in As tolerance and transport. Based on the homology search with the mammalian AQP9 and yeast Fps1 arsenite transporters, we identified and cloned five rice PIP gene subfamily members. qRT-PCR analysis of PIPs in rice root and shoot tissues revealed a significant down regulation of transcripts encoding OsPIP1;2, OsPIP1;3, OsPIP2;4, OsPIP2;6, and OsPIP2;7 in response to arsenite treatment. Heterologous expression of OsPIP2;4, OsPIP2;6, and OsPIP2;7 in Xenopus laevis oocytes significantly increased the uptake of arsenite. Overexpression of OsPIP2;4, OsPIP2;6, and OsPIP2;7 in Arabidopsis yielded enhanced arsenite tolerance and higher biomass accumulation. Further, these transgenic plants showed no significant accumulation of As in shoot and root tissues in long term uptake assays. Whereas, short duration exposure to arsenite caused both active influx and efflux of As in the roots. The data suggests a bidirectional arsenite permeability of rice PIPs in plants. These rice PIPs genes will be highly useful for engineering important food and biofuel crops for enhanced crop productivity on contaminated soils without increasing the accumulation of toxic As in the biomass or edible tissues. PMID:22350764

  19. 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.

  20. VAMP (synaptobrevin) is present in the plasma membrane of nerve terminals.

    PubMed

    Taubenblatt, P; Dedieu, J C; Gulik-Krzywicki, T; Morel, N

    1999-10-01

    Synaptic vesicle docking and exocytosis require the specific interaction of synaptic vesicle proteins (such as VAMP/synaptobrevin) with presynaptic plasma membrane proteins (such as syntaxin and SNAP 25). These proteins form a stable, SDS-resistant, multimolecular complex, the SNARE complex. The subcellular distribution of VAMP and syntaxin within Torpedo electric organ nerve endings was studied by immunogoldlabeling of SDS-digested freeze-fracture replicas (Fujimoto, 1995). This technique allowed us to visualize large surface areas of the presynaptic plasma membrane and numerous synaptic vesicles from rapidly frozen nerve endings and synaptosomes. VAMP was found associated with synaptic vesicles, as also shown by conventional electron microscopy immunolabeling, and to the presynaptic plasma membrane (P leaflet). Syntaxin was also detected in the nerve ending plasma membrane, without gold labeling of synaptic vesicles. Comparison of gold particle densities suggests that the presynaptic plasma membrane contains 3 VAMP molecules per molecule of syntaxin. After biotinylation of intact synaptosomes, the synaptosomal plasma membrane was isolated on Streptavidin coated magnetic beads. Its antigenic content was compared to that of purified synaptic vesicles. VAMP was present in both membranes whereas syntaxin and SNAP 25 were highly enriched in the synaptosomal plasma membrane. This membrane has a low content of classical synaptic vesicle proteins (synaptophysin, SV2 and the vesicular acetylcholine transporter). The VAMP to syntaxin stoichiometry in the isolated synaptosomal membrane was estimated by comparison with purified antigens and close to 2, in accordance with morphological data. SDS-resistant SNARE complexes were detected in the isolated presynaptic membrane but absent in purified synaptic vesicles. Taken together, these results show that the presence of VAMP in the plasma membrane of nerve endings cannot result from exocytosis of synaptic vesicles, a process

  1. Removal of toxic substances by a selective membrane plasma separator.

    PubMed

    Nakae, Hajime; Hattori, Tomoko; Igarashi, Toshiko; Okuyama, Manabu; Tajimi, Kimitaka

    2014-06-01

    We devised a method of plasma exchange with dialysis (PED), in which selective plasma exchange (sPE) is performed using a selective membrane plasma separator (EC-2A) with an albumin-sieving coefficient of 0.3 while the dialysate flows outside the hollow fibers, and reported the usefulness of the system for treating acute liver failure. Thereafter, EC-4A with an albumin-sieving coefficient of 0.6 was developed, which was expected to be even more effective for removing protein-bound substances. In order to examine whether or not EC-4A might be applicable to blood purification therapy against drug poisoning, we compared the efficacies of sPE, PED, and direct hemoperfusion (DHP) using an activated carbon column for the removal of phenobarbital and lithium. Subjects undergoing the extracorporeal circulation study were assigned to the sPE group, PED group, or DHP group, and the changes in the blood concentrations of phenobarbital and lithium were measured over 180 min. A significant decrease of the phenobarbital concentration over time was seen in the PED group, as compared to that in the sPE group (P < 0.0001), while no significant difference in the concentration was observed between the PED and DHP groups. The PED group showed a significant decrease of the lithium concentration over time, as compared to the DHP group (P < 0.0001), while no significant difference in the concentration was observed between the PED and sPE groups. Thus, PED was as effective as DHP for removing phenobarbital and was as effective as sPE for removing lithium. These results suggest that PED therapy using EC-4A may be a feasible modality for the treatment of drug poisoning. PMID:24965293

  2. 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

    2016-05-01

    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. PMID:26866566

  3. Tetracyclines increase lipid phosphate phosphatase expression on plasma membranes and turnover of plasma lysophosphatidate.

    PubMed

    Tang, Xiaoyun; Zhao, Yuan Y; Dewald, Jay; Curtis, Jonathan M; Brindley, David N

    2016-04-01

    Extracellular lysophosphatidate and sphingosine 1-phosphate (S1P) are important bioactive lipids, which signal through G-protein-coupled receptors to stimulate cell growth and survival. The lysophosphatidate and S1P signals are terminated partly by degradation through three broad-specificity lipid phosphate phosphatases (LPPs) on the cell surface. Significantly, the expression of LPP1 and LPP3 is decreased in many cancers, and this increases the impact of lysophosphatidate and S1P signaling. However, relatively little is known about the physiological or pharmacological regulation of the expression of the different LPPs. We now show that treating several malignant and nonmalignant cell lines with 1 μg/ml tetracycline, doxycycline, or minocycline significantly increased the extracellular degradation of lysophosphatidate. S1P degradation was also increased in cells that expressed high LPP3 activity. These results depended on an increase in the stabilities of the three LPPs and increased expression on the plasma membrane. We tested the physiological significance of these results and showed that treating rats with doxycycline accelerated the clearance of lysophosphatidate, but not S1P, from the circulation. However, administering 100 mg/kg/day doxycycline to mice decreased plasma concentrations of lysophosphatidate and S1P. This study demonstrates a completely new property of tetracyclines in increasing the plasma membrane expression of the LPPs. PMID:26884614

  4. Properties of Plasma Membrane from Pea Root Seedlings under Altered Gravity

    NASA Astrophysics Data System (ADS)

    Klymchuk, D.; Baranenko, V.; Vorobyova, T. V.; Kurylenko, I.; Chyzhykova, O.; Dubovoy, V.

    In this study, the properties of pea (Pisum sativum L.) plasma membrane were examined to determine how the membrane structure and functions are regulated in response to clinorotation (2 rev/min) conditions. Membrane preparations enriched by plasma membrane vesicles were obtained by aqueous two-phase partitioning from 6-day seedling roots. The specific characteristics of H^+-ATPase, lípid composition and peroxidation intensity as well as fluidity of lipid bilayer were analysed. ATP hydrolytic activity was inhibited by ortovanadate and was insensitive to aside and nitrate in sealed plasma membrane vesicles isolated from both clinorotated and control seedlings. Plasma membrane vesicles from clinorotated seedlings in comparison to controls were characterised by increase in the total lipid/protein ratio, ATP hydrolytic activity and intensifying of lipid peroxidation. Sitosterol and campesterol were the predominant free sterol species. Clinorotated seedlings contained a slightly higher level of unsaturated fatty acid than controls. Plasma membrane vesicles were labelled with pyrene and fluorescence originating from monomeric (I_M) molecules and excimeric (I_E) aggregates were measured. The calculated I_E/I_M values were higher in clinorotated seedlings compared with controls reflecting the reduction in membrane microviscosity. The involvement of the changes in plasma membrane lipid content and composition, fluidity and H^+-ATPase activity in response of pea seedlings to altered gravity is discussed.

  5. 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 plasma membranes make them more resilient towards pore formation and lysis of membrane.

  6. The Liver Connexin32 Interactome Is a Novel Plasma Membrane-Mitochondrial Signaling Nexus

    PubMed Central

    2013-01-01

    Connexins are the structural subunits of gap junctions and act as protein platforms for signaling complexes. Little is known about tissue-specific connexin signaling nexuses, given significant challenges associated with affinity-purifying endogenous channel complexes to the level required for interaction analyses. Here, we used multiple subcellular fractionation techniques to isolate connexin32-enriched membrane microdomains from murine liver. We show, for the first time, that connexin32 localizes to both the plasma membrane and inner mitochondrial membrane of hepatocytes. Using a combination of immunoprecipitation-high throughput mass spectrometry, reciprocal co-IP, and subcellular fractionation methodologies, we report a novel interactome validated using null mutant controls. Eighteen connexin32 interacting proteins were identified. The majority represent resident mitochondrial proteins, a minority represent plasma membrane, endoplasmic reticulum, or cytoplasmic partners. In particular, connexin32 interacts with connexin26 and the mitochondrial protein, sideroflexin-1, at the plasma membrane. Connexin32 interaction enhances connexin26 stability. Converging bioinformatic, biochemical, and confocal analyses support a role for connexin32 in transiently tethering mitochondria to connexin32-enriched plasma membrane microdomains through interaction with proteins in the outer mitochondrial membrane, including sideroflexin-1. Complex formation increases the pool of sideroflexin-1 that is present at the plasma membrane. Together, these data identify a novel plasma membrane/mitochondrial signaling nexus in the connexin32 interactome. PMID:23590695

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

    PubMed

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

    2008-12-01

    Plasma membrane repair in animal cells uses synaptotagmin 7, a Ca(2+)-activated membrane fusion protein that mediates delivery of intracellular membranes to wound sites by a mechanism resembling neuronal Ca(2+)-regulated exocytosis. Here, we show that loss of function of the homologous Arabidopsis thaliana Synaptotagmin 1 protein (SYT1) reduces the viability of cells as a consequence of a decrease in the integrity of the plasma membrane. This reduced integrity is enhanced in the syt1-2 null mutant in conditions of osmotic stress likely caused by a defective plasma membrane repair. Consistent with a role in plasma membrane repair, SYT1 is ubiquitously expressed, is located at the plasma membrane, and shares all domains characteristic of animal synaptotagmins (i.e., an N terminus-transmembrane domain and a cytoplasmic region containing two C2 domains with phospholipid binding activities). Our analyses support that membrane trafficking mediated by SYT1 is important for plasma membrane integrity and plant fitness. PMID:19088329

  8. Effects of Vanadate on the Plasma Membrane ATPase of Red Beet and Corn 1

    PubMed Central

    O'Neill, Sharman D.; Spanswick, Roger M.

    1984-01-01

    The effect of vanadate on the plant plasma membrane ATPase were investigated in plasma membrane fractions derived from corn roots (Zea mays L.) and red beets (Beta vulgaris L.). The Ki for vanadate inhibition of the plasma membrane ATPase from corn roots and red beets was between 6 and 15 micromolar vanadate. In both membrane fractions, 80% to 90% of the total ATPase was inhibited at vanadate concentrations below 100 micromolar. Vanadate inhibition was optimal at pH 6.5, enhanced by the presence of K+, and was partially reversed by 1 millimolar EDTA. The Mg:ATP kinetics for the plasma membrane ATPase were hyperbolic in both the absence and presence of vanadate. Vanadate decreased both the Km and Vmax of the red beet plasma membrane ATPase, indicating that vanadate inhibits the ATPase uncompetitively. These results indicate many similarities with respect to vanadate inhibition between the plant plasma membrane ATPase and other major iontranslocating ATPases from fungal and animal cells. The high sensitivity to vanadate reported here, however, differs from other reports of vanadate inhibition of the plant plasma membrane ATPase from corn, beets, and in some instances oats. PMID:16663670

  9. Surface Modification of Polypropylene Membrane by RF Methane/Oxygen Mixture Plasma Treatment

    NASA Astrophysics Data System (ADS)

    Tsai, Ching-Yuan; Juang, Ruey-Shin; Huang, Chun

    2011-08-01

    The hydrophilic surface modification of micro-porous polypropylene (PP) membranes is achieved by low-pressure 13.56 MHz RF methane (CH4)/oxygen (O2) gas mixture plasma treatment. The changes in surface wettability and surface free energy were examined by static contact angle analysis. The static water contact angle of the plasma modified membrane notably decreased with increases in treatment time and plasma power. The obvious increase in the surface energy of polypropylene membranes due to CH4/O2 mixture gas plasma treatments was also observed. Optical emission spectroscopy (OES) was used to analyze the chemical species of CH4/O2 mixture gas plasma treatment. The variations in the surface morphology and chemical structure of the micro-porous PP membranes were confirmed by confocal laser scanning microscopy (CLSM), Fourier transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS) measurements. XPS analysis showed significantly higher surface concentrations of oxygen functional groups for CH4/O2 mixture gas plasma-modified polypropylene membrane surfaces than for the originally unmodified polypropylene membrane surface. The experimental results show the important role of chemical species in the interaction between a CH4/O2 mixture gas plasma and a membrane surface, which can be controlled by surface modification to tailor the hydrophilicity of the membrane to the requirements of various applications.

  10. Responses of hybrid aspen over-expressing a PIP2;5 aquaporin to low root temperature.

    PubMed

    Ranganathan, Kapilan; El Kayal, Walid; Cooke, Janice E K; Zwiazek, Janusz J

    2016-03-15

    Aquaporins mediate the movement of water across cell membranes. Plasma membrane intrinsic protein 2;5 from Populus trichocarpa×deltoides (PtdPIP2;5) was previously demonstrated to be a functionally important water conducting aquaporin. To study the relevance of aquaporin-mediated root water transport at low temperatures, we generated transgenic Populus tremula×alba over-expressing PtdPIP2;5 under control of the maize ubiquitin promoter, and compared the physiological responses and water transport properties of the PtdPIP2;5 over-expressing lines (PtdPIP2;5ox) with wild-type plants. We hypothesized that over-expression of PtdPIP2;5 would reduce temperature sensitivity of root water transport and gas exchange. Decreasing root temperatures to 10 and 5°C significantly decreased hydraulic conductivities (Lp) in wild-type plants, but had no significant effect on Lp in PtdPIP2;5ox plants. Recovery of Lp in the transgenic lines returned to 20°C from 5°C was faster than in the wild-type plants. Low root temperature did not induce major changes in transcript levels for other PIPs. When roots were exposed to 5°C in solution culture and shoots were exposed to 20°C, wild-type plants had significantly lower net photosynthetic and transpiration rates compared to PtdPIP2;5ox plants. Taken together, our results demonstrate that over-expression of PtdPIP2;5 in P. tremula×alba was effective in alleviating the effects of low root temperature on Lp and gas exchange. PMID:26895330

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

    PubMed Central

    Prada, Ilaria; Meldolesi, Jacopo

    2016-01-01

    Exosomes and ectosomes, extracellular vesicles of two types generated by all cells at multivesicular bodies and the plasma membrane, respectively, play critical roles in physiology and pathology. A key mechanism of their function, analogous for both types of vesicles, is the fusion of their membrane to the plasma membrane of specific target cells, followed by discharge to the cytoplasm of their luminal cargo containing proteins, RNAs, and DNA. Here we summarize the present knowledge about the interactions, binding and fusions of vesicles with the cell plasma membrane. The sequence initiates with dynamic interactions, during which vesicles roll over the plasma membrane, followed by the binding of specific membrane proteins to their cell receptors. Membrane binding is then converted rapidly into fusion by mechanisms analogous to those of retroviruses. Specifically, proteins of the extracellular vesicle membranes are structurally rearranged, and their hydrophobic sequences insert into the target cell plasma membrane which undergoes lipid reorganization, protein restructuring and membrane dimpling. Single fusions are not the only process of vesicle/cell interactions. Upon intracellular reassembly of their luminal cargoes, vesicles can be regenerated, released and fused horizontally to other target cells. Fusions of extracellular vesicles are relevant also for specific therapy processes, now intensely investigated. PMID:27517914

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

    PubMed

    Prada, Ilaria; Meldolesi, Jacopo

    2016-01-01

    Exosomes and ectosomes, extracellular vesicles of two types generated by all cells at multivesicular bodies and the plasma membrane, respectively, play critical roles in physiology and pathology. A key mechanism of their function, analogous for both types of vesicles, is the fusion of their membrane to the plasma membrane of specific target cells, followed by discharge to the cytoplasm of their luminal cargo containing proteins, RNAs, and DNA. Here we summarize the present knowledge about the interactions, binding and fusions of vesicles with the cell plasma membrane. The sequence initiates with dynamic interactions, during which vesicles roll over the plasma membrane, followed by the binding of specific membrane proteins to their cell receptors. Membrane binding is then converted rapidly into fusion by mechanisms analogous to those of retroviruses. Specifically, proteins of the extracellular vesicle membranes are structurally rearranged, and their hydrophobic sequences insert into the target cell plasma membrane which undergoes lipid reorganization, protein restructuring and membrane dimpling. Single fusions are not the only process of vesicle/cell interactions. Upon intracellular reassembly of their luminal cargoes, vesicles can be regenerated, released and fused horizontally to other target cells. Fusions of extracellular vesicles are relevant also for specific therapy processes, now intensely investigated. PMID:27517914

  13. 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

  14. Genome-wide expression analysis of rice aquaporin genes and development of a functional gene network mediated by aquaporin expression in roots.

    PubMed

    Nguyen, Minh Xuan; Moon, Sunok; Jung, Ki-Hong

    2013-10-01

    The world population continually faces challenges of water scarcity for agriculture. A common strategy called water-balance control has evolved to adapt plant growth to these challenges. Aquaporins are a family of integral membrane proteins that play a central role in water-balance control. In this study, we identified 34 members of the rice aquaporin gene family, adding a novel member to the previous list. A combination of phylogenetic tree and anatomical meta-expression profiling data consisting of 983 Affymetrix arrays and 209 Agilent 44 K arrays was used to identify tissue-preferred aquaporin genes and evaluate functional redundancy among aquaporin family members. Eight aquaporins showed root-preferred expression in the vegetative growth stage, while 4 showed leaf/shoot-preferred expression. Integrating stress-induced expression patterns into phylogenetic tree and semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed that 3 rice aquaporin genes were markedly downregulated and 4 were upregulated by water deficiency in the root, suggesting that these candidate genes are key regulators of water uptake from the soil. Finally, we constructed a functional network of genes mediated by water stress and refined the network by confirming the differential expression using RT-PCR and real-time PCR. Our data will be useful to elucidate the molecular mechanism of water-balance control in rice root. PMID:23801298

  15. Electrostatics of aquaporin and aquaglyceroporin channels correlates with their transport selectivity

    PubMed Central

    Oliva, Romina; Calamita, Giuseppe; Thornton, Janet M.; Pellegrini-Calace, Marialuisa

    2010-01-01

    Aquaporins are homotetrameric channel proteins, which allow the diffusion of water and small solutes across biological membranes. According to their transport function, aquaporins can be divided into “orthodox aquaporins”, which allow the flux of water molecules only, and “aquaglyceroporins”, which facilitate the diffusion of glycerol and other small solutes in addition to water. The contribution of individual residues in the pore to the selectivity of orthodox aquaporins and aquaglyceroporins is not yet fully understood. To gain insights into aquaporin selectivity, we focused on the sequence variation and electrostatics of their channels. The continuum Poisson-Boltzmann electrostatic potential along the channel was calculated and compared for ten three-dimensional-structures which are representatives of different aquaporin subfamilies, and a panel of functionally characterized mutants, for which high-accuracy three-dimensional-models could be derived. Interestingly, specific electrostatic profiles associated with the main selectivity to water or glycerol could be identified. In particular: (i) orthodox aquaporins showed a distinctive electrostatic potential maximum at the periplasmic side of the channel around the aromatic/Arg (ar/R) constriction site; (ii) aquaporin-0 (AQP0), a mammalian aquaporin with considerably low water permeability, had an additional deep minimum at the cytoplasmic side; (iii) aquaglyceroporins showed a rather flat potential all along the channel; and (iv) the bifunctional protozoan PfAQP had an unusual all negative profile. Evaluation of electrostatics of the mutants, along with a thorough sequence analysis of the aquaporin pore-lining residues, illuminated the contribution of specific residues to the electrostatics of the channels and possibly to their selectivity. PMID:20147624

  16. 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

    2012-01-01

    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

  17. 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

  18. Alterations in the activities of hepatic plasma-membrane and microsomal enzymes during liver regeneration.

    PubMed Central

    Deliconstantinos, G; Ramantanis, G

    1983-01-01

    A marked increase in the activities of rat liver plasma-membrane (Na+ + K+)-stimulated ATPase and microsomal Ca2+-stimulated ATPase was observed 18h after partial hepatectomy. Lipid analyses for both membrane preparations reveal that in partially hepatectomized rats the cholesterol and sphingomyelin content are decreased with a subsequent decrease in the cholesterol/phospholipid molar ratio compared with those of sham-operated animals. Changes in the allosteric properties of plasma-membrane (Na+ + K+)-stimulated ATPase by F- (as reflected by changes in the Hill coefficient) indicated a fluidization of the lipid bilayer of both membrane preparations in 18 h-regenerating liver. The amphipathic dodecyl glucoside incorporated into the hepatic plasma membranes evoked a marked increase in the (Na+ + K+)-stimulated ATPase and 5'-nucleotidase activities. The lack of effect of the glucoside on the Lubrol-PX-solubilized 5'-nucleotidase indicates that changes in the activities of the membrane-bound enzymes caused by the glucoside are due to modulation of the membrane fluidity. Dodecyl glucoside appears to increase the membrane fluidity, evaluated through changes in the Hill coefficient for plasma-membrane (Na+ + K+)-stimulated ATPase. The biological significance of these data is discussed in terms of the differences and changes in the interaction of membrane-bound enzymes with membrane lipids during liver regeneration. PMID:6309144

  19. Optimizing water permeability through the hourglass shape of aquaporins

    PubMed Central

    Gravelle, Simon; Joly, Laurent; Detcheverry, François; Ybert, Christophe; Cottin-Bizonne, Cécile; Bocquet, Lydéric

    2013-01-01

    The ubiquitous aquaporin channels are able to conduct water across cell membranes, combining the seemingly antagonist functions of a very high selectivity with a remarkable permeability. Whereas molecular details are obvious keys to perform these tasks, the overall efficiency of transport in such nanopores is also strongly limited by viscous dissipation arising at the connection between the nanoconstriction and the nearby bulk reservoirs. In this contribution, we focus on these so-called entrance effects and specifically examine whether the characteristic hourglass shape of aquaporins may arise from a geometrical optimum for such hydrodynamic dissipation. Using a combination of finite-element calculations and analytical modeling, we show that conical entrances with suitable opening angle can indeed provide a large increase of the overall channel permeability. Moreover, the optimal opening angles that maximize the permeability are found to compare well with the angles measured in a large variety of aquaporins. This suggests that the hourglass shape of aquaporins could be the result of a natural selection process toward optimal hydrodynamic transport. Finally, in a biomimetic perspective, these results provide guidelines to design artificial nanopores with optimal performances. PMID:24067650

  20. Optimizing water permeability through the hourglass shape of aquaporins.

    PubMed

    Gravelle, Simon; Joly, Laurent; Detcheverry, François; Ybert, Christophe; Cottin-Bizonne, Cécile; Bocquet, Lydéric

    2013-10-01

    The ubiquitous aquaporin channels are able to conduct water across cell membranes, combining the seemingly antagonist functions of a very high selectivity with a remarkable permeability. Whereas molecular details are obvious keys to perform these tasks, the overall efficiency of transport in such nanopores is also strongly limited by viscous dissipation arising at the connection between the nanoconstriction and the nearby bulk reservoirs. In this contribution, we focus on these so-called entrance effects and specifically examine whether the characteristic hourglass shape of aquaporins may arise from a geometrical optimum for such hydrodynamic dissipation. Using a combination of finite-element calculations and analytical modeling, we show that conical entrances with suitable opening angle can indeed provide a large increase of the overall channel permeability. Moreover, the optimal opening angles that maximize the permeability are found to compare well with the angles measured in a large variety of aquaporins. This suggests that the hourglass shape of aquaporins could be the result of a natural selection process toward optimal hydrodynamic transport. Finally, in a biomimetic perspective, these results provide guidelines to design artificial nanopores with optimal performances. PMID:24067650

  1. MzPIP2;1: An Aquaporin Involved in Radial Water Movement in Both Water Uptake and Transportation, Altered the Drought and Salt Tolerance of Transgenic Arabidopsis

    PubMed Central

    Lei, Qiong; Feng, Chao; Gao, Yinan; Zheng, Xiaodong; Zhao, Yu; Wang, Zhi; Kong, Jin

    2015-01-01

    Background Plants are unavoidably subjected to various abiotic stressors, including high salinity, drought and low temperature, which results in water deficit and even death. Water uptake and transportation play a critical role in response to these stresses. Many aquaporin proteins, localized at different tissues, function in various transmembrane water movements. We targeted at the key aquaporin in charge of both water uptake in roots and radial water transportation from vascular tissues through the whole plant. Results The MzPIP2;1 gene encoding a plasma membrane intrinsic protein was cloned from salt-tolerant apple rootstock Malus zumi Mats. The GUS gene was driven by MzPIP2;1 promoter in transgenic Arabidopsis. It indicated that MzPIP2;1 might function in the epidermal and vascular cells of roots, parenchyma cells around vessels through the stems and vascular tissues of leaves. The ectopically expressed MzPIP2;1 conferred the transgenic Arabidopsis plants enhanced tolerance to slight salt and drought stresses, but sensitive to moderate salt stress, which was indicated by root length, lateral root number, fresh weight and K+/Na+ ratio. In addition, the possible key cis-elements in response to salt, drought and cold stresses were isolated by the promoter deletion experiment. Conclusion The MzPIP2;1 protein, as a PIP2 aquaporins subgroup member, involved in radial water movement, controls water absorption and usage efficiency and alters transgenic plants drought and salt tolerance. PMID:26562158

  2. The speed of swelling kinetics modulates cell volume regulation and calcium signaling in astrocytes: A different point of view on the role of aquaporins.

    PubMed

    Mola, Maria Grazia; Sparaneo, Angelo; Gargano, Concetta Domenica; Spray, David C; Svelto, Maria; Frigeri, Antonio; Scemes, Eliana; Nicchia, Grazia Paola

    2016-01-01

    Regulatory volume decrease (RVD) is a process by which cells restore their original volume in response to swelling. In this study, we have focused on the role played by two different Aquaporins (AQPs), Aquaporin-4 (AQP4), and Aquaporin-1 (AQP1), in triggering RVD and in mediating calcium signaling in astrocytes under hypotonic stimulus. Using biophysical techniques to measure water flux through the plasma membrane of wild-type (WT) and AQP4 knockout (KO) astrocytes and of an astrocyte cell line (DI TNC1) transfected with AQP4 or AQP1, we here show that AQP-mediated fast swelling kinetics play a key role in triggering and accelerating RVD. Using calcium imaging, we show that AQP-mediated fast swelling kinetics also significantly increases the amplitude of calcium transients inhibited by Gadolinium and Ruthenium Red, two inhibitors of the transient receptor potential vanilloid 4 (TRPV4) channels, and prevented by removing extracellular calcium. Finally, inhibition of TRPV4 or removal of extracellular calcium does not affect RVD. All together our study provides evidence that (1) AQP influenced swelling kinetics is the main trigger for RVD and in mediating calcium signaling after hypotonic stimulus together with TRPV4, and (2) calcium influx from the extracellular space and/or TRPV4 are not essential for RVD to occur in astrocytes. PMID:26413835

  3. Controlled change of transport properties of poly(ethylene terephthalate) track membranes by plasma method

    NASA Astrophysics Data System (ADS)

    Kravets, L. I.; Dmitriev, S. N.; Drachev, A. I.; Gilman, A. B.; Lazea, A.; Dinescu, G.

    2007-04-01

    A process of plasma polymerization of dimethylaniline and acrylic acid vapours on the surface of poly(ethylene terephthalate) track membranes has been investigated. The surface and hydrodynamic properties of the composite membranes produced in this case have been studied. It is shown that the water permeability of the obtained polymeric membranes can be controlled by changing the filtrate pH. Membranes with such properties can be used for controllable drug delivery and in sensor control.

  4. Detergent-free domain isolated from Xenopus egg plasma membrane with properties similar to those of detergent-resistant membranes.

    PubMed

    Luria, Ayala; Vegelyte-Avery, Vaida; Stith, Brad; Tsvetkova, Nelly M; Wolkers, Willem F; Crowe, John H; Tablin, Fern; Nuccitelli, Richard

    2002-11-01

    Microdomains known as "rafts" have been isolated from many cell types as detergent-resistant membranes (DRMs) and are enriched in sphingolipids and cholesterol. However, there has been considerable controversy over whether such domains are found in native membranes or are artificially generated by the purification procedure. This controversy is based at least in part on the fact that raft membranes were first detected following detergent extraction in the cold. We isolated two plasma membrane fractions, without detergent treatment, using a discontinuous sucrose density gradient. One fraction was designated "light" and the other "heavy." These fractions were compared with DRMs, which were isolated in the presence of 1% Triton X-100. We found that Xenopus DRMs are enriched with sphingomyelin and cholesterol and exhibit a phase state similar to the liquid-ordered phase. Comparison of DRM complexes with the light and heavy plasma membrane fractions revealed some physical and biochemical similarities between the light fraction of the plasma membrane and the DRM complexes, based on (1) the phosphatidylcholine/sphingomyelin ratio and (2) the protein composition visualized on a two-dimensional gel. These two fractions are also quite similar in their thermotropic phase behavior, and their high levels of ganglioside GM1. We conclude that the light membrane fraction isolated in a detergent-free environment has many of the characteristics normally associated with DRMs. PMID:12403620

  5. Plasma membrane growth during the cell cycle: unsolved mysteries and recent progress

    PubMed Central

    McCusker, Derek; Kellogg, Douglas R.

    2012-01-01

    Growth of the plasma membrane is as fundamental to cell reproduction as DNA replication, chromosome segregation and ribosome biogenesis, yet little is known about the underlying mechanisms. Membrane growth during the cell cycle requires mechanisms that control the initiation, location, and extent of membrane growth, as well as mechanisms that coordinate membrane growth with cell cycle progression. Recent experiments have established links between membrane growth and core cell cycle regulators. Further analysis of these links will yield insights into conserved and fundamental mechanisms of cell growth. A better understanding of the post-Golgi pathways by which membrane growth occurs will be essential for future progress. PMID:23141634

  6. LIPID RAFTS, FLUID/FLUID PHASE SEPARATION, AND THEIR RELEVANCE TO PLASMA MEMBRANE STRUCTURE AND FUNCTION

    PubMed Central

    Sengupta, Prabuddha; Baird, Barbara; Holowka, David

    2007-01-01

    Novel biophysical approaches combined with modeling and new biochemical data have helped to recharge the lipid raft field and have contributed to the generation of a refined model of plasma membrane organization. In this review, we summarize new information in the context of previous literature to provide new insights into the spatial organization and dynamics of lipids and proteins in the plasma membrane of live cells. Recent findings of large-scale separation of liquid-ordered and liquid-disordered phases in plasma membrane vesicles demonstrate this capacity within the complex milieu of plasma membrane proteins and lipids. Roles for membrane heterogeneity and reorganization in immune cell activation are discussed in light of this new information. PMID:17764993

  7. Effect of plasma membrane fluidity on serotonin transport by endothelial cells

    SciTech Connect

    Block, E.R.; Edwards, D. )

    1987-11-01

    To evaluate the effect of plasma membrane fluidity of lung endothelial cells on serotonin transport, porcine pulmonary artery endothelial cells were incubated for 3 h with either 0.1 mM cholesterol hemisuccinate, 0.1 mM cis-vaccenic acid, or vehicle (control), after which plasma membrane fluidity and serotinin transport were measured. Fluorescence spectroscopy was used to measure fluidity in the plasma membrane. Serotonin uptake was calculated from the disappearance of ({sup 14}C)-serotonin from the culture medium. Cholesterol decreased fluidity in the subpolar head group and central and midacyl side-chain regions of the plasma membrane and decreased serotonin transport, whereas cis-vaccenic acid increased fluidity in the central and midacyl side-chain regions of the plasma membrane and also increased serotonin transport. Cis-vaccenic acid had no effect of fluidity in the subpolar head group region of the plasma membrane. These results provide evidence that the physical state of the central and midacyl chains within the pulmonary artery endothelial cell plasma membrane lipid bilayer modulates transmembrane transport of serotonin by these cells.

  8. The Arabidopsis Abiotic Stress-Induced TSPO-Related Protein Reduces Cell-Surface Expression of the Aquaporin PIP2;7 through Protein-Protein Interactions and Autophagic Degradation[C][W][OPEN

    PubMed Central

    Hachez, Charles; Veljanovski, Vasko; Reinhardt, Hagen; Guillaumot, Damien; Vanhee, Celine; Chaumont, François

    2014-01-01

    The Arabidopsis thaliana multi-stress regulator TSPO is transiently induced by abiotic stresses. The final destination of this polytopic membrane protein is the Golgi apparatus, where its accumulation is strictly regulated, and TSPO is downregulated through a selective autophagic pathway. TSPO-related proteins regulate the physiology of the cell by generating functional protein complexes. A split-ubiquitin screen for potential TSPO interacting partners uncovered a plasma membrane aquaporin, PIP2;7. Pull-down assays and fluorescence imaging approaches revealed that TSPO physically interacts with PIP2;7 at the endoplasmic reticulum and Golgi membranes in planta. Intriguingly, constitutive expression of fluorescently tagged PIP2;7 in TSPO-overexpressing transgenic lines resulted in patchy distribution of the fluorescence, reminiscent of the pattern of constitutively expressed yellow fluorescent protein-TSPO in Arabidopsis. Mutational stabilization of TSPO or pharmacological inhibition of the autophagic pathway affected concomitantly the detected levels of PIP2;7, suggesting that the complex containing both proteins is degraded through the autophagic pathway. Coexpression of TSPO and PIP2;7 resulted in decreased levels of PIP2;7 in the plasma membrane and abolished the membrane water permeability mediated by transgenic PIP2;7. Taken together, these data support a physiological role for TSPO in regulating the cell-surface expression of PIP2;7 during abiotic stress conditions through protein-protein interaction and demonstrate an aquaporin regulatory mechanism involving TSPO. PMID:25538184

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

    SciTech Connect

    Hayashi, Masami; Shimada, Yukiko; Inomata, Mitsushi; Ohno-Iwashita, Yoshiko . E-mail: iwashita@tmig.or.jp

    2006-12-22

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

  10. Microtubule Motors Power Plasma Membrane Tubulation in Clathrin-Independent Endocytosis

    PubMed Central

    Day, Charles A; Baetz, Nicholas W; Copeland, Courtney A; Kraft, Lewis J; Han, Bing; Tiwari, Ajit; Drake, Kimberly R; De Luca, Heidi; Chinnapen, Daniel J-F; Davidson, Michael W; Holmes, Randall K; Jobling, Michael G; Schroer, Trina A; Lencer, Wayne I; Kenworthy, Anne K

    2015-01-01

    How the plasma membrane is bent to accommodate clathrin-independent endocytosis remains uncertain. Recent studies suggest Shiga and cholera toxin induce membrane curvature required for their uptake into clathrin-independent carriers by binding and cross-linking multiple copies of their glycosphingolipid receptors on the plasma membrane. But it remains unclear if toxin-induced sphingolipid crosslinking provides sufficient mechanical force for deforming the plasma membrane, or if host cell factors also contribute to this process. To test this, we imaged the uptake of cholera toxin B-subunit into surface-derived tubular invaginations. We found that cholera toxin mutants that bind to only one glycosphingolipid receptor accumulated in tubules, and that toxin binding was entirely dispensable for membrane tubulations to form. Unexpectedly, the driving force for tubule extension was supplied by the combination of microtubules, dynein and dynactin, thus defining a novel mechanism for generating membrane curvature during clathrin-independent endocytosis. PMID:25690058

  11. Organization of cGMP sensing structures on the rod photoreceptor outer segment plasma membrane

    PubMed Central

    Nemet, Ina; Tian, Guilian; Imanishi, Yoshikazu

    2014-01-01

    A diffusion barrier segregates the plasma membrane of the rod photoreceptor outer segment into 2 domains; one which is optimized for the conductance of ions in the phototransduction cascade and another for disk membrane synthesis. We propose the former to be named “phototransductive plasma membrane domain," and the latter to be named “disk morphogenic plasma membrane domain." Within the phototransductive plasma membrane, cGMP-gated channels are concentrated in striated membrane features, which are proximally located to the sites of active cGMP production within the disk membranes. For proper localization of cGMP-gated channel to the phototransductive plasma membrane, the glutamic acid-rich protein domain encoded in the β subunit plays a critical role. Quantitative study suggests that the disk morphogenic domain likely plays an important role in enriching rhodopsin prior to its sequestration into closed disk membranes. Thus, this and our previous studies provide new insight into the mechanism that spatially organizes the vertebrate phototransduction cascade. PMID:25616687

  12. Urinary concentrating mechanism and Aquaporin-2 abundance in rats chronically treated with aluminum lactate.

    PubMed

    Mahieu, Stella; Millen, Néstor; Contini, María del Carmen; Gonzalez, Marcela; Molinas, Sara M; Elías, María Mónica

    2006-06-15

    The aim of this work was to study the effects of chronic administration of aluminum (Al) on the urinary concentrating and diluting mechanisms in the distal tubules and collecting ducts. Male Wistar rats were chronically treated with aluminum lactate for 12 weeks (0.575 mg Al/100g of body weight, i.p., three times per week). After 12 weeks, renal function of control and Al-treated rats was evaluated by clearance techniques. To study urinary concentrating mechanisms, renal function was also measured in control and Al-treated rats deprived of water, after the administration of desmopressin (vasopressin agonist) and after the infusion of hypertonic saline at increasing infusion rates. Sodium and water balance were impaired. We found decreased urinary concentrating ability in situations in which endogenous (thirst or infusion of hypertonic saline) or exogenous plasma antidiuretic hormone was increased. Solute-free water formation, measured during the infusion of hypotonic saline showed normal transport in the thick ascending limb. Aquaporin-2 (AQP2) expression was measured by Western blot to evaluate water permeability in collecting ducts. We found that Al produced downregulation of AQP2 in plasma membranes and intracellular vesicles, that could account for the impaired water handling. Administration of desmopressin increased AQP2 in plasma membranes, suggesting that Al did not impair trafficking of this protein, but could interfere with AQP2 synthesis. PMID:16675087

  13. Role of Phosphatidylinositol 4,5-Bisphosphate in Regulating EHD2 Plasma Membrane Localization

    PubMed Central

    Simone, Laura C.; Caplan, Steve; Naslavsky, Naava

    2013-01-01

    The four mammalian C-terminal Eps15 homology domain-containing proteins (EHD1-EHD4) play pivotal roles in endocytic membrane trafficking. While EHD1, EHD3 and EHD4 associate with intracellular tubular/vesicular membranes, EHD2 localizes to the inner leaflet of the plasma membrane. Currently, little is known about the regulation of EHD2. Thus, we sought to define the factors responsible for EHD2’s association with the plasma membrane. The subcellular localization of endogenous EHD2 was examined in HeLa cells using confocal microscopy. Although EHD partner proteins typically mediate EHD membrane recruitment, EHD2 was targeted to the plasma membrane independent of two well-characterized binding proteins, syndapin2 and EHBP1. Additionally, the EH domain of EHD2, which facilitates canonical EHD protein interactions, was not required to direct overexpressed EHD2 to the cell surface. On the other hand, several lines of evidence indicate that the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) plays a crucial role in regulating EHD2 subcellular localization. Pharmacologic perturbation of PIP2 metabolism altered PIP2 plasma membrane distribution (as assessed by confocal microscopy), and caused EHD2 to redistribute away from the plasma membrane. Furthermore, overexpressed EHD2 localized to PIP2-enriched vacuoles generated by active Arf6. Finally, we show that although cytochalasin D caused actin microfilaments to collapse, EHD2 was nevertheless maintained at the plasma membrane. Intriguingly, cytochalasin D induced relocalization of both PIP2 and EHD2 to actin aggregates, supporting a role of PIP2 in controlling EHD2 subcellular localization. Altogether, these studies emphasize the significance of membrane lipid composition for EHD2 subcellular distribution and offer new insights into the regulation of this important endocytic protein. PMID:24040268

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

    NASA Astrophysics Data System (ADS)

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

    2011-09-01

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

  15. Inhibition of Acid Sphingomyelinase Depletes Cellular Phosphatidylserine and Mislocalizes K-Ras from the Plasma Membrane.

    PubMed

    Cho, Kwang-Jin; van der Hoeven, Dharini; Zhou, Yong; Maekawa, Masashi; Ma, Xiaoping; Chen, Wei; Fairn, Gregory D; Hancock, John F

    2015-01-01

    K-Ras must localize to the plasma membrane for biological activity; thus, preventing plasma membrane interaction blocks K-Ras signal output. Here we show that inhibition of acid sphingomyelinase (ASM) mislocalizes both the K-Ras isoforms K-Ras4A and K-Ras4B from the plasma membrane to the endomembrane and inhibits their nanoclustering. We found that fendiline, a potent ASM inhibitor, reduces the phosphatidylserine (PtdSer) and cholesterol content of the inner plasma membrane. These lipid changes are causative because supplementation of fendiline-treated cells with exogenous PtdSer rapidly restores K-Ras4A and K-Ras4B plasma membrane binding, nanoclustering, and signal output. Conversely, supplementation with exogenous cholesterol restores K-Ras4A but not K-Ras4B nanoclustering. These experiments reveal different operational pools of PtdSer on the plasma membrane. Inhibition of ASM elevates cellular sphingomyelin and reduces cellular ceramide levels. Concordantly, delivery of recombinant ASM or exogenous ceramide to fendiline-treated cells rapidly relocalizes K-Ras4B and PtdSer to the plasma membrane. K-Ras4B mislocalization is also recapitulated in ASM-deficient Neimann-Pick type A and B fibroblasts. This study identifies sphingomyelin metabolism as an indirect regulator of K-Ras4A and K-Ras4B signaling through the control of PtdSer plasma membrane content. It also demonstrates the critical and selective importance of PtdSer to K-Ras4A and K-Ras4B plasma membrane binding and nanoscale spatial organization. PMID:26572827

  16. Inhibition of Acid Sphingomyelinase Depletes Cellular Phosphatidylserine and Mislocalizes K-Ras from the Plasma Membrane

    PubMed Central

    Cho, Kwang-jin; van der Hoeven, Dharini; Zhou, Yong; Maekawa, Masashi; Ma, Xiaoping; Chen, Wei

    2015-01-01

    K-Ras must localize to the plasma membrane for biological activity; thus, preventing plasma membrane interaction blocks K-Ras signal output. Here we show that inhibition of acid sphingomyelinase (ASM) mislocalizes both the K-Ras isoforms K-Ras4A and K-Ras4B from the plasma membrane to the endomembrane and inhibits their nanoclustering. We found that fendiline, a potent ASM inhibitor, reduces the phosphatidylserine (PtdSer) and cholesterol content of the inner plasma membrane. These lipid changes are causative because supplementation of fendiline-treated cells with exogenous PtdSer rapidly restores K-Ras4A and K-Ras4B plasma membrane binding, nanoclustering, and signal output. Conversely, supplementation with exogenous cholesterol restores K-Ras4A but not K-Ras4B nanoclustering. These experiments reveal different operational pools of PtdSer on the plasma membrane. Inhibition of ASM elevates cellular sphingomyelin and reduces cellular ceramide levels. Concordantly, delivery of recombinant ASM or exogenous ceramide to fendiline-treated cells rapidly relocalizes K-Ras4B and PtdSer to the plasma membrane. K-Ras4B mislocalization is also recapitulated in ASM-deficient Neimann-Pick type A and B fibroblasts. This study identifies sphingomyelin metabolism as an indirect regulator of K-Ras4A and K-Ras4B signaling through the control of PtdSer plasma membrane content. It also demonstrates the critical and selective importance of PtdSer to K-Ras4A and K-Ras4B plasma membrane binding and nanoscale spatial organization. PMID:26572827

  17. Use of Aquaporins to Achieve Needed Water Purity On ISS for the EMU Space Suit System

    NASA Technical Reports Server (NTRS)

    Hill, Terry; Taylor ,Brandon W.

    2012-01-01

    Use of Aquaporins to Achieve Needed Water Purity On ISS for the EMU Space Suit System. With the U.S. Space Shuttle fleet retired, the supply of extremely high-quality water "super-Q" - required for the EMU Space suit cooling on this ISS - will become a significant operational hardware challenge in the very near future. A proposed potential solution is the use of a filtration system consisting of a semi-permeable membrane embedded with aquaporin proteins. Aquaporins are a special class of trans-membrane proteins that facilitate passive transport of water and other substances across a membrane. The specificity of these proteins is such that only water is allowed through the protein structure, and this novel property invites their adaptation for use in water filtration systems, specifically usage on the ISS for the EMU space suit system. These proteins are found in many living systems and have been developed for commercial use today.

  18. Deposition of Lanthanum Strontium Cobalt Ferrite (LSCF) Using Suspension Plasma Spraying for Oxygen Transport Membrane Applications

    NASA Astrophysics Data System (ADS)

    Fan, E. S. C.; Kesler, O.

    2015-08-01

    Suspension plasma spray deposition was utilized to fabricate dense lanthanum strontium cobalt ferrite oxygen separation membranes (OSMs) on porous metal substrates for mechanical support. The as-sprayed membranes had negligible and/or reversible material decomposition. At the longer stand-off distance (80 mm), smooth and dense membranes could be manufactured using a plasma with power below approximately 81 kW. Moreover, a membrane of 55 μm was observed to have very low gas leakage rates desirable for OSM applications. This thickness could potentially be decreased further to improve oxygen diffusion by using metal substrates with finer surface pores.

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

    PubMed Central

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

    2014-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-07-01

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

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

    SciTech Connect

    Hasan, Nazarul; Hu, Chuan

    2010-01-01

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

  2. Aquaporins and Brain Tumors.

    PubMed

    Maugeri, Rosario; Schiera, Gabriella; Di Liegro, Carlo Maria; Fricano, Anna; Iacopino, Domenico Gerardo; Di Liegro, Italia

    2016-01-01

    Brain primary tumors are among the most diverse and complex human cancers, and they are normally classified on the basis of the cell-type and/or the grade of malignancy (the most malignant being glioblastoma multiforme (GBM), grade IV). Glioma cells are able to migrate throughout the brain and to stimulate angiogenesis, by inducing brain capillary endothelial cell proliferation. This in turn causes loss of tight junctions and fragility of the blood-brain barrier, which becomes leaky. As a consequence, the most serious clinical complication of glioblastoma is the vasogenic brain edema. Both glioma cell migration and edema have been correlated with modification of the expression/localization of different isoforms of aquaporins (AQPs), a family of water channels, some of which are also involved in the transport of other small molecules, such as glycerol and urea. In this review, we discuss relationships among expression/localization of AQPs and brain tumors/edema, also focusing on the possible role of these molecules as both diagnostic biomarkers of cancer progression, and therapeutic targets. Finally, we will discuss the possibility that AQPs, together with other cancer promoting factors, can be exchanged among brain cells via extracellular vesicles (EVs). PMID:27367682

  3. Aquaporins and Brain Tumors

    PubMed Central

    Maugeri, Rosario; Schiera, Gabriella; Di Liegro, Carlo Maria; Fricano, Anna; Iacopino, Domenico Gerardo; Di Liegro, Italia

    2016-01-01

    Brain primary tumors are among the most diverse and complex human cancers, and they are normally classified on the basis of the cell-type and/or the grade of malignancy (the most malignant being glioblastoma multiforme (GBM), grade IV). Glioma cells are able to migrate throughout the brain and to stimulate angiogenesis, by inducing brain capillary endothelial cell proliferation. This in turn causes loss of tight junctions and fragility of the blood–brain barrier, which becomes leaky. As a consequence, the most serious clinical complication of glioblastoma is the vasogenic brain edema. Both glioma cell migration and edema have been correlated with modification of the expression/localization of different isoforms of aquaporins (AQPs), a family of water channels, some of which are also involved in the transport of other small molecules, such as glycerol and urea. In this review, we discuss relationships among expression/localization of AQPs and brain tumors/edema, also focusing on the possible role of these molecules as both diagnostic biomarkers of cancer progression, and therapeutic targets. Finally, we will discuss the possibility that AQPs, together with other cancer promoting factors, can be exchanged among brain cells via extracellular vesicles (EVs). PMID:27367682

  4. Aquaporin-4 autoimmunity

    PubMed Central

    Zekeridou, Anastasia

    2015-01-01

    Neuromyelitis optica (NMO) and a related spectrum of inflammatory CNS disorders are unified by detection of a serum autoantibody specific for the aquaporin-4 (AQP4) water channel, which is abundant in astrocytic foot processes. The classic clinical manifestations of NMO are optic neuritis and longitudinally extensive transverse myelitis. Newly recognized manifestations of AQP4 autoimmunity include lesions of circumventricular organs and skeletal muscle. NMO is commonly relapsing, is frequently accompanied by other autoimmune disorders, and sometimes occurs in a paraneoplastic context. The goals of treatment are to minimize neurologic disability in the acute attack and thereafter to prevent relapses and cumulative disability. The disease specificity of AQP4 immunoglobulin (Ig) G approaches 100% using optimized molecular-based detection assays. Clinical, immunohistopathologic, and in vitro evidence support this antibody being central to NMO pathogenesis. Current animal models yield limited histopathologic characteristics of NMO, with no clinical deficits to date. Recent descriptions of a myelin oligodendrocyte glycoprotein autoantibody in a minority of patients with NMO spectrum phenotype who lack AQP4-IgG predict serologic delineation of additional distinctive disease entities. PMID:26185772

  5. Tonoplast Aquaporins Facilitate Lateral Root Emergence1[OPEN

    PubMed Central

    Hachez, Charles; Bienert, Manuela Désirée; Beebo, Azeez; Swarup, Kamal

    2016-01-01

    Aquaporins (AQPs) are water channels allowing fast and passive diffusion of water across cell membranes. It was hypothesized that AQPs contribute to cell elongation processes by allowing water influx across the plasma membrane and the tonoplast to maintain adequate turgor pressure. Here, we report that, in Arabidopsis (Arabidopsis thaliana), the highly abundant tonoplast AQP isoforms AtTIP1;1, AtTIP1;2, and AtTIP2;1 facilitate the emergence of new lateral root primordia (LRPs). The number of lateral roots was strongly reduced in the triple tip mutant, whereas the single, double, and triple tip mutants showed no or minor reduction in growth of the main root. This phenotype was due to the retardation of LRP emergence. Live cell imaging revealed that tight spatiotemporal control of TIP abundance in the tonoplast of the different LRP cells is pivotal to mediating this developmental process. While lateral root emergence is correlated to a reduction of AtTIP1;1 and AtTIP1;2 protein levels in LRPs, expression of AtTIP2;1 is specifically needed in a restricted cell population at the base, then later at the flanks, of developing LRPs. Interestingly, the LRP emergence phenotype of the triple tip mutants could be fully rescued by expressing AtTIP2;1 under its native promoter. We conclude that TIP isoforms allow the spatial and temporal fine-tuning of cellular water transport, which is critically required during the highly regulated process of LRP morphogenesis and emergence. PMID:26802038

  6. Using plasma membrane nanoclusters to build better signaling circuits.

    PubMed

    Harding, Angus S; Hancock, John F

    2008-08-01

    Cellular signaling pathways do not simply transmit data; they integrate and process signals to operate as switches, oscillators, logic gates, memory modules and many other types of control system. These complex processing capabilities enable cells to respond appropriately to the myriad of external cues that direct growth and development. The idea that crosstalk and feedback loops are used as control systems in biological signaling networks is well established. Signaling networks are also subject to exquisite spatial regulation, yet how spatial control modulates signal outputs is less well understood. Here, we explore the spatial organization of two different signal transduction circuits: receptor tyrosine kinase activation of the mitogen-activated protein kinase module; and glycosylphosphatidylinositol-anchored receptor activation of phospholipase C. With regards to these pathways, recent results have refocused attention on the crucial role of lipid rafts and plasma membrane nanodomains in signal transmission. We identify common design principals that highlight how the spatial organization of signal transduction circuits can be used as a fundamental control mechanism to modulate system outputs in vivo. PMID:18620858

  7. Cocaine induction of dopamine transporter trafficking to the plasma membrane.

    PubMed

    Little, Karley Y; Elmer, Lawrence W; Zhong, Huailing; Scheys, Joshua O; Zhang, Lian

    2002-02-01

    Several previous human postmortem experiments have detected an increase in striatal [(3)H]WIN 35428 binding to the dopamine transporter (DAT) in chronic cocaine users. However, animal experiments have found considerable variability in DAT radioligand binding levels in brain after cocaine administration, perhaps caused by length and dose of treatment and type of radioligand used. The present experiments tested the hypothesis that [(3)H]WIN 35428 binding and [(3)H]dopamine uptake would be increased by exposure to cocaine through alterations in DAT cellular trafficking, rather than increased protein synthesis. Experiments were conducted in stably hDAT-transfected N2A cells and assessed the dose response and time course of cocaine effects on [(3)H]WIN 35428 binding to the DAT, [(3)H]dopamine uptake, measures of DAT protein and mRNA, as well as DAT subcellular location. Cocaine doses of 10(-6) M caused statistically significant increases in [(3)H]WIN 35428 binding and [(3)H]dopamine uptake after 12 and 3 h, respectively. Despite these increases in DAT function, there was no change in DAT total protein or mRNA. Immunofluorescence and biotinylation experiments indicated that cocaine treatment induced increases in plasma membrane DAT immunoreactivity and intracellular decreases. The present model system may further our understanding of regulatory alterations in DAT radioligand binding and function caused by cocaine exposure. PMID:11809869

  8. Plant cell plasma membrane structure and properties under clinostatting

    NASA Astrophysics Data System (ADS)

    Polulakh, Yu. A.; Zhadko, S. I.; Klimchuk, D. A.; Baraboy, V. A.; Alpatov, A. N.; Sytnik, K. M.

    Structural-functional organization of plasma membrane of pea roots seedling was investigated by methods of chemiluminescence, fluorescence probes, chromatography and freeze-fracture studies under normal conditions and clinostatting. Phase character of lipid peroxidation intensity was fixed. The initial phase of this process is characterized by lipid peroxidation decreasing with its next induction. The primary changes depending on free-radical mechanisms of lipid peroxidation were excellently revealed by chemiluminescence. Plasmalemma microviscosity increased on the average of 15-20 % under microgravity at the initial stages of its phenomenon. There were major changes of phosphatidilcholine and phosphatidilethanolamine contents. The total quantity of phospholipids remained rather stable. Changes of phosphatide acid concentration point to degradation and phospholipids biosynthesis. There were increases of unsaturated fatty acids mainly at the expense of linoleic and linolenic acids and also a decrease of saturated fatty acid content at the expense of palmitic and stearic acids. Unsaturation index of fatty acids increased as well. On the whole fatty acid composition was variable in comparison with phospholipids. Probably it is one of mechanisms of maintaining of microviscosity within definite limits. Considerable structural changes in organization of plasmalemma protein-lipid complex were not revealed by the freeze-fracture studies.

  9. Inhomogeneity Based Characterization of Distribution Patterns on the Plasma Membrane.

    PubMed

    Paparelli, Laura; Corthout, Nikky; Pavie, Benjamin; Wakefield, Devin L; Sannerud, Ragna; Jovanovic-Talisman, Tijana; Annaert, Wim; Munck, Sebastian

    2016-09-01

    Cell surface protein and lipid molecules are organized in various patterns: randomly, along gradients, or clustered when segregated into discrete micro- and nano-domains. Their distribution is tightly coupled to events such as polarization, endocytosis, and intracellular signaling, but challenging to quantify using traditional techniques. Here we present a novel approach to quantify the distribution of plasma membrane proteins and lipids. This approach describes spatial patterns in degrees of inhomogeneity and incorporates an intensity-based correction to analyze images with a wide range of resolutions; we have termed it Quantitative Analysis of the Spatial distributions in Images using Mosaic segmentation and Dual parameter Optimization in Histograms (QuASIMoDOH). We tested its applicability using simulated microscopy images and images acquired by widefield microscopy, total internal reflection microscopy, structured illumination microscopy, and photoactivated localization microscopy. We validated QuASIMoDOH, successfully quantifying the distribution of protein and lipid molecules detected with several labeling techniques, in different cell model systems. We also used this method to characterize the reorganization of cell surface lipids in response to disrupted endosomal trafficking and to detect dynamic changes in the global and local organization of epidermal growth factor receptors across the cell surface. Our findings demonstrate that QuASIMoDOH can be used to assess protein and lipid patterns, quantifying distribution changes and spatial reorganization at the cell surface. An ImageJ/Fiji plugin of this analysis tool is provided. PMID:27603951

  10. Molecular characterisation of plasma membrane-derived vesicles.

    PubMed

    Antwi-Baffour, Samuel S

    2015-01-01

    Plasma membrane-derived vesicles (PMVs) are released into circulation in response to normal and stress/pathogenic conditions. They are of tremendous significance for the prediction, diagnosis, and observation of the therapeutic success of many diseases. Knowledge of their molecular characteristics and therefore functional properties would contribute to a better understanding of the pathological mechanisms leading to various diseases in which their levels are raised. The review aims at outlining and discussing the molecular characteristics of PMVs in order to bring to the fore some aspects/characteristics of PMVs that will assist the scientific community to properly understand the role of PMVs in various physiological and pathological processes. The review covers PMVs characterisation and discusses how distinct they are from exosomes and endosomes. Also, methods of PMVs analysis, importance of proper PMV level estimation/characterisation, PMVs and their constituents as well as their therapeutic significance are discussed. The review concludes by drawing attention to the importance of further study into the functions of the characteristics discussed which will lead to understanding the general role of PMVs both in health and in disease states. PMID:26259622

  11. Plasma membrane coenzyme Q: evidence for a role in autism

    PubMed Central

    Crane, Frederick L; Löw, Hans; Sun, Iris; Navas, Placido; Gvozdjáková, Anna

    2014-01-01

    Background The Voltage Dependent Anion Channel (VDAC) is involved in control of autism. Treatments, including coenzyme Q, have had some success on autism control. Data sources Correlation of porin redox activity and expression of autism is based on extensive literature, especially studies of antibodies, identification of cytosolic nicotinamide adenine dinucleotide reduced (NADH) dehydrogenase activity in the VDAC, and evidence for extreme sensitivity of the dehydrogenase to a mercurial. Evidence for a coenzyme Q requirement came from extraction and analog inhibition of NADH ferricyanide reductase in the erythrocyte plasma membrane, done in 1994, and reinterpreted when it was identified in VDAC in 2004. The effects of ubiquinol (the QH2 – reduced form of coenzyme Q) in children with autism were studied. Results A new role for coenzyme Q in the porin channels has implications on autism. Ubiquinol, the more active form of coenzyme Q, produces favorable response in children with autism. Agents which affected electron transport in porin show parallel effects in autism. Conclusion We propose a hypothesis that autism is controlled by a coenzyme Q-dependent redox system in the porin channels; this conclusion is based on the effects of agents that positively or negatively affect electron transport and the symptoms of autism. The full understanding of the mechanism of their control needs to be established. PMID:24920882

  12. Structural Rearrangements in CHO Cells After Disruption of Individual Cytoskeletal Elements and Plasma Membrane.

    PubMed

    Jokhadar, Špela Zemljič; Derganc, Jure

    2015-04-01

    Cellular structural integrity is provided primarily by the cytoskeleton, which comprises microtubules, actin filaments, and intermediate filaments. The plasma membrane has been also recognized as a mediator of physical forces, yet its contribution to the structural integrity of the cell as a whole is less clear. In order to investigate the relationship between the plasma membrane and the cytoskeleton, we selectively disrupted the plasma membrane and each of the cytoskeletal elements in Chinese hamster ovary cells and assessed subsequent changes in cellular structural integrity. Confocal microscopy was used to visualize cytoskeletal rearrangements, and optical tweezers were utilized to quantify membrane tether extraction. We found that cholesterol depletion from the plasma membrane resulted in rearrangements of all cytoskeletal elements. Conversely, the state of the plasma membrane, as assessed by tether extraction, was affected by disruption of any of the cytoskeletal elements, including microtubules and intermediate filaments, which are located mainly in the cell interior. The results demonstrate that, besides the cytoskeleton, the plasma membrane is an important contributor to cellular integrity, possibly by acting as an essential framework for cytoskeletal anchoring. In agreement with the tensegrity model of cell mechanics, our results support the notion of the cell as a prestressed structure. PMID:25395197

  13. Plasma Membrane ATPase Activity following Reversible and Irreversible Freezing Injury 1

    PubMed Central

    Iswari, S.; Palta, Jiwan P.

    1989-01-01

    Plasma membrane ATPase has been proposed as a site of functional alteration during early stages of freezing injury. To test this, plasma membrane was purified from Solanum leaflets by a single step partitioning of microsomes in a dextran-polyethylene glycol two phase system. Addition of lysolecithin in the ATPase assay produced up to 10-fold increase in ATPase activity. ATPase activity was specific for ATP with a Km around 0.4 millimolar. Presence of the ATPase enzyme was identified by immunoblotting with oat ATPase antibodies. Using the phase partitioning method, plasma membrane was isolated from Solanum commersonii leaflets which had four different degrees of freezing damage, namely, slight (reversible), partial (partially reversible), substantial and total (irreversible). With slight (reversible) damage the plasma membrane ATPase specific activity increased 1.5- to 2-fold and its Km was decreased by about 3-fold, whereas the specific activity of cytochrome c reductase and cytochrome c oxidase in the microsomes were not different from the control. However, with substantial (lethal, irreversible) damage, there was a loss of membrane protein, decrease in plasma membrane ATPase specific activity and decrease in Km, while cytochrome c oxidase and cytochrome c reductase were unaffected. These results support the hypothesis that plasma membrane ATPase is altered by slight freeze-thaw stress. Images Figure 1 Figure 2 PMID:16666856

  14. Plasma Membrane Lesions In Anthracycline-Resistant Tumor Cells Probed Using A Fluorescent Dye

    NASA Astrophysics Data System (ADS)

    Burke, Thomas G.; Doroshow, James H.

    1989-06-01

    Human cancer cells selected for resistance to several structurally unrelated cytotoxic drugs are known to display plasma membrane alterations such as amplified levels of a variety of glycoproteins, modifications in lipid composition, alterations in membrane fluidity and increased cellular fragility to osmotic shock. We have studied the plasma membrane fluidity of HL60 human leukemia cells and MCF-7 human breast cancer cells that have been selected for acquired resistance against the cytocidal effects of the anthracycline anticancer drug Adriamycin. Fluidity measurements were accomplished by evaluating the fluorescence anisotropy of the plasma membrane specific probe trimethylamino-1,6-dipihenylhexatriene (TMA.DPH) bound to whole, living cells. TMA.DPH anisotropy values for MCF-7 sensitive and 12-fold resistant cells were 0.306 and 0.285, respectively, while anisotropy values for HL-60 sensitive and 80-fold resistant cells lines were 0.310 and 0.295, respectively. In all cases, cell viability exceeded 97% and anisotropy values were subject to a day-to-day uncertainty of +/-2%. Our results demonstrate that increased plasma membrane fluidity apparently accompanies the development of resistance in both cell lines. Because it is known that increased membrane fluidity results in significantly decreased Adriamycin binding in artificial membrane systems, we propose here that decreased drug associations with fluidized, plasma membrane lipid bilayer regions may be a mechanism which contributes, in part, to the reduced rates of drug accumulation observed in HL60 and MCF-7 cells resistant to Adriamycin.

  15. Plasma membrane microorganization of LR73 multidrug-resistant cells revealed by FCS

    NASA Astrophysics Data System (ADS)

    Winckler, Pascale; Jaffiol, Rodolphe; Cailler, Aurélie; Morjani, Hamid; Jeannesson, Pierre; Deturche, Régis

    2011-03-01

    Tumoral cells could present a multidrug resistance (MDR) to chemotherapeutic treatments. This drug resistance would be associated to biomechanisms occurring at the plasma membrane level, involving modification of membrane fluidity, drug permeability, presence of microdomains (rafts, caveolae...), and membrane proteins overexpression such as Pglycoprotein. Fluorescence correlation spectroscopy (FCS) is the relevant method to investigate locally the fluidity of biological membranes through the lateral diffusion of a fluorescent membrane probe. Thus, we use FCS to monitor the plasma membrane local organization of LR73 carcinoma cells and three derived multidrug-resistant cancer cells lines. Measurements were conducted at the single cell level, which enabled us to get a detailed overview of the plasma membrane microviscosity distribution of each cell line studied. Moreover, we propose 2D diffusion simulation based on a Monte Carlo model to investigate the membrane organisation in terms of microdomains. This simulation allows us to relate the differences in the fluidity distributions with microorganization changes in plasma membrane of MDR cells.

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

    PubMed Central

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

    2015-01-01

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

  17. Aquaporins Contribute to ABA-Triggered Stomatal Closure through OST1-Mediated Phosphorylation.

    PubMed

    Grondin, Alexandre; Rodrigues, Olivier; Verdoucq, Lionel; Merlot, Sylvain; Leonhardt, Nathalie; Maurel, Christophe

    2015-07-01

    Stomatal movements in response to environmental stimuli critically control the plant water status. Although these movements are governed by osmotically driven changes in guard cell volume, the role of membrane water channels (aquaporins) has remained hypothetical. Assays in epidermal peels showed that knockout Arabidopsis thaliana plants lacking the Plasma membrane Intrinsic Protein 2;1 (PIP2;1) aquaporin have a defect in stomatal closure, specifically in response to abscisic acid (ABA). ABA induced a 2-fold increase in osmotic water permeability (Pf) of guard cell protoplasts and an accumulation of reactive oxygen species in guard cells, which were both abrogated in pip2;1 plants. Open stomata 1 (OST1)/Snf1-related protein kinase 2.6 (SnRK2.6), a protein kinase involved in guard cell ABA signaling, was able to phosphorylate a cytosolic PIP2;1 peptide at Ser-121. OST1 enhanced PIP2;1 water transport activity when coexpressed in Xenopus laevis oocytes. Upon expression in pip2;1 plants, a phosphomimetic form (Ser121Asp) but not a phosphodeficient form (Ser121Ala) of PIP2;1 constitutively enhanced the Pf of guard cell protoplasts while suppressing its ABA-dependent activation and was able to restore ABA-dependent stomatal closure in pip2;1. This work supports a model whereby ABA-triggered stomatal closure requires an increase in guard cell permeability to water and possibly hydrogen peroxide, through OST1-dependent phosphorylation of PIP2;1 at Ser-121. PMID:26163575

  18. Plasma membrane receptor-like kinase leaf panicle 2 acts downstream of the DROUGHT AND SALT TOLERANCE transcription factor to regulate drought sensitivity in rice

    PubMed Central

    Wu, Fuqing; Sheng, Peike; Tan, Junjie; Chen, Xiuling; Lu, Guangwen; Ma, Weiwei; Heng, Yueqin; Lin, Qibing; Zhu, Shanshan; Wang, Jiulin; Wang, Jie; Guo, Xiuping; Zhang, Xin; Lei, Cailin; Wan, Jianmin

    2015-01-01

    Drought is a recurring climatic hazard that reduces the crop yields. To avoid the negative effects of drought on crop production, extensive efforts have been devoted to investigating the complex mechanisms of gene expression and signal transduction during drought stress. Receptor-like kinases (RLKs) play important roles in perceiving extracellular stimuli and activating downstream signalling responses. The rice genome contains >1100 RLK genes, of which only two are reported to function in drought stress. A leucine-rich repeat (LRR)-RLK gene named Leaf Panicle 2 (LP2) was previously found to be strongly expressed in leaves and other photosynthetic tissues, but its function remains unclear. In the present study, it was shown that the expression of LP2 was down-regulated by drought and abscisic acid (ABA). Transgenic plants overexpressing LP2 accumulated less H2O2, had more open stomata in leaves, and showed hypersensitivity to drought stress. Further investigation revealed that transcription of LP2 was directly regulated by the zinc finger transcription factor DROUGHT AND SALT TOLERANCE (DST). In addition, LP2 was identified as a functional kinase localized to the plasma membrane and interacted with the drought-responsive aquaporin proteins OsPIP1; 1, OsPIP1; 3, and OsPIP2; 3. Thus, the findings provided evidence that the LRR-RLK LP2, transcriptionally regulated by the drought-related transcription factor DST, served as a negative regulator in drought response. PMID:25385766

  19. Plasma membrane receptor-like kinase leaf panicle 2 acts downstream of the DROUGHT AND SALT TOLERANCE transcription factor to regulate drought sensitivity in rice.

    PubMed

    Wu, Fuqing; Sheng, Peike; Tan, Junjie; Chen, Xiuling; Lu, Guangwen; Ma, Weiwei; Heng, Yueqin; Lin, Qibing; Zhu, Shanshan; Wang, Jiulin; Wang, Jie; Guo, Xiuping; Zhang, Xin; Lei, Cailin; Wan, Jianmin

    2015-01-01

    Drought is a recurring climatic hazard that reduces the crop yields. To avoid the negative effects of drought on crop production, extensive efforts have been devoted to investigating the complex mechanisms of gene expression and signal transduction during drought stress. Receptor-like kinases (RLKs) play important roles in perceiving extracellular stimuli and activating downstream signalling responses. The rice genome contains >1100 RLK genes, of which only two are reported to function in drought stress. A leucine-rich repeat (LRR)-RLK gene named Leaf Panicle 2 (LP2) was previously found to be strongly expressed in leaves and other photosynthetic tissues, but its function remains unclear. In the present study, it was shown that the expression of LP2 was down-regulated by drought and abscisic acid (ABA). Transgenic plants overexpressing LP2 accumulated less H₂O₂, had more open stomata in leaves, and showed hypersensitivity to drought stress. Further investigation revealed that transcription of LP2 was directly regulated by the zinc finger transcription factor DROUGHT AND SALT TOLERANCE (DST). In addition, LP2 was identified as a functional kinase localized to the plasma membrane and interacted with the drought-responsive aquaporin proteins OsPIP1; 1, OsPIP1; 3, and OsPIP2; 3. Thus, the findings provided evidence that the LRR-RLK LP2, transcriptionally regulated by the drought-related transcription factor DST, served as a negative regulator in drought response. PMID:25385766

  20. Purification of rat liver plasma membranes by wheat-germ-agglutinin affinity partitioning.

    PubMed Central

    Persson, A; Johansson, B; Olsson, H; Jergil, B

    1991-01-01

    Rat liver plasma membranes were separated from other cellular membranes by affinity partitioning in an aqueous polymer two-phase system by using the lectin wheat-germ agglutinin covalently bound to dextran as the affinity ligand. In borate buffer the bulk of membranes partitioned in the poly(ethylene glycol)-rich top phase, whereas plasma membranes were pulled selectively into the dextran-rich bottom phase in the presence of ligand. The purity and yield of plasma membranes prepared by lectin affinity partitioning and by conventional sucrose-density-gradient centrifugation was similar, as judged from marker-enzyme activities. The affinity procedure, not dependent on lengthy centrifugations, is fast and gentle and will be advantageous when studying labile components. PMID:1703408

  1. NPA binding activity is peripheral to the plasma membrane and is associated with the cytoskeleton.

    PubMed Central

    Cox, D N; Muday, G K

    1994-01-01

    N-1-Naphthylphthalamic acid (NPA) binding activity is released into the supernatant when plasma membranes are subjected to high-salt treatment, indicating that this activity is peripherally associated with the membrane. Extraction of plasma membrane vesicles with Triton X-100 resulted in retention of NPA binding activity in the detergent-insoluble cytoskeletal pellet. Treatment of this pellet with KI released NPA binding activity, actin, and alpha-tubulin. Dialysis to remove KI led to the repolymerization of cytoskeletal elements and movement of NPA binding activity into an insoluble cytoskeletal pellet. NPA binding activity partitioned into the detergent-insoluble cytoskeletal pellet obtained from both zucchini and maize membranes and was released from these pellets by KI treatment. Treatment of a cytoskeletal pellet with cytochalasin B doubled NPA binding activity in the resulting supernatant. Together, these experiments indicate that NPA binding activity is peripherally associated with the plasma membrane and interacts with the cytoskeleton in vitro. PMID:11536654

  2. Rapid preparation of plasma membranes from avian lymphoid cells and fibroblasts for virus binding studies.

    PubMed

    Nieper, H; Müller, H

    1998-06-01

    A simple and rapid protocol for the preparation of plasma membranes from chicken embryo fibroblasts and chicken lymphoid cells was developed. Characterization of the preparations by morphological, biochemical and serological methods indicated the specific enrichment of the plasma membranes as well as cell surface proteins. Binding of infectious bursal disease virus (IBDV) particles was demonstrated after immobilization of the plasma membranes, and cell type-specific differences were observed. Although the results of these studies reflect the interaction between IBDV and isolated cells only partially, the advantages of these plasma membrane preparations, the specific enrichment of cell surface proteins, their constant quality and the possibility to store aliquots over several months, make them a useful tool for virus binding studies with avian cells. PMID:9694323

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

    DOE PAGESBeta

    Frisz, Jessica F.; Klitzing, Haley A.; Lou, Kaiyan; Hutcheon, Ian D.; Weber, Peter K.; Zimmerberg, Joshua; Kraft, Mary L.

    2013-04-22

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

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

    SciTech Connect

    Frisz, Jessica F.; Klitzing, Haley A.; Lou, Kaiyan; Hutcheon, Ian D.; Weber, Peter K.; Zimmerberg, Joshua; Kraft, Mary L.

    2013-04-22

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

  5. Modification of pro-inflammatory signaling by dietary components: The plasma membrane as a target.

    PubMed

    Ciesielska, Anna; Kwiatkowska, Katarzyna

    2015-07-01

    You are what you eat - this well-known phrase properly describes the phenomenon of the effects of diet on acute and chronic inflammation. Several lipids and lipophilic compounds that are delivered with food or are produced in situ in pathological conditions exert immunomodulatory activity due to their interactions with the plasma membrane. This group of compounds includes cholesterol and its oxidized derivatives, fatty acids, α-tocopherol, and polyphenols. Despite their structural heterogeneity, all these compounds ultimately induce changes in plasma membrane architecture and fluidity. By doing this, they modulate the dynamics of plasma membrane receptors, such as TLR4. This receptor is activated by lipopolysaccharide, triggering acute inflammation during bacterial infection, which often leads to sepsis and is linked with diverse chronic inflammatory diseases. In this review, we discuss how the impact on plasma membrane properties contributes to the immunomodulatory activity of dietary compounds, pointing to the therapeutic potential of some of them. Also watch the Video Abstract. PMID:25966354

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

    PubMed

    Douglas, Lois M; Konopka, James B

    2016-03-01

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

  7. Hemagglutinin clusters in the plasma membrane are not enriched with cholesterol and sphingolipids.

    PubMed

    Wilson, Robert L; Frisz, Jessica F; Klitzing, Haley A; Zimmerberg, Joshua; Weber, Peter K; Kraft, Mary L

    2015-04-01

    The clusters of the influenza envelope protein, hemagglutinin, within the plasma membrane are hypothesized to be enriched with cholesterol and sphingolipids. Here, we directly tested this hypothesis by using high-resolution secondary ion mass spectrometry to image the distributions of antibody-labeled hemagglutinin and isotope-labeled cholesterol and sphingolipids in the plasma membranes of fibroblast cells that stably express hemagglutinin. We found that the hemagglutinin clusters were neither enriched with cholesterol nor colocalized with sphingolipid domains. Thus, hemagglutinin clustering and localization in the plasma membrane is not controlled by cohesive interactions between hemagglutinin and liquid-ordered domains enriched with cholesterol and sphingolipids, or from specific binding interactions between hemagglutinin, cholesterol, and/or the majority of sphingolipid species in the plasma membrane. PMID:25863057

  8. Sphingolipid Domains in the Plasma Membranes of Fibroblasts Are Not Enriched with Cholesterol*

    PubMed Central

    Frisz, Jessica F.; Klitzing, Haley A.; Lou, Kaiyan; Hutcheon, Ian D.; Weber, Peter K.; Zimmerberg, Joshua; Kraft, Mary L.

    2013-01-01

    The plasma membranes of mammalian cells are widely expected to contain domains that are enriched with cholesterol and sphingolipids. In this work, we have used high-resolution secondary ion mass spectrometry to directly map the distributions of isotope-labeled cholesterol and sphingolipids in the plasma membranes of intact fibroblast cells. Although acute cholesterol depletion reduced sphingolipid domain abundance, cholesterol was evenly distributed throughout the plasma membrane and was not enriched within the sphingolipid domains. Thus, we rule out favorable cholesterol-sphingolipid interactions as dictating plasma membrane organization in fibroblast cells. Because the sphingolipid domains are disrupted by drugs that depolymerize the cells actin cytoskeleton, cholesterol must instead affect the sphingolipid organization via an indirect mechanism that involves the cytoskeleton. PMID:23609440

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

    PubMed Central

    Wilson, Robert L.; Frisz, Jessica F.; Klitzing, Haley A.; Zimmerberg, Joshua; Weber, Peter K.; Kraft, Mary L.

    2015-01-01

    The clusters of the influenza envelope protein, hemagglutinin, within the plasma membrane are hypothesized to be enriched with cholesterol and sphingolipids. Here, we directly tested this hypothesis by using high-resolution secondary ion mass spectrometry to image the distributions of antibody-labeled hemagglutinin and isotope-labeled cholesterol and sphingolipids in the plasma membranes of fibroblast cells that stably express hemagglutinin. We found that the hemagglutinin clusters were neither enriched with cholesterol nor colocalized with sphingolipid domains. Thus, hemagglutinin clustering and localization in the plasma membrane is not controlled by cohesive interactions between hemagglutinin and liquid-ordered domains enriched with cholesterol and sphingolipids, or from specific binding interactions between hemagglutinin, cholesterol, and/or the majority of sphingolipid species in the plasma membrane. PMID:25863057

  10. Membrane-based Therapeutic Plasma Exchange: A New Frontier for Nephrologists.

    PubMed

    Gashti, Casey N

    2016-09-01

    Therapeutic plasma exchange has long been utilized to manage a variety of immune-mediated diseases. The underlying principle is the removal of a circulating pathogenic substance from the plasma and substitution with a replacement fluid. Different methodologies of plasma separation include the use of centrifuge, which relies on the variation in the specific gravity of blood components, and membrane-based separation, which relies on particle size. With advancements in technology and clinical insight into disease pathophysiology, membrane technology has become more biocompatible, safer, and more adaptable to conventional hemodialysis and hemofiltration machines. As such, nephrologists, who are familiar with management of extracorporeal blood purification systems, are increasingly involved with membrane-based plasma separation. This review aims to highlight the technical aspects of membrane-based separation, review the prescription for therapy, and draw comparisons with the centrifuge-based technique when applicable. PMID:27062015

  11. Immunoprecipitation of Plasma Membrane Receptor-Like Kinases for Identification of Phosphorylation Sites and Associated Proteins.

    PubMed

    Kadota, Yasuhiro; Macho, Alberto P; Zipfel, Cyril

    2016-01-01

    Membrane proteins are difficult to study for numerous reasons. The surface of membrane proteins is relatively hydrophobic and sometimes very unstable, additionally requiring detergents for their extraction from the membrane. This leads to challenges at all levels, including expression, solubilization, purification, identification of associated proteins, and the identification of post-translational modifications. However, recent advances in immunoprecipitation technology allow to isolate membrane proteins efficiently, facilitating the study of protein-protein interactions, the identification of novel associated proteins, and to identify post-translational modifications, such as phosphorylation. Here, we describe an optimized immunoprecipitation protocol for plant plasma membrane receptor-like kinases. PMID:26577786

  12. Heterologous Expression of Two Jatropha Aquaporins Imparts Drought and Salt Tolerance and Improves Seed Viability in Transgenic Arabidopsis thaliana

    PubMed Central

    Khan, Kasim; Agarwal, Pallavi; Shanware, Arti; Sane, Vidhu Aniruddha

    2015-01-01

    Drought and high salinity are environmental conditions that cause adverse effects on the growth and productivity of crops. Aquaporins are small integral membrane proteins that belong to the family of the major intrinsic proteins (MIPs), with members in animals, plants and microbes, where they facilitate the transport of water and/or small neutral solutes thereby affecting water balance. In this study we characterized two aquaporin genes namely, plasma membrane intrinsic protein (PIP2;7) and tonoplast intrinsic protein TIP1;3 from Jatropha curcas that are localised to the plasma membrane and vacuole respectively. Transgenic Arabidopsis thaliana lines over-expressing JcPIP2;7 and JcTIP1;3 under a constitutive promoter show improved germination under high salt and mannitol compared to control seeds. These transgenic plants also show increased root length under abiotic stress conditions compared to wild type Col-0 plants. Transgenic lines exposed to drought conditions by withholding water for 20 days, were able to withstand water stress and attained normal growth after re-watering unlike control plants which could not survive. Transgenic lines also had better seed yield than control under salt stress. Importantly, seed viability of transgenic plants grown under high salt concentration was 35%-45% compared to less than 5% for control seeds obtained from plants growing under salt stress. The effect of JcPIP2;7 and JcTIP1;3 on improving germination and seed viability in drought and salinity make these important candidates for genetic manipulation of plants for growth in saline soils. PMID:26067295

  13. Heterologous Expression of Two Jatropha Aquaporins Imparts Drought and Salt Tolerance and Improves Seed Viability in Transgenic Arabidopsis thaliana.

    PubMed

    Khan, Kasim; Agarwal, Pallavi; Shanware, Arti; Sane, Vidhu Aniruddha

    2015-01-01

    Drought and high salinity are environmental conditions that cause adverse effects on the growth and productivity of crops. Aquaporins are small integral membrane proteins that belong to the family of the major intrinsic proteins (MIPs), with members in animals, plants and microbes, where they facilitate the transport of water and/or small neutral solutes thereby affecting water balance. In this study we characterized two aquaporin genes namely, plasma membrane intrinsic protein (PIP2;7) and tonoplast intrinsic protein TIP1;3 from Jatropha curcas that are localised to the plasma membrane and vacuole respectively. Transgenic Arabidopsis thaliana lines over-expressing JcPIP2;7 and JcTIP1;3 under a constitutive promoter show improved germination under high salt and mannitol compared to control seeds. These transgenic plants also show increased root length under abiotic stress conditions compared to wild type Col-0 plants. Transgenic lines exposed to drought conditions by withholding water for 20 days, were able to withstand water stress and attained normal growth after re-watering unlike control plants which could not survive. Transgenic lines also had better seed yield than control under salt stress. Importantly, seed viability of transgenic plants grown under high salt concentration was 35%-45% compared to less than 5% for control seeds obtained from plants growing under salt stress. The effect of JcPIP2;7 and JcTIP1;3 on improving germination and seed viability in drought and salinity make these important candidates for genetic manipulation of plants for growth in saline soils. PMID:26067295

  14. Hypoxia directly increases serotonin transport by porcine pulmonary artery endothelial cell (PAEC) plasma membrane vesicles

    SciTech Connect

    Bhat, G.B.; Block, E.R. )

    1990-02-26

    Alterations in the physical state and composition of membrane lipids have been shown to interfere with a number of critical cellular and membrane functions including transmembrane transport. The authors have reported that hypoxia has profound effects upon the physical state and lipid composition of the PAEC plasma membrane bilayer and have suggested that this is responsible for increased serotonin uptake by these cells. In order to determine whether hypoxia has a direct effect on the plasma membrane transport of serotonin, they measured serotonin transport activity (1) in plasma membrane vesicles isolated from normoxic (20% O{sub 2}-5% CO{sub 2}) and hypoxic (0% O{sub 2}-5% CO{sub 2}) PAEC and (2) in PAEC plasma membrane vesicles that were exposed directly to normoxia or hypoxia. A 24-h exposure of PAEC to hypoxia resulted in a 40% increase in specific serotonin transport by plasma membrane vesicles derived from these cells. When plasma membrane vesicles were isolated and then directly exposed to normoxia or hypoxia for 1 h at 37C, a 31% increase in specific 5-HT transport was observed in hypoxic vesicles. Hypoxia did not alter the Km of serotonin transport (normoxia = 3.47 {mu}M versus hypoxia = 3.76 {mu}M) but markedly increased the maximal rate of transport (V{sup max}) (normoxia = 202.4 pmol/min/mg protein versus hypoxia = 317.9 pmol/min/mg protein). These results indicate that hypoxia increases serotonin transport in PAEC by a direct effect on the plasma membrane leading to an increase in the effective number of transporter molecules without alteration in transporter affinity for serotonin.

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

    PubMed Central

    Taylor, Alison R.; Brownlee, Colin

    1992-01-01

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

  16. Laser Microsurgery Permits Fungal Plasma Membrane Single-Ion-Channel Resolution at the Hyphal Tip

    PubMed Central

    Véry, Anne-Aliénor; Davies, Julia M.

    1998-01-01

    A method for formation of high-electrical-resistance seals on the Neurospora crassa plasma membrane, allowing resolution of single-ion-channel activity by patch clamp electrophysiology, is reported. Laser microsurgery permits access to the hyphal apex without enzymatic cell wall digestion and loss of morphological polarity. Cell wall reformation is delayed by brefeldin. This method can allow full characterization of apical plasma membrane channels, which are implicated in tip growth. PMID:16349556

  17. Barriers to diffusion of plasma membrane proteins form early during guinea pig spermiogenesis.

    PubMed Central

    Cowan, A E; Nakhimovsky, L; Myles, D G; Koppel, D E

    1997-01-01

    The plasma membrane of the mature guinea pig sperm is segregated into at least four domains of different composition. Previous studies have shown that some proteins localized within these domains are free to diffuse laterally, suggesting that barriers to protein diffusion are responsible for maintaining the nonuniform distribution of at least some surface proteins in mature sperm. The different membrane domains appear sequentially during sperm morphogenesis in the testis and during later passage through the epididymis. To determine when diffusion barriers become functional during sperm development, we examined the diffusion of two proteins that are expressed on the cell surface of developing spermatids and become segregated to different plasma membrane domains during the course of spermiogenesis. Both proteins exhibited rapid lateral diffusion throughout spermiogenesis, even after they become localized to specific regions of the surface membrane. These results suggest that barriers to membrane diffusion form concomitantly with membrane domains during spermiogenesis. Images FIGURE 1 FIGURE 2 PMID:9199813

  18. Chronic and acute ethanol treatment modifies fluidity and composition in plasma membranes of a human hepatic cell line (WRL-68).

    PubMed

    Gutiérrez-Ruiz, M C; Gómez, J L; Souza, V; Bucio, L

    1995-04-01

    The aim of this study was to compare the effects of chronic (0.1 mol/L ethanol exposure during 30 days) and acute (0.5 mol/L ethanol exposure during 24 h) ethanol treatment on the physical properties and the lipid composition of plasma membranes of the WRL-68 cells (fetal human hepatic cell line). Using fluorescence polarization we found that ethanol treatment reduced membrane anisotropy due to disorganization of acyl chains in plasma membranes and consequently increased fluidity, as measured with the diphenylhexatriene probe. Addition of ethanol in vitro reduced anisotropy in control plasma membranes, whereas chronically ethanol-treated plasma membranes were relatively tolerant to the in vitro addition of ethanol. Acutely ethanol-treated plasma membranes exhibited a smaller anisotropy parameter value than control plasma membranes. We found a decrease in total phospholipid content in acute ethanol WRL-68 plasma membranes. Cholesterol content was increased in both ethanol treatments, and we also found a significant decrease in phosphatidylinositol and phosphatidylcholine and an increase in phosphatidylethanolamine content in ethanol-treated plasma membranes. Our data showed that ethanol treatment decreased the anisotropy parameter consistently with increased fluidity, while increasing the cholesterol/phospholipid ratio of plasma membranes of WRL-68 cells, but only chronically ethanol-treated plasma membranes exhibited tolerance to the in vitro addition of ethanol. It is important to note that some changes that were interpreted as a result of chronic ethanol treatment were also present in short-period ethanol treatments. PMID:7583873

  19. Yeast cell wall integrity sensors form specific plasma membrane microdomains important for signalling.

    PubMed

    Kock, Christian; Arlt, Henning; Ungermann, Christian; Heinisch, Jürgen J

    2016-09-01

    The cell wall integrity (CWI) pathway of the yeast Saccharomyces cerevisiae relies on the detection of cell surface stress by five sensors (Wsc1, Wsc2, Wsc3, Mid2, Mtl1). Each sensor contains a single transmembrane domain and a highly mannosylated extracellular region, and probably detects mechanical stress in the cell wall or the plasma membrane. We here studied the distribution of the five sensors at the cell surface by using fluorescently tagged variants in conjunction with marker proteins for established membrane compartments. We find that each of the sensors occupies a specific microdomain at the plasma membrane. The novel punctate 'membrane compartment occupied by Wsc1' (MCW) shows moderate overlap with other Wsc-type sensors, but not with those of the Mid-type sensors or other established plasma membrane domains. We further observed that sensor density and formation of the MCW compartment depends on the cysteine-rich head group near the N-terminus of Wsc1. Yet, signalling capacity depends more on the sensor density in the plasma membrane than on clustering within its microcompartment. We propose that the MCW microcompartment provides a quality control mechanism for retaining functional sensors at the plasma membrane to prevent them from endocytosis. PMID:27337501

  20. Directing membrane chromatography to manufacture α1-antitrypsin from human plasma fraction IV.

    PubMed

    Fan, Jinxin; Luo, Jianquan; Song, Weijie; Chen, Xiangrong; Wan, Yinhua

    2015-12-01

    The surging demand for plasma proteins, mainly driven by the growing market and the development of new therapeutic indications, is promoting manufacturers to improve the throughput of plasma proteins. Due to the inherent convective mass transfer, membrane chromatography has been proved to be an efficient approach for extracting a small amount of target proteins from large-volume feed. In this study, α1-antitrypsin (AAT) was extracted from human plasma fraction IV by a two-step membrane chromatography. An anion-exchange membrane chromatography (AEMC) was used to capture the plasma proteins in bind/elute mode, and the obtained effluent was further polished by a hydrophobic interaction membrane chromatography (HIMC) in flow-through mode. Under optimal conditions, the recovery and purity of AAT achieved 87.0% and 0.58 AAT/protein (g/g) by AEMC, respectively. After the precise polishing by HIMC, the purity of AAT was 1.22 AAT/protein (g/g). The comparison results showed that membrane chromatography outperformed column chromatography in both steps because of its high throughput. This two-step membrane chromatography could obtain an AAT recovery of 83.3% and an activity recovery of 91.4%. The outcome of this work not only offers an alternative process for protein purification from plasma, but also provides guidelines for manufacturing product from a large-volume feed with multi-components by membrane chromatography. PMID:26518493

  1. Oxytocin regulates the plasma membrane Ca2+ transport in rat myometrium.

    PubMed Central

    Enyedi, A; Brandt, J; Minami, J; Penniston, J T

    1989-01-01

    Development of myometrium in young female rats was stimulated by administration of diethylstilboestrol. Plasma membrane and sarcoplasmic reticulum from rat myometrium were separated by a new and rapid method using a Percoll gradient. Calcium uptake was inhibited in plasma membrane vesicles isolated from oxytocin-treated myometrium, while no consistent effect of oxytocin was found on the Ca2+ uptake in the sarcoplasmic reticulum. Oxytocin regulated the plasma membrane Ca2+ pump by decreasing its apparent affinity for Ca2+ without affecting its maximal velocity. The K1/2 for Ca2+ in the absence of calmodulin was 0.41 +/- 0.04 microM in normal membranes; this was increased to 0.93 +/- 0.12 microM in oxytocin-treated membranes. Calmodulin decreased the K1/2 for Ca2+ to 0.27 +/- 0.027 microM and oxytocin also increased this, to 0.46 +/- 0.061 microM. The effect of oxytocin on the plasma membrane Ca2+ pump was highly dependent on the hormonal status of the animals. When the diethylstilboestrol was administered together with progesterone, the inhibitory action of oxytocin was totally suppressed, consistent with the expected action of this agent. The results suggest that regulation of the plasma membrane Ca2+ pump may be important in the prolonged elevation of intracellular Ca2+ caused by oxytocin. PMID:2775210

  2. Sorting Nexin 11 Regulates Lysosomal Degradation of Plasma Membrane TRPV3.

    PubMed

    Li, Caiyue; Ma, Wenbo; Yin, Shikui; Liang, Xin; Shu, Xiaodong; Pei, Duanqing; Egan, Terrance M; Huang, Jufang; Pan, Aihua; Li, Zhiyuan

    2016-05-01

    The trafficking of ion channels to/from the plasma membrane is considered an important mechanism for cellular activity and an interesting approach for disease therapies. The transient receptor potential vanilloid 3 (TRPV3) ion channel is widely expressed in skin keratinocytes, and its trafficking mechanism to/from the plasma membrane is unknown. Here, we report that the vesicular trafficking protein sorting nexin 11 (SNX11) downregulates the level of the TRPV3 plasma membrane protein. Overexpression of SNX11 causes a decrease in the level of TRPV3 current and TRPV3 plasma membrane protein in TRPV3-transfected HEK293T cells. Subcellular localizations and western blots indicate that SNX11 interacts with TRPV3 and targets it to lysosomes for degradation, which is blocked by the lysosomal inhibitors chloroquine and leupeptin. Both TRPV3 and SNX11 are highly expressed in HaCaT cells. We show that TRPV3 agonists-activated Ca(2+) influxes and the level of native TRPV3 total protein in HaCaT cells are decreased by overexpression of SNX11 and increased by knockdown of SNX11. Our findings reveal that SNX11 promotes the trafficking of TRPV3 from the plasma membrane to lysosomes for degradation via protein-protein interactions, which demonstrates a previously unknown function of SNX11 as a regulator of TRPV3 trafficking from the plasma membrane to lysosomes. PMID:26818531

  3. Plasma membrane phosphoinositide balance regulates cell shape during Drosophila embryo morphogenesis

    PubMed Central

    Reversi, Alessandra; Loeser, Eva; Subramanian, Devaraj; Schultz, Carsten

    2014-01-01

    Remodeling of cell shape during morphogenesis is driven by the coordinated expansion and contraction of specific plasma membrane domains. Loss of this coordination results in abnormal cell shape and embryonic lethality. Here, we show that plasma membrane lipid composition plays a key role in coordinating plasma membrane contraction during expansion. We found that an increase in PI(4,5)P2 levels caused premature actomyosin contraction, resulting in the formation of shortened cells. Conversely, acute depletion of PI(4,5)P2 blocked plasma membrane expansion and led to premature actomyosin disassembly. PI(4,5)P2-mediated contractility is counteracted by PI(3,4,5)P3 and the zygotic gene bottleneck, which acts by limiting myosin recruitment during plasma membrane expansion. Collectively, these data support a model in which the ratio of PI(4,5)P2/PI(3,4,5)P3 coordinates actomyosin contractility and plasma membrane expansion during tissue morphogenesis, thus ensuring proper cell shape. PMID:24798734

  4. High heterogeneity of plasma membrane microfluidity in multidrug-resistant cancer cells

    NASA Astrophysics Data System (ADS)

    Boutin, Céline; Roche, Yann; Millot, Christine; Deturche, Régis; Royer, Pascal; Manfait, Michel; Plain, Jérôme; Jeannesson, Pierre; Millot, Jean-Marc; Jaffiol, Rodolphe

    2009-05-01

    Diffusion-time distribution analysis (DDA) has been used to explore the plasma membrane fluidity of multidrug-resistant cancer cells (LR73 carcinoma cells) and also to characterize the influence of various membrane agents present in the extracellular medium. DDA is a recent single-molecule technique, based on fluorescence correlation spectroscopy (FCS), well suited to retrieve local organization of cell membrane. The method was conducted on a large number of living cells, which enabled us to get a detailed overview of plasma membrane microviscosity, and plasma membrane micro-organization, between the cells of the same line. Thus, we clearly reveal the higher heterogeneity of plasma membrane in multidrug-resistant cancer cells in comparison with the nonresistant ones (denoted sensitive cells). We also display distinct modifications related to a membrane fluidity modulator, benzyl alcohol, and two revertants of multidrug resistance, verapamil and cyclosporin-A. A relation between the distribution of the diffusion-time values and the modification of membrane lateral heterogeneities is proposed.

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

    PubMed

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

    2014-05-20

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

  6. Bright fluorogenic squaraines with tuned cell entry for selective imaging of plasma membrane vs. endoplasmic reticulum.

    PubMed

    Collot, Mayeul; Kreder, Rémy; Tatarets, Anatoliy L; Patsenker, Leonid D; Mely, Yves; Klymchenko, Andrey S

    2015-12-14

    A rational design of squaraine dyes with lipophilic and zwitterionic groups tunes cell entry, allowing for selective far-red/near-infrared imaging of plasma membrane vs. endoplasmic reticulum. They exhibit up to 110-fold fluorescence enhancement in biomembranes and enable cellular imaging at 1 nM concentration, which make them the brightest membrane probes to date. PMID:26455447

  7. [Isolation and characteristics of the plasma membrane fraction from the swine myometrium].

    PubMed

    Kondratiuk, T P; Bychenok, S F; Prishchepa, L A; Babich, L G; Kurskiĭ, M D

    1986-01-01

    An accelerated method is developed for isolating a fraction of plasma membranes of pig myometrium using ultracentrifugation within the sucrose density gradient (15% and 30%). The membranes possessed the high activity of 5'-nucleotidase and Na+, K+-ATPase and the low activity of rhotenon-insensitive NADH-cytochrome c reductase. The vesicularized preparations of plasma membranes are able of ATP-dependent accumulation of Ca2+ (7.5 +/- 0.3 nmol. 45Ca2+ per 1 mg of protein for 15 min). Phosphate increases the calcium accumulation in the presence of ATP and Mg2+. Ionophore A 23187 promotes a complete and rapid release of the previously active-accumulated calcium. The release of 45Ca2+ accumulated by the membrane fraction may be reached by introduction of 1 mM EGTA or DS-Na into the incubation medium, that evidences for the cation accumulation inside closed structures. Using concanavalin-A-sepharose 4B it is shown that 60% of membrane vesicles are turned inside out. The low saponine concentrations (0.0005%) which inhibit Ca2+-accumulation by plasma membranes but not by the endoplasmic reticulum inhibit this process by 60-70% in preparations of the isolated membrane fraction. The method has certain advantages over the previously applied methods used for isolating of plasma membrane fragments from smooth muscles. PMID:3016962

  8. Effect of ion bombardment on plasma-driven superpermeation of hydrogen isotopes through a niobium membrane

    NASA Astrophysics Data System (ADS)

    Notkin, M. E.; Livshits, A. I.; Bruneteau, A. M.; Bacal, M.

    2001-08-01

    Hydrogen plasma-driven permeation through the superpermeable niobium membrane was investigated under bombardment of the input membrane surface with hydrogen, deuterium and helium ions with energy 0-250 eV over the range of membrane temperature 910-1420 K. The membrane surface was covered with a nonmetal monolayer generating a potential barrier responsible for the superpermeability to suprathermal hydrogen particles. Both an increase of ion energy and an increase of mass of sputtering ions result in a significant decrease of permeability due to destruction of the nonmetal monolayer, when the ion energy is higher than the threshold energy of surface film sputtering. On the contrary, the increase of the membrane temperature results in the decrease of the ion bombardment effect and in the increase of the membrane permeability due to recovery of the surface barrier through segregation of impurities dissolved in the membrane bulk onto the membrane surface. To increase the membrane ability to recover the potential barrier, oxygen was dissolved in the membrane bulk up to a concentration of 2.5 at.%. This resulted in a significant decrease of the damaging effect of ion bombardment and in the extension of the range of the membrane temperature and ion energy over which plasma-driven superpermeability was observed.

  9. Computational analysis of the tether pulling experiment to probe plasma membrane - cytoskeleton interaction in cells

    PubMed Central

    Schumacher, Kristopher R.; Popel, Aleksander S.; Anvari, Bahman; Brownell, William E.; Spector, Alexander A.

    2016-01-01

    Tethers are thin membrane tubes that can be formed when relatively small and localized forces are applied to cellular membranes and lipid bilayers. Tether pulling experiments have been used to better understand the fine membrane properties. These include the interaction between the plasma membrane and the underlying cytoskeleton, which is an important factor affecting membrane mechanics. We use a computational method aimed at the interpretation and design of tether pulling experiments in cells with a strong membrane-cytoskeleton attachment. In our model, we take into account the detailed information on the topology of bonds connecting the plasma membrane and the cytoskeleton. We compute the force-dependent piecewise membrane deflection and bending as well as modes of stored energy in three major regions of the system: body of the tether, membrane-cytoskeleton attachment zone, and the transition zone between the two. We apply our method to three cells: cochlear outer hair cells (OHCs), human embryonic kidney (HEK) cells, and Chinese hamster ovary (CHO) cells. OHCs have a special system of pillars connecting the membrane and the cytoskeleton, and HEK and CHO cells have a bond arrangement via bonds (e.g., PIP2) which is common to many other cells. We also present a validation of our model by using experimental data on CHO and HEK cells. The proposed method can be an effective tool in the analyses of experiments to probe the properties of cellular membranes. PMID:19905340

  10. Aquaporins in the hepatobiliary tract. Which, where and what they do in health and disease.

    PubMed

    Portincasa, P; Palasciano, G; Svelto, M; Calamita, G

    2008-01-01

    The biological importance of the aquaporin family of water channels was recently acknowledged by the 2003 Nobel Prize for Chemistry awarded to the discovering scientist Peter Agre. Among the pleiotropic roles exerted by aquaporins in nature in both health and disease, the review addresses the latest acquisitions about the expression and regulation, as well as physiology and pathophysiology of aquaporins in the hepatobiliary tract. Of note, at least seven out of the thirteen mammalian aquaporins are expressed in the liver, bile ducts and gallbladder. Aquaporins are essential for bile water secretion and reabsorption, as well as for plasma glycerol uptake by the hepatocyte and its conversion to glucose during starvation. Novel data are emerging regarding the physio-pathological involvement of aquaporins in multiple diseases such as cholestases, liver cirrhosis, obesity and insulin resistance, fatty liver, gallstone formation and even microparasite invasion of intrahepatic bile ducts. This body of knowledge represents the mainstay of present and future research in a rapidly expanding field. PMID:18173545

  11. Overexpression of the wheat aquaporin gene, TaAQP7, enhances drought tolerance in transgenic tobacco.

    PubMed

    Zhou, Shiyi; Hu, Wei; Deng, Xiaomin; Ma, Zhanbing; Chen, Lihong; Huang, Chao; Wang, Chen; Wang, Jie; He, Yanzhen; Yang, Guangxiao; He, Guangyuan

    2012-01-01

    Aquaporin (AQP) proteins have been shown to transport water and other small molecules through biological membranes, which is crucial for plants to combat stress caused by drought. However, the precise role of AQPs in drought stress response is not completely understood in plants. In this study, a PIP2 subgroup gene AQP, designated as TaAQP7, was cloned and characterized from wheat. Expression of TaAQP7-GFP fusion protein revealed its localization in the plasma membrane. TaAQP7 exhibited high water channel activity in Xenopus laevis oocytes and TaAQP7 transcript was induced by dehydration, and treatments with polyethylene glycol (PEG), abscisic acid (ABA) and H(2)O(2). Further, TaAQP7 was upregulated after PEG treatment and was blocked by inhibitors of ABA biosynthesis, implying that ABA signaling was involved in the upregulation of TaAQP7 after PEG treatment. Overexpression of TaAQP7 increased drought tolerance in tobacco. The transgenic tobacco lines had lower levels of malondialdehyde (MDA) and H(2)O(2), and less ion leakage (IL), but higher relative water content (RWC) and superoxide dismutase (SOD) and catalase (CAT) activities when compared with the wild type (WT) under drought stress. Taken together, our results show that TaAQP7 confers drought stress tolerance in transgenic tobacco by increasing the ability to retain water, reduce ROS accumulation and membrane damage, and enhance the activities of antioxidants. PMID:23285044

  12. A 39-kD plasma membrane protein (IP39) is an anchor for the unusual membrane skeleton of Euglena gracilis

    SciTech Connect

    Rosiere, T.K.; Marrs, J.A.; Bouck, G.B. )

    1990-04-01

    The major integral plasma membrane protein (IP39) of Euglena gracilis was radiolabeled, peptide mapped, and dissected with proteases to identify cytoplasmic domains that bind and anchor proteins of the cell surface. When plasma membranes were radioiodinated and extracted with octyl glucoside, 98% of the extracted label was found in IP39 or the 68- and 110-kD oligomers of IP39. The octyl glucoside extracts were incubated with unlabeled cell surface proteins immobilized on nitrocellulose (overlays). Radiolabel from the membrane extract bound one (80 kD) of the two (80 and 86 kD) major membrane skeletal protein bands. Resolubilization of the bound label yielded a radiolabeled polypeptide identical in Mr to IP39. Intact plasma membranes were also digested with papain before or after radioiodination, thereby producing a cytoplasmically truncated IP39. The octyl glucoside extract of truncated IP39 no longer bound to the 80-kD membrane skeletal protein in the nitrocellulose overlays. EM of intact or trypsin digested plasma membranes incubated with membrane skeletal proteins under stringent conditions similar to those used in the nitrocellulose overlays revealed a partially reformed membrane skeletal layer. Little evidence of a membrane skeletal layer was found, however, when plasma membranes were predigested with papain before reassociation. A candidate 80-kD binding domain of IP39 has been tentatively identified as a peptide fragment that was present after trypsin digestion of plasma membranes, but was absent after papain digestion in two-dimensional peptide maps of IP39. Together, these data suggest that the unique peripheral membrane skeleton of Euglena binds to the plasma membrane through noncovalent interactions between the major 80-kD membrane skeletal protein and a small, papain sensitive cytoplasmic domain of IP39.

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

    SciTech Connect

    Briskin, D.P.

    1993-12-31

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

  14. Hydraulic conductivity and aquaporin transcription in roots of trembling aspen (Populus tremuloides) seedlings colonized by Laccaria bicolor.

    PubMed

    Xu, Hao; Cooke, Janice E K; Kemppainen, Minna; Pardo, Alejandro G; Zwiazek, Janusz J

    2016-07-01

    Ectomycorrhizal fungi have been reported to increase root hydraulic conductivity (L pr) by altering apoplastic and plasma membrane intrinsic protein (PIP)-mediated cell-to-cell water transport pathways in associated roots, or to have little effect on root water transport, depending on the interacting species and imposed stresses. In this study, we investigated the water transport properties and PIP transcription in roots of aspen (Populus tremuloides) seedlings colonized by the wild-type strain of Laccaria bicolor and by strains overexpressing a major fungal water-transporting aquaporin JQ585595. Inoculation of aspen seedlings with L. bicolor resulted in about 30 % colonization rate of root tips, which developed dense mantle and the Hartig net that was restricted in the modified root epidermis. Transcript abundance of the aspen aquaporins PIP1;2, PIP2;1, and PIP2;2 decreased in colonized root tips. Root colonization by JQ585595-overexpressing strains had no significant impact on seedling shoot water potentials, gas exchange, or dry mass; however, it led to further decrease in transcript abundance of PIP1;2 and PIP2;3 and the significantly lower L pr than in non-inoculated roots. These results, taken together with our previous study that showed enhanced root water hydraulics of L. bicolor-colonized white spruce (Picea glauca), suggest that the impact of L. bicolor on root hydraulics varies by the ectomycorrhiza-associated tree species. PMID:26861480

  15. Interplay between phosphorylation and palmitoylation mediates plasma membrane targeting and sorting of GAP43

    PubMed Central

    Gauthier-Kemper, Anne; Igaev, Maxim; Sündermann, Frederik; Janning, Dennis; Brühmann, Jörg; Moschner, Katharina; Reyher, Hans-Jürgen; Junge, Wolfgang; Glebov, Konstantin; Walter, Jochen; Bakota, Lidia; Brandt, Roland

    2014-01-01

    Phosphorylation and lipidation provide posttranslational mechanisms that contribute to the distribution of cytosolic proteins in growing nerve cells. The growth-associated protein GAP43 is susceptible to both phosphorylation and S-palmitoylation and is enriched in the tips of extending neurites. However, how phosphorylation and lipidation interplay to mediate sorting of GAP43 is unclear. Using a combination of biochemical, genetic, and imaging approaches, we show that palmitoylation is required for membrane association and that phosphorylation at Ser-41 directs palmitoylated GAP43 to the plasma membrane. Plasma membrane association decreased the diffusion constant fourfold in neuritic shafts. Sorting to the neuritic tip required palmitoylation and active transport and was increased by phosphorylation-mediated plasma membrane interaction. Vesicle tracking revealed transient association of a fraction of GAP43 with exocytic vesicles and motion at a fast axonal transport rate. Simulations confirmed that a combination of diffusion, dynamic plasma membrane interaction and active transport of a small fraction of GAP43 suffices for efficient sorting to growth cones. Our data demonstrate a complex interplay between phosphorylation and lipidation in mediating the localization of GAP43 in neuronal cells. Palmitoylation tags GAP43 for global sorting by piggybacking on exocytic vesicles, whereas phosphorylation locally regulates protein mobility and plasma membrane targeting of palmitoylated GAP43. PMID:25165142

  16. Tuning the resistance of polycarbonate membranes by plasma-induced graft surface modification

    NASA Astrophysics Data System (ADS)

    Baumann, Lukas; Hegemann, Dirk; de Courten, Damien; Wolf, Martin; Rossi, René M.; Meier, Wolfgang P.; Scherer, Lukas J.

    2013-03-01

    To tune the permeability resistance of porous polycarbonate (PC) membranes for caffeine, their surfaces were plasma modified with different monomers in a grafting from process. These coatings provided characteristic surface hydrophilicities. It was found that membranes with more hydrophilic surfaces have lower resistances to let caffeine pass through than membranes with hydrophobic surfaces. Additionally, it was possible to post-modify a poly(2-aminoethyl methacrylate) (AEMA) coated PC membrane with octanoic acid (Oct) under mild conditions. This post modification allowed transforming a slightly hydrophilic PC-AEMA membrane with a moderate permeability resistance into a hydrophobic PC-AEMA-Oct membrane with a high permeability resistance. Overall, it was possible to tune the PC membrane resistance for caffeine in a range from 5100 up to 15,100 s/cm.

  17. Neomycin inhibits the phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate stimulation of plasma membrane ATPase activity

    SciTech Connect

    Chen, Qiuyun; Boss, W.F. )

    1991-05-01

    The inositol phospholipids, phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP{sub 2}), have been shown to increase the vanadate-sensitive ATPase activity of plant plasma membranes. In this paper, the authors show the effect of various concentrations of phosphatidyinositol, PIP, and PIP{sub 2} on the plasma membrane vanadate-sensitive ATPase activity. PIP and PIP{sub 2} at concentrations at 10 nanomoles per 30 microgram membrane protein per milliliter of reaction mixture caused a twofold and 1.8-fold increase in the ATPase activity, respectively. The effect of these negatively charged phospholipids on the ATPase activity was inhibited by adding the positively charged aminoglycoside, neomycin. Neomycin did not affect the endogenous plasma membrane ATPase activity in the absence of exogenous lipids.

  18. Role of C-terminal domain and transmembrane helices 5 and 6 in function and quaternary structure of major intrinsic proteins: analysis of aquaporin/glycerol facilitator chimeric proteins.

    PubMed

    Duchesne, Laurence; Pellerin, Isabelle; Delamarche, Christian; Deschamps, Stephane; Lagree, Valerie; Froger, Alexandrine; Bonnec, Georgette; Thomas, Daniel; Hubert, Jean-Francois

    2002-06-01

    We previously observed that aquaporins and glycerol facilitators exhibit different oligomeric states when studied by sedimentation on density gradients following nondenaturing detergent solubilization. To determine the domains of major intrinsic protein (MIP) family proteins involved in oligomerization, we constructed protein chimeras corresponding to the aquaporin AQPcic substituted in the loop E (including the proximal part of transmembrane domain (TM) 5) and/or the C-terminal part (including the distal part of TM 6) by the equivalent domain of the glycerol channel aquaglyceroporin (GlpF) (chimeras called AGA, AAG, and AGG). The analogous chimeras of GlpF were also constructed (chimeras GAG, GGA, and GAA). cRNA corresponding to all constructs were injected into Xenopus oocytes. AQPcic, GlpF, AAG, AGG, and GAG were targeted to plasma membranes. Water or glycerol membrane permeability measurements demonstrated that only the AAG chimera exhibited a channel function corresponding to water transport. Analysis of all proteins expressed either in oocytes or in yeast by velocity sedimentation on sucrose gradients following solubilization by 2% n-octyl glucoside indicated that only AQPcic and AAG exist in tetrameric forms. GlpF, GAG, and GAA sediment in a monomeric form, whereas GGA and AGG were found mono/dimeric. These data bring new evidence that, within the MIP family, aquaporins and GlpFs behave differently toward nondenaturing detergents. We demonstrate that the C-terminal part of AQPcic, including the distal half of TM 6, can be substituted by the equivalent domain of GlpF (AAG chimera) without modifying the transport specificity. Our results also suggest that interactions of TM 5 of one monomer with TM 1 of the adjacent monomer are crucial for aquaporin tetramer stability. PMID:11927589

  19. Plasma Membrane Repair in Health and Disease.

    PubMed

    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-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 Ca(2+), 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

  20. GPI-anchored proteins do not reside in ordered domains in the live cell plasma membrane

    PubMed Central

    Sevcsik, Eva; Brameshuber, Mario; Fölser, Martin; Weghuber, Julian; Honigmann, Alf; Schütz, Gerhard J.

    2015-01-01

    The organization of proteins and lipids in the plasma membrane has been subject of a long-lasting debate. Membrane rafts of higher lipid chain order were proposed to mediate protein interactions, but have thus far not been directly observed. Here, we use protein micropatterning combined with single-molecule tracking to put current models to the test: we rearranged lipid-anchored raft proteins (glycosylphosphatidylinositol(GPI)-anchored mGFP) directly in the live cell plasma membrane and measured the effect on the local membrane environment. Intriguingly, this treatment does neither nucleate the formation of an ordered membrane phase, nor result in any enrichment of nanoscopic ordered domains within the micropatterned regions. In contrast, we find that immobilized mGFP-GPIs behave as inert obstacles to the diffusion of other membrane constituents without influencing their membrane environment over distances beyond their physical size. Our results indicate that phase partitioning is not a fundamental element of protein organization in the plasma membrane. PMID:25897971

  1. GPI-anchored proteins do not reside in ordered domains in the live cell plasma membrane.

    PubMed

    Sevcsik, Eva; Brameshuber, Mario; Fölser, Martin; Weghuber, Julian; Honigmann, Alf; Schütz, Gerhard J

    2015-01-01

    The organization of proteins and lipids in the plasma membrane has been the subject of a long-lasting debate. Membrane rafts of higher lipid chain order were proposed to mediate protein interactions, but have thus far not been directly observed. Here we use protein micropatterning combined with single-molecule tracking to put current models to the test: we rearranged lipid-anchored raft proteins (glycosylphosphatidylinositol(GPI)-anchored-mGFP) directly in the live cell plasma membrane and measured the effect on the local membrane environment. Intriguingly, this treatment does neither nucleate the formation of an ordered membrane phase nor result in any enrichment of nanoscopic-ordered domains within the micropatterned regions. In contrast, we find that immobilized mGFP-GPIs behave as inert obstacles to the diffusion of other membrane constituents without influencing their membrane environment over distances beyond their physical size. Our results indicate that phase partitioning is not a fundamental element of protein organization in the plasma membrane. PMID:25897971

  2. Inositol 1,4,5-trisphosphate-induced calcium release from platelet plasma membrane vesicles

    SciTech Connect

    Rengasamy, A.; Feinberg, H.

    1988-02-15

    A platelet membrane preparation, enriched in plasma membrane markers, took up /sup 45/Ca/sup 2 +/ in exchange for intravesicular Na+ and released it after the addition of inositol 1,4,5-trisphosphate (IP3). The possibility that contaminating dense tubular membrane (DTS) vesicles contributed the Ca/sup 2 +/ released by IP3 was eliminated by the addition of vanadate to inhibit Ca/sup +/-ATPase-mediated DTS Ca/sup 2 +/ sequestration and by the finding that only plasma membrane vesicles exhibit Na/sup +/-dependent Ca/sup 2 +/ uptake. Ca/sup 2 +/ released by IP3 was dependent on low extravesicular Ca/sup 2 +/ concentrations. IP3-induced Ca/sup 2 +/ release was additive to that released by Na/sup +/ addition while GTP or polyethylene glycol (PEG) had no effect. These results strongly suggest that IP3 facilitates extracellular Ca/sup 2 +/ influx in addition to release from DTS membranes.

  3. Effects of non-thermal plasma on the electrical properties of an erythrocyte membrane

    NASA Astrophysics Data System (ADS)

    Lee, Jin Young; Baik, Ku Youn; Kim, Tae Soo; Lim, Jaekwan; Uhm, Han S.; Choi, Eun Ha

    2015-09-01

    Non-thermal plasma is used here for membrane oxidation and permeabilization in which the electrical properties of an erythrocyte membrane are investigated after treatments. The zeta potential as measured by electrophoresis shows the increased negativity of the membrane surface potential (Ψs). The secondary electron emission coefficient ( γ) measured by a focused ion beam shows a decrease in the dipole potential (Ψd) of lipid molecules. The voltage-sensitive fluorescent intensity as measured by flow cytometry shows a decrease in the trans-membrane potential (ΔΨ) through the lipid bilayer membrane. These results allow us to take a step forward to unveil the complex events occurring in plasma-treated cells.

  4. Structural determinants of proton blockage in aquaporins.

    PubMed

    Chakrabarti, Nilmadhab; Roux, Benoît; Pomès, Régis

    2004-10-15

    Aquaporins are an important class of membrane channels selective for water and linear polyols but impermeable to ions, including protons. Recent computational studies have revealed that the relay of protons through the water-conduction pathway of aquaporin channels is opposed by a substantial free energy barrier peaking at the signature NPA motifs. Here, free-energy simulations and continuum electrostatic calculations are combined to examine the nature and the magnitude of the contribution of specific structural elements to proton blockage in the bacterial glycerol uptake facilitator, GlpF. Potential of mean-force profiles for both hop and turn steps of structural diffusion in the narrow pore are obtained for artificial variants of the GlpF channel in which coulombic interactions between the pore contents and conserved residues Asn68 and Asn203 at the NPA signature motifs, Arg206 at the selectivity filter, and the peptidic backbone of the two half-helices M3 and M7, which are arranged in head-to-head fashion around the NPA motifs, are turned off selectively. A comparison of these results with electrostatic energy profiles for the translocation of a probe cation throughout the water permeation pathway indicates that the free-energy profile for proton movement inside the narrow pore is dominated by static effects arising from the distribution of charged and polar groups of the channel, whereas dielectric effects contribute primarily to opposing the access of H+ to the pore mouths (desolvation penalty). The single most effective way to abolish the free-energy gradients opposing the movement of H+ around the NPA motif is to turn off the dipole moments of helices M3 and M7. Mutation of either of the two NPA Asn residues to Asp compensates for charge-dipole and dipole-dipole effects opposing the hop and turn steps of structural diffusion, respectively, and dramatically reduces the free energy barrier of proton translocation, suggesting that these single mutants could

  5. Characterization of plasma-induced cell membrane permeabilization: focus on OH radical distribution

    NASA Astrophysics Data System (ADS)

    Sasaki, Shota; Honda, Ryosuke; Hokari, Yutaro; Takashima, Keisuke; Kanzaki, Makoto; Kaneko, Toshiro

    2016-08-01

    Non-equilibrium atmospheric-pressure plasma (APP) is used medically for plasma-induced cell permeabilization. However, how plasma irradiation specifically triggers permeabilization remains unclear. In an attempt to identify the dominant factor(s), the distribution of plasma-produced reactive species was investigated, primarily focusing on OH radicals. A stronger plasma discharge, which produced more OH radicals in the gas phase, also produced more OH radicals in the liquid phase (OHaq), enhancing the cell membrane permeability. In addition, plasma irradiation-induced enhancement of cell membrane permeability decreased markedly with increased solution thickness (<1 mm), and the plasma-produced OHaq decayed in solution (diffusion length on the order of several hundred micrometers). Furthermore, the horizontally center-localized distribution of OHaq corresponded with the distribution of the permeabilized cells by plasma irradiation, while the overall plasma-produced oxidizing species in solution (detected by iodine-starch reaction) exhibited a doughnut-shaped horizontal distribution. These results suggest that OHaq, among the plasma-produced oxidizing species, represents the dominant factor in plasma-induced cell permeabilization. These results enhance the current understanding of the mechanism of APP as a cell-permeabilization tool.

  6. A banana aquaporin gene, MaPIP1;1, is involved in tolerance to drought and salt stresses

    PubMed Central

    2014-01-01

    Background Aquaporin (AQP) proteins function in transporting water and other small molecules through the biological membranes, which is crucial for plants to survive in drought or salt stress conditions. However, the precise role of AQPs in drought and salt stresses is not completely understood in plants. Results In this study, we have identified a PIP1 subfamily AQP (MaPIP1;1) gene from banana and characterized it by overexpression in transgenic Arabidopsis plants. Transient expression of MaPIP1;1-GFP fusion protein indicated its localization at plasma membrane. The expression of MaPIP1;1 was induced by NaCl and water deficient treatment. Overexpression of MaPIP1;1 in Arabidopsis resulted in an increased primary root elongation, root hair numbers and survival rates compared to WT under salt or drought conditions. Physiological indices demonstrated that the increased salt tolerance conferred by MaPIP1;1 is related to reduced membrane injury and high cytosolic K+/Na+ ratio. Additionally, the improved drought tolerance conferred by MaPIP1;1 is associated with decreased membrane injury and improved osmotic adjustment. Finally, reduced expression of ABA-responsive genes in MaPIP1;1-overexpressing plants reflects their improved physiological status. Conclusions Our results demonstrated that heterologous expression of banana MaPIP1;1 in Arabidopsis confers salt and drought stress tolerances by reducing membrane injury, improving ion distribution and maintaining osmotic balance. PMID:24606771

  7. The anti-inflammatory drug indomethacin alters nanoclustering in synthetic and cell plasma membranes.

    PubMed

    Zhou, Yong; Plowman, Sarah J; Lichtenberger, Lenard M; Hancock, John F

    2010-11-01

    The nonsteroidal anti-inflammatory drug indomethacin exhibits diverse biological effects, many of which have no clear molecular mechanism. Membrane-bound receptors and enzymes are sensitive to their phospholipid microenvironment. Amphipathic indomethacin could therefore potentially modulate cell signaling by changing membrane properties. Here we examined the effect of indomethacin on membrane lateral heterogeneity. Fluorescence lifetime imaging of cells expressing lipid-anchored probes revealed that treatment of BHK cells with therapeutic levels of indomethacin enhances cholesterol-dependent nanoclustering, but not cholesterol-independent nanoclustering. Immuno-electron microscopy and quantitative spatial mapping of intact plasma membrane sheets similarly showed a selective effect of indomethacin on promoting cholesterol-dependent, but not cholesterol-independent, nanoclustering. To further evaluate the biophysical effects of indomethacin, we measured fluorescence polarization of the phase-sensitive probe Laurdan and FRET between phase-partitioning probes in model bilayers. Therapeutic levels of indomethacin enhanced phase separation in DPPC/DOPC/Chol (1:1:1) and DPPC/Chol membranes in a temperature-dependent manner, but had minimal effect on the phase behavior of pure DOPC at any temperature. Taken together, the imaging results on intact epithelial cells and the biophysical assays of model membranes suggest that indomethacin can enhance phase separation and stabilize cholesterol-dependent nanoclusters in biological membranes. These effects on membrane lateral heterogeneity may have significant consequences for cell signaling cascades that are assembled on the plasma membrane. PMID:20826816

  8. The Anti-inflammatory Drug Indomethacin Alters Nanoclustering in Synthetic and Cell Plasma Membranes*

    PubMed Central

    Zhou, Yong; Plowman, Sarah J.; Lichtenberger, Lenard M.; Hancock, John F.

    2010-01-01

    The nonsteroidal anti-inflammatory drug indomethacin exhibits diverse biological effects, many of which have no clear molecular mechanism. Membrane-bound receptors and enzymes are sensitive to their phospholipid microenvironment. Amphipathic indomethacin could therefore potentially modulate cell signaling by changing membrane properties. Here we examined the effect of indomethacin on membrane lateral heterogeneity. Fluorescence lifetime imaging of cells expressing lipid-anchored probes revealed that treatment of BHK cells with therapeutic levels of indomethacin enhances cholesterol-dependent nanoclustering, but not cholesterol-independent nanoclustering. Immuno-electron microscopy and quantitative spatial mapping of intact plasma membrane sheets similarly showed a selective effect of indomethacin on promoting cholesterol-dependent, but not cholesterol-independent, nanoclustering. To further evaluate the biophysical effects of indomethacin, we measured fluorescence polarization of the phase-sensitive probe Laurdan and FRET between phase-partitioning probes in model bilayers. Therapeutic levels of indomethacin enhanced phase seperation in DPPC/DOPC/Chol (1:1:1) and DPPC/Chol membranes in a temperature-dependent manner, but had minimal effect on the phase behavior of pure DOPC at any temperature. Taken together, the imaging results on intact epithelial cells and the biophysical assays of model membranes suggest that indomethacin can enhance phase separation and stabilize cholesterol-dependent nanoclusters in biological membranes. These effects on membrane lateral heterogeneity may have significant consequences for cell signaling cascades that are assembled on the plasma membrane. PMID:20826816

  9. Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies

    PubMed Central

    Zidovetzki, Raphael

    2007-01-01

    The physiological importance of cholesterol in the cell plasma membrane has attracted increased attention in recent years. Consequently, the use of methods of controlled manipulation of membrane cholesterol content has also increased sharply, especially as a method of studying putative cholesterol-enriched cell membrane domains (rafts). The most common means of modifying the cholesterol content of cell membranes is the incubation of cells or model membranes with cyclodextrins, a family of compounds, which, due to the presence of relatively hydrophobic cavity, can be used to extract cholesterol from cell membranes. However, the mechanism of this activity of cyclodextrins is not completely established. Moreover, under conditions commonly used for cholesterol extraction, cyclodextrins may remove cholesterol from both raft and non-raft domains of the membrane as well as alter the distribution of cholesterol between plasma and intracellular membranes. In addition, other hydrophobic molecules such as phospholipids may also be extracted from the membranes by cyclodextrins. We review the evidence for the specific and non-specific effects of cyclodextrins and what is known about the mechanisms for cyclodextrin-induced cholesterol and phospholipid extraction. Finally, we discuss useful control strategies that may help to verify that the observed effects are due specifically to cyclodextrin-induced changes in cellular cholesterol. PMID:17493580

  10. Increased Differentiation Capacity of Bone Marrow-Derived Mesenchymal Stem Cells in Aquaporin-5 Deficiency

    PubMed Central

    Yi, Fei; Khan, Muhammad; Gao, Hongwen; Hao, Feng; Sun, Meiyan; Zhong, Lili; Lu, Changzheng; Feng, Xuechao

    2012-01-01

    Mesenchymal stem cells (MSCs) are adult stem cells with a self-renewal and multipotent capability and express extensively in multitudinous tissues. We found that water channel aquaporin-5 (AQP5) is expressed in bone marrow-derived MSCs (BMMSCs) in the plasma membrane pattern. BMMSCs from AQP5−/− mice showed significantly lower plasma membrane water permeability than those from AQP5+/+ mice. In characterizing the cultured BMMSCs from AQP5−/− and AQP5+/+ mice, we found no obvious differences in morphology and proliferation between the 2 genotypes. However, the multiple differentiation capacity was significantly higher in AQP5−/− than AQP5+/+ BMMSCs as revealed by representative staining by Oil Red O (adipogenesis); Alizarin Red S and alkaline phosphatase (ALP; osteogenesis); and type II collagen and Safranin O (chondrogenesis) after directional induction. Relative mRNA expression levels of 3 lineage differentiation markers, including PPARγ2, C/EBPα, adipsin, collagen 1a, osteopontin, ALP, collagen 11a, collagen 2a, and aggrecan, were significantly higher in AQP5−/− -differentiating BMMSCs, supporting an increased differentiation capacity of AQP5−/− BMMSCs. Furthermore, a bone-healing process was accelerated in AQP5−/− mice in a drill-hole injury model. Mechanistic studies indicated a significantly lower apoptosis rate in AQP5−/− than AQP5+/+ BMMSCs. Apoptosis inhibitor Z-VAD-FMK increased the differentiation capacity to a greater extent in AQP5+/+ than AQP5−/− BMMSCs. We conclude that AQP5-mediated high plasma membrane water permeability enhances the apoptosis rate of differentiating BMMSCs, thus decreasing their differentiation capacity. These data implicate AQP5 as a novel determinant of differentiation of BMMSCs and therefore a new molecular target for regulating differentiation of BMMSCs during tissue repair and regeneration. PMID:22420587

  11. Characterization of the Red Beet Plasma Membrane H+-ATPase Reconstituted in a Planar Bilayer System.

    PubMed Central

    Briskin, D. P.; Basu, S.; Assmann, S. M.

    1995-01-01

    The transport activity of the red beet (Beta vulgaris L.) plasma membrane H+-ATPase was examined following reconstitution into a planar bilayer membrane. Fusion of partially purified plasma membrane H+-ATPase with the bilayer membrane was accomplished by perfusion of proteoliposomes against the bilayer under hypoosmotic conditions. Following incorporation into the bilayer, an ATP-dependent current was measured that demonstrated properties consistent with those of the plasma membrane H+-ATPase. Current production was substrate specific for ATP, inhibited by orthovanadate, and insensitive to 200 nM erythrosin B but inhibited by 100 [mu]M erythrosin B. When current production was measured as a function of Mg:ATP concentration, a simple Michaelis-Menten relationship was observed and a Km of 0.62 mM was estimated. Current-voltage analysis of ATP-dependent current in the presence of 0.5 mM ATP, 20 mM ADP, 40 mM orthophosphate, and an opposing 2.5-unit [delta]pH revealed a reversal potential of about -149 mV. Based on the free energy available from ATP hydrolysis, this reversal potential is consistent with an H+/ATP stoichiometry of 1. This study demonstrates the usefulness of a planar bilayer system for investigation of energy coupling to H+ transport by the plasma membrane H+-ATPase. PMID:12228483

  12. Deposition of polymeric perfluored thin films in proton ionic membranes by plasma processes

    NASA Astrophysics Data System (ADS)

    Polak, Peter Lubomir; Mousinho, Ana Paula; Ordonez, Nelson; da Silva Zambom, Luis; Mansano, Ronaldo Domingues

    2007-10-01

    In this work the surfaces of polymeric membranes based on Nafion (proton conducting material), used in proton exchange membranes fuel cells (PEMFC) had been modified by plasma deposition of perfluored polymers, in order to improve its functioning in systems of energy generation (fuel cells). The deposition increases the chemical resistance of the proton ionic polymers without losing the electrical properties. The processing of the membranes also reduces the permeability of the membranes to the alcohols (methanol and ethanol), thus preventing poisoning of the fuel cell. The processing of the membranes of Nafion was carried through in a system of plasma deposition using a mixture of CF 4 and H 2 gases. The plasma processing was made mainly to increase the chemical resistance and result in hydrophobic surfaces. The Fourier transformed infrared (FTIR) technique supplies a spectrum with information about the CF n bond formation. Through the Rutherford back scattering (RBS) technique it was possible to verify the deposition rate of the polymeric layer. The plasma process with composition of 60% of CF 4 and 40% of H 2 presented the best deposition rate. By the spectrum analysis for the optimized configuration, it was possible to verify that the film deposition occurred with a thickness of 90 nm, and fluorine concentration was nearly 30%. Voltammetry made possible to verify that the fluorination increases the membranes chemical resistance, improving the stability of Nafion, becoming an attractive process for construction of fuel cells.

  13. Oligomerization and Pore Formation by Equinatoxin II Inhibit Endocytosis and Lead to Plasma Membrane Reorganization*

    PubMed Central

    García-Sáez, Ana J.; Buschhorn, Sabine B.; Keller, Heiko; Anderluh, Gregor; Simons, Kai; Schwille, Petra

    2011-01-01

    Pore-forming toxins have evolved to induce membrane injury by formation of pores in the target cell that alter ion homeostasis and lead to cell death. Many pore-forming toxins use cholesterol, sphingolipids, or other raft components as receptors. However, the role of plasma membrane organization for toxin action is not well understood. In this study, we have investigated cellular dynamics during the attack of equinatoxin II, a pore-forming toxin from the sea anemone Actinia equina, by combining time lapse three-dimensional live cell imaging, fluorescence recovery after photobleaching, FRET, and fluorescence cross-correlation spectroscopy. Our results show that membrane binding by equinatoxin II is accompanied by extensive plasma membrane reorganization into microscopic domains that resemble coalesced lipid rafts. Pore formation by the toxin induces Ca2+ entry into the cytosol, which is accompanied by hydrolysis of phosphatidylinositol 4,5-bisphosphate, plasma membrane blebbing, actin cytoskeleton reorganization, and inhibition of endocytosis. We propose that plasma membrane reorganization into stabilized raft domains is part of the killing strategy of equinatoxin II. PMID:21885440

  14. The relationship between cAMP, Ca(2)+, and transport of CFTR to the plasma membrane.

    PubMed

    Chen, P; Hwang, T C; Gillis, K D

    2001-08-01

    The mechanism whereby cAMP stimulates Cl(-) flux through CFTR ion channels in secretory epithelia remains controversial. It is generally accepted that phosphorylation by cAMP-dependent protein kinase increases the open probability of the CFTR channel. A more controversial hypothesis is that cAMP triggers the translocation of CFTR from an intracellular pool to the cell surface. We have monitored membrane turnover in Calu-3 cells, a cell line derived from human airway submucosal glands that expresses high levels of CFTR using membrane capacitance and FM1-43 fluorescence measurements. Using a conventional capacitance measurement technique, we observe an apparent increase in membrane capacitance in most cells that exhibit an increase in Cl(-) current. However, after we carefully correct our recordings for changes in membrane conductance, the apparent changes in capacitance are eliminated. Measurements using the fluorescent membrane marker FM1-43 also indicate that no changes in membrane turnover accompany the activation of CFTR. Robust membrane insertion can be triggered with photorelease of caged Ca(2)+ in Calu-3 cells. However, no increase in Cl(-) current accompanies Ca(2)+-evoked membrane fusion. We conclude that neither increases in cAMP or Ca(2)+ lead to transport of CFTR to the plasma membrane in Calu-3 cells. In addition, we conclude that membrane capacitance measurements must be interpreted with caution when large changes in membrane conductance occur. PMID:11479341

  15. The Relationship between Camp, Ca2+, and Transport of Cftr to the Plasma Membrane

    PubMed Central

    Chen, Peng; Hwang, Tzyh-Chang; Gillis, Kevin D.

    2001-01-01

    The mechanism whereby cAMP stimulates Cl− flux through CFTR ion channels in secretory epithelia remains controversial. It is generally accepted that phosphorylation by cAMP-dependent protein kinase increases the open probability of the CFTR channel. A more controversial hypothesis is that cAMP triggers the translocation of CFTR from an intracellular pool to the cell surface. We have monitored membrane turnover in Calu-3 cells, a cell line derived from human airway submucosal glands that expresses high levels of CFTR using membrane capacitance and FM1–43 fluorescence measurements. Using a conventional capacitance measurement technique, we observe an apparent increase in membrane capacitance in most cells that exhibit an increase in Cl− current. However, after we carefully correct our recordings for changes in membrane conductance, the apparent changes in capacitance are eliminated. Measurements using the fluorescent membrane marker FM1–43 also indicate that no changes in membrane turnover accompany the activation of CFTR. Robust membrane insertion can be triggered with photorelease of caged Ca2+ in Calu-3 cells. However, no increase in Cl− current accompanies Ca2+-evoked membrane fusion. We conclude that neither increases in cAMP or Ca2+ lead to transport of CFTR to the plasma membrane in Calu-3 cells. In addition, we conclude that membrane capacitance measurements must be interpreted with caution when large changes in membrane conductance occur. PMID:11479341

  16. Towards Enhanced Performance Thin-film Composite Membranes via Surface Plasma Modification

    PubMed Central

    Reis, Rackel; Dumée, Ludovic F.; Tardy, Blaise L.; Dagastine, Raymond; Orbell, John D.; Schutz, Jürg A.; Duke, Mikel C.

    2016-01-01

    Advancing the design of thin-film composite membrane surfaces is one of the most promising pathways to deal with treating varying water qualities and increase their long-term stability and permeability. Although plasma technologies have been explored for surface modification of bulk micro and ultrafiltration membrane materials, the modification of thin film composite membranes is yet to be systematically investigated. Here, the performance of commercial thin-film composite desalination membranes has been significantly enhanced by rapid and facile, low pressure, argon plasma activation. Pressure driven water desalination tests showed that at low power density, flux was improved by 22% without compromising salt rejection. Various plasma durations and excitation powers have been systematically evaluated to assess the impact of plasma glow reactions on the physico-chemical properties of these materials associated with permeability. With increasing power density, plasma treatment enhanced the hydrophilicity of the surfaces, where water contact angles decreasing by 70% were strongly correlated with increased negative charge and smooth uniform surface morphology. These results highlight a versatile chemical modification technique for post-treatment of commercial membrane products that provides uniform morphology and chemically altered surface properties. PMID:27363670

  17. The exocyst complex is required for targeting of Glut4 to the plasma membrane by insulin.

    PubMed

    Inoue, Mayumi; Chang, Louise; Hwang, Joseph; Chiang, Shian-Huey; Saltiel, Alan R

    2003-04-10

    Insulin stimulates glucose transport by promoting exocytosis of the glucose transporter Glut4 (refs 1, 2). The dynamic processes involved in the trafficking of Glut4-containing vesicles, and in their targeting, docking and fusion at the plasma membrane, as well as the signalling processes that govern these events, are not well understood. We recently described tyrosine-phosphorylation events restricted to subdomains of the plasma membrane that result in activation of the G protein TC10 (refs 3, 4). Here we show that TC10 interacts with one of the components of the exocyst complex, Exo70. Exo70 translocates to the plasma membrane in response to insulin through the activation of TC10, where it assembles a multiprotein complex that includes Sec6 and Sec8. Overexpression of an Exo70 mutant blocked insulin-stimulated glucose uptake, but not the trafficking of Glut4 to the plasma membrane. However, this mutant did block the extracellular exposure of the Glut4 protein. So, the exocyst might have a crucial role in the targeting of the Glut4 vesicle to the plasma membrane, perhaps directing the vesicle to the precise site of fusion. PMID:12687004

  18. Visualization of the exocyst complex dynamics at the plasma membrane of Arabidopsis thaliana.

    PubMed

    Fendrych, Matyás; Synek, Lukás; Pecenková, Tamara; Drdová, Edita Janková; Sekeres, Juraj; de Rycke, Riet; Nowack, Moritz K; Zársky, Viktor

    2013-02-01

    The exocyst complex, an effector of Rho and Rab GTPases, is believed to function as an exocytotic vesicle tether at the plasma membrane before soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation. Exocyst subunits localize to secretory-active regions of the plasma membrane, exemplified by the outer domain of Arabidopsis root epidermal cells. Using variable-angle epifluorescence microscopy, we visualized the dynamics of exocyst subunits at this domain. The subunits colocalized in defined foci at the plasma membrane, distinct from endocytic sites. Exocyst foci were independent of cytoskeleton, although prolonged actin disruption led to changes in exocyst localization. Exocyst foci partially overlapped with vesicles visualized by VAMP721 v-SNARE, but the majority of the foci represent sites without vesicles, as indicated by electron microscopy and drug treatments, supporting the concept of the exocyst functioning as a dynamic particle. We observed a decrease of SEC6-green fluorescent protein foci in an exo70A1 exocyst mutant. Finally, we documented decreased VAMP721 trafficking to the plasma membrane in exo70A1 and exo84b mutants. Our data support the concept that the exocyst-complex subunits dynamically dock and undock at the plasma membrane to create sites primed for vesicle tethering. PMID:23283982

  19. Towards Enhanced Performance Thin-film Composite Membranes via Surface Plasma Modification.

    PubMed

    Reis, Rackel; Dumée, Ludovic F; Tardy, Blaise L; Dagastine, Raymond; Orbell, John D; Schutz, Jürg A; Duke, Mikel C

    2016-01-01

    Advancing the design of thin-film composite membrane surfaces is one of the most promising pathways to deal with treating varying water qualities and increase their long-term stability and permeability. Although plasma technologies have been explored for surface modification of bulk micro and ultrafiltration membrane materials, the modification of thin film composite membranes is yet to be systematically investigated. Here, the performance of commercial thin-film composite desalination membranes has been significantly enhanced by rapid and facile, low pressure, argon plasma activation. Pressure driven water desalination tests showed that at low power density, flux was improved by 22% without compromising salt rejection. Various plasma durations and excitation powers have been systematically evaluated to assess the impact of plasma glow reactions on the physico-chemical properties of these materials associated with permeability. With increasing power density, plasma treatment enhanced the hydrophilicity of the surfaces, where water contact angles decreasing by 70% were strongly correlated with increased negative charge and smooth uniform surface morphology. These results highlight a versatile chemical modification technique for post-treatment of commercial membrane products that provides uniform morphology and chemically altered surface properties. PMID:27363670

  20. Visualization of the exocyst complex dynamics at the plasma membrane of Arabidopsis thaliana

    PubMed Central

    Fendrych, Matyáš; Synek, Lukáš; Pečenková, Tamara; Drdová, Edita Janková; Sekereš, Juraj; de Rycke, Riet; Nowack, Moritz K.; Žárský, Viktor

    2013-01-01

    The exocyst complex, an effector of Rho and Rab GTPases, is believed to function as an exocytotic vesicle tether at the plasma membrane before soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complex formation. Exocyst subunits localize to secretory-active regions of the plasma membrane, exemplified by the outer domain of Arabidopsis root epidermal cells. Using variable-angle epifluorescence microscopy, we visualized the dynamics of exocyst subunits at this domain. The subunits colocalized in defined foci at the plasma membrane, distinct from endocytic sites. Exocyst foci were independent of cytoskeleton, although prolonged actin disruption led to changes in exocyst localization. Exocyst foci partially overlapped with vesicles visualized by VAMP721 v-SNARE, but the majority of the foci represent sites without vesicles, as indicated by electron microscopy and drug treatments, supporting the concept of the exocyst functioning as a dynamic particle. We observed a decrease of SEC6–green fluorescent protein foci in an exo70A1 exocyst mutant. Finally, we documented decreased VAMP721 trafficking to the plasma membrane in exo70A1 and exo84b mutants. Our data support the concept that the exocyst-complex subunits dynamically dock and undock at the plasma membrane to create sites primed for vesicle tethering. PMID:23283982

  1. Towards Enhanced Performance Thin-film Composite Membranes via Surface Plasma Modification

    NASA Astrophysics Data System (ADS)

    Reis, Rackel; Dumée, Ludovic F.; Tardy, Blaise L.; Dagastine, Raymond; Orbell, John D.; Schutz, Jürg A.; Duke, Mikel C.

    2016-07-01

    Advancing the design of thin-film composite membrane surfaces is one of the most promising pathways to deal with treating varying water qualities and increase their long-term stability and permeability. Although plasma technologies have been explored for surface modification of bulk micro and ultrafiltration membrane materials, the modification of thin film composite membranes is yet to be systematically investigated. Here, the performance of commercial thin-film composite desalination membranes has been significantly enhanced by rapid and facile, low pressure, argon plasma activation. Pressure driven water desalination tests showed that at low power density, flux was improved by 22% without compromising salt rejection. Various plasma durations and excitation powers have been systematically evaluated to assess the impact of plasma glow reactions on the physico-chemical properties of these materials associated with permeability. With increasing power density, plasma treatment enhanced the hydrophilicity of the surfaces, where water contact angles decreasing by 70% were strongly correlated with increased negative charge and smooth uniform surface morphology. These results highlight a versatile chemical modification technique for post-treatment of commercial membrane products that provides uniform morphology and chemically altered surface properties.

  2. Eisosomes promote the ability of Sur7 to regulate plasma membrane organization in Candida albicans

    PubMed Central

    Wang, Hong X.; Douglas, Lois M.; Veselá, Petra; Rachel, Reinhard; Malinsky, Jan; Konopka, James B.

    2016-01-01

    The plasma membrane of the fungal pathogen Candida albicans forms a protective barrier that also mediates many processes needed for virulence, including cell wall synthesis, invasive hyphal morphogenesis, and nutrient uptake. Because compartmentalization of the plasma membrane is believed to coordinate these diverse activities, we examined plasma membrane microdomains termed eisosomes or membrane compartment of Can1 (MCC), which correspond to ∼200-nm-long furrows in the plasma membrane. A pil1∆ lsp1∆ mutant failed to form eisosomes and displayed strong defects in plasma membrane organization and morphogenesis, including extensive cell wall invaginations. Mutation of eisosome proteins Slm2, Pkh2, and Pkh3 did not cause similar cell wall defects, although pkh2∆ cells formed chains of furrows and pkh3∆ cells formed wider furrows, identifying novel roles for the Pkh protein kinases in regulating furrows. In contrast, the sur7∆ mutant formed cell wall invaginations similar to those for the pil1∆ lsp1∆ mutant even though it could form eisosomes and furrows. A PH-domain probe revealed that the regulatory lipid phosphatidylinositol 4,5-bisphosphate was enriched at sites of cell wall invaginations in both the sur7∆ and pil1∆ lsp1∆ cells, indicating that this contributes to the defects. The sur7∆ and pil1∆ lsp1∆ mutants displayed differential susceptibility to various types of stress, indicating that they affect overlapping but distinct functions. In support of this, many mutant phenotypes of the pil1∆ lsp1∆ cells were rescued by overexpressing SUR7. These results demonstrate that C. albicans eisosomes promote the ability of Sur7 to regulate plasma membrane organization. PMID:27009204

  3. The plasma membrane as a capacitor for energy and metabolism.

    PubMed

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

    2016-02-01

    When considering which components of the cell are the most critical to function and physiology, we naturally focus on the nucleus, the mitochondria that regulate energy and apoptotic signaling, or other organelles such as the endoplasmic reticulum, Golgi, ribosomes, etc. Few people will suggest that the membrane is the most critical element of a cell in terms of function and physiology. Those that consider the membrane critical will point to its obvious barrier function regulated by the lipid bilayer and numerous ion channels that regulate homeostatic gradients. What becomes evident upon closer inspection is that not all membranes are created equal and that there are lipid-rich microdomains that serve as platforms of signaling and a means of communication with the intracellular environment. In this review, we explore the evolution of membranes, focus on lipid-rich microdomains, and advance the novel concept that membranes serve as "capacitors for energy and metabolism." Within this framework, the membrane then is the primary and critical regulator of stress and disease adaptation of the cell. PMID:26771520

  4. Large Pore Ion and Metabolite-Permeable Channel Regulation of Postnatal Ventricular Zone Neural Stem and Progenitor Cells: Interplay between Aquaporins, Connexins, and Pannexins?

    PubMed Central

    Wicki-Stordeur, Leigh E.; Swayne, Leigh Anne

    2012-01-01

    The birth of new neurons from unspecialized neural stem and progenitor cells surrounding the lateral ventricles occurs throughout postnatal life. This process, termed neurogenesis, is complex and multistepped, encompassing several types of cellular behaviours, such as proliferation, differentiation, and migration. These behaviours are influenced by numerous factors present in the unique, permissive microenvironment. A major cellular mechanism for sensing the plethora of environmental cues directing this process is the presence of different channel forming proteins spanning the plasma membrane. So-called large pore membrane channels, which are selective for the passage of specific types of small molecules and ions, are emerging as an important subgroup of channel proteins. Here, we focus on the roles of three such large pore channels, aquaporin 4, connexin 43, and pannexin 1. We highlight both their independent functions as well as the accumulating evidence for crosstalk between them. PMID:22754577

  5. D184E mutation in aquaporin-4 gene impairs water permeability and links to deafness.

    PubMed

    Nicchia, G P; Ficarella, R; Rossi, A; Giangreco, I; Nicolotti, O; Carotti, A; Pisani, F; Estivill, X; Gasparini, P; Svelto, M; Frigeri, A

    2011-12-01

    Aquaporins (AQPs) play a physiological role in several organs and tissues, and their alteration is associated with disorders of water regulation. The identification of molecular interactions, which are crucial in determining the rate of water flux through the channel, is of pivotal role for the discovery of molecules able to target those interactions and therefore to be used for pathologies ascribable to an altered AQP-dependent water balance. In the present study, a mutational screening of human aquaporin-4 (AQP4) gene was performed on subjects with variable degrees of hearing loss. One heterozygous missense mutation was identified in a Spanish sporadic case, leading to an Asp/Glu amino acid substitution at position 184 (D184E). A BLAST analysis revealed that the amino acid D184 is conserved across species, consistently with a crucial role in the structure/function of AQP4 water channels. The mutation induces a significant reduction in water permeability as measured by the Xenopus laevis oocytes swelling assay and by the use of mammalian cells by total internal reflection microscopy. By Western blot, immunofluorescence and 2D Blue Native/SDS-PAGE we show that the reduction in water permeability is not ascribable to a reduced expression of AQP4 mutant protein or to its incorrect plasma membrane targeting and aggregation into orthogonal arrays of particles. Molecular dynamics simulation provided a molecular explanation of the mechanism whereby the mutation induces a loss of function of the channel. Substituting glutamate for aspartate affects the mobility of the D loop, which acquires a higher propensity to equilibrate in a "closed conformation", thus affecting the rate of water flux. We speculate that this mutation, combined with other genetic defects or concurrently with certain environmental stimuli, could confer a higher susceptibility to deafness. PMID:21952128

  6. Role of the aquaporin PIP1 subfamily in the chilling tolerance of rice.

    PubMed

    Matsumoto, Tadashi; Lian, Hong-Li; Su, Wei-Ai; Tanaka, Daisuke; Liu, Cheng wei; Iwasaki, Ikuko; Kitagawa, Yoshichika

    2009-02-01

    Although an association between chilling tolerance and aquaporins has been reported, the exact mechanisms involved in this relationship remain unclear. We compared the expression profiles of aquaporin genes between a chilling-tolerant and a low temperature-sensitive rice variety using real-time PCR and identified seven genes that closely correlated with chilling tolerance. Chemical treatment experiments, by which rice plants were induced to lose their chilling tolerance, implicated the PIP1 (plasma membrane intrinsic protein 1) subfamily member genes in chilling tolerance. Of these members, changes in expression of the OsPIP1;3 gene suggested this to be the most closely related to chilling tolerance. Although OsPIP1;3 showed a much lower water permeability than members of the OsPIP2 family, OsPIP1;3 enhanced the water permeability of OsPIP2;2 and OsPIP2;4 when co-expressed with either of these proteins in oocytes. Transgenic rice plants (OE1) overexpressing OsPIP1;3 showed an enhanced level of chilling tolerance and the ability to maintain high OsPIP1;3 expression levels under low temperature treatment, similar to that of chilling-tolerant rice plants. We assume that OsPIP1;3, constitutively overexpressed in the leaf and root of transgenic OE1 plants, interacts with members of the OsPIP2 subfamily, thereby improving the plants' water balance under low temperatures and resulting in the observed chilling tolerance of the plants. PMID:19098326

  7. An aquaporin PvTIP4;1 from Pteris vittata may mediate arsenite uptake.

    PubMed

    He, Zhenyan; Yan, Huili; Chen, Yanshan; Shen, Hongling; Xu, Wenxiu; Zhang, Haiyan; Shi, Lei; Zhu, Yong-Guan; Ma, Mi

    2016-01-01

    The fern Pteris vittata is an arsenic hyperaccumulator. The genes involved in arsenite (As(III)) transport are not yet clear. Here, we describe the isolation and characterization of a new P. vittata aquaporin gene, PvTIP4;1, which may mediate As(III) uptake. PvTIP4;1 was identified from yeast functional complement cDNA library of P. vittata. Arsenic toxicity and accumulating activities of PvTIP4;1 were analyzed in Saccharomyces cerevisiae and Arabidopsis. Subcellular localization of PvTIP4;1-GFP fusion protein in P. vittata protoplast and callus was conducted. The tissue expression of PvTIP4;1 was investigated by quantitative real-time PCR. Site-directed mutagenesis of the PvTIP4;1 aromatic/arginine (Ar/R) domain was studied. Heterologous expression in yeast demonstrates that PvTIP4;1 was able to facilitate As(III) diffusion. Transgenic Arabidopsis showed that PvTIP4;1 increases arsenic accumulation and induces arsenic sensitivity. Images and FM4-64 staining suggest that PvTIP4;1 localizes to the plasma membrane in P. vittata cells. A tissue location study shows that PvTIP4;1 transcripts are mainly expressed in roots. Site-directed mutation in yeast further proved that the cysteine at the LE1 position of PvTIP4;1 Ar/R domain is a functional site. PvTIP4;1 is a new represented tonoplast intrinsic protein (TIP) aquaporin from P. vittata and the function and location results imply that PvTIP4;1 may be involved in As(III) uptake. PMID:26372374

  8. Fluorinated carboxylic membranes deposited by plasma enhanced chemical vapour deposition for fuel cell applications

    NASA Astrophysics Data System (ADS)

    Thery, J.; Martin, S.; Faucheux, V.; Le Van Jodin, L.; Truffier-Boutry, D.; Martinent, A.; Laurent, J.-Y.

    Among the fuel cell technologies, the polymer electrolyte membrane fuel cells (PEMFCs) are particularly promising because they are energy-efficient, clean, and fuel-flexible (i.e., can use hydrogen or methanol). The great majority of PEM fuel cells rely on a polymer electrolyte from the family of perfluorosulfonic acid membranes, nevertheless alternative materials are currently being developed, mainly to offer the alternative workout techniques which are required for the portable energy sources. Plasma polymerization represents a good solution, as it offers the possibility to deposit thin layer with an accurate and homogeneous thickness, even on 3D surfaces. In this paper, we present the results for the growth of proton conductive fluoro carboxylic membranes elaborated by plasma enhanced chemical vapour deposition. These membranes present conductivity values of the same order than the one of Nafion ®. The properties of the membrane, such as the chemical composition, the ionic conductivity, the swelling behaviour and the permeability were correlated to the plasma process parameters. The membranes were integrated in fuel cells on porous substrates and we present here the results regarding the barrier effect and the power output. Barrier effect similar to those of 40 μm Nafion ® layers was reached for 10 μm thick carboxylic membranes. Power outputs around 3 mW cm -2 were measured. We discuss the results regarding the gas barrier effect and the power outputs.

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

    NASA Astrophysics Data System (ADS)

    Andoh, Yoshimichi; Aoki, Noriyuki; Okazaki, Susumu

    2016-02-01

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

  10. Modification of plasma membrane organization in tobacco cells elicited by cryptogein.

    PubMed

    Gerbeau-Pissot, Patricia; Der, Christophe; Thomas, Dominique; Anca, Iulia-Andra; Grosjean, Kevin; Roche, Yann; Perrier-Cornet, Jean-Marie; Mongrand, Sébastien; Simon-Plas, Françoise

    2014-01-01

    Lipid mixtures within artificial membranes undergo a separation into liquid-disordered and liquid-ordered phases. However, the existence of this segregation into microscopic liquid-ordered phases has been difficult to prove in living cells, and the precise organization of the plasma membrane into such phases has not been elucidated in plant cells. We developed a multispectral confocal microscopy approach to generate ratiometric images of the plasma membrane surface of Bright Yellow 2 tobacco (Nicotiana tabacum) suspension cells labeled with an environment sensitive fluorescent probe. This allowed the in vivo characterization of the global level of order of this membrane, by which we could demonstrate that an increase in its proportion of ordered phases transiently occurred in the early steps of the signaling triggered by cryptogein and flagellin, two elicitors of plant defense reactions. The use of fluorescence recovery after photobleaching revealed an increase in plasma membrane fluidity induced by cryptogein, but not by flagellin. Moreover, we characterized the spatial distribution of liquid-ordered phases on the membrane of living plant cells and monitored their variations induced by cryptogein elicitation. We analyze these results in the context of plant defense signaling, discuss their meaning within the framework of the "membrane raft" hypothesis, and propose a new mechanism of signaling platform formation in response to elicitor treatment. PMID:24235133

  11. Modification of Plasma Membrane Organization in Tobacco Cells Elicited by Cryptogein1[W

    PubMed Central

    Gerbeau-Pissot, Patricia; Der, Christophe; Thomas, Dominique; Anca, Iulia-Andra; Grosjean, Kevin; Roche, Yann; Perrier-Cornet, Jean-Marie; Mongrand, Sébastien; Simon-Plas, Françoise

    2014-01-01

    Lipid mixtures within artificial membranes undergo a separation into liquid-disordered and liquid-ordered phases. However, the existence of this segregation into microscopic liquid-ordered phases has been difficult to prove in living cells, and the precise organization of the plasma membrane into such phases has not been elucidated in plant cells. We developed a multispectral confocal microscopy approach to generate ratiometric images of the plasma membrane surface of Bright Yellow 2 tobacco (Nicotiana tabacum) suspension cells labeled with an environment sensitive fluorescent probe. This allowed the in vivo characterization of the global level of order of this membrane, by which we could demonstrate that an increase in its proportion of ordered phases transiently occurred in the early steps of the signaling triggered by cryptogein and flagellin, two elicitors of plant defense reactions. The use of fluorescence recovery after photobleaching revealed an increase in plasma membrane fluidity induced by cryptogein, but not by flagellin. Moreover, we characterized the spatial distribution of liquid-ordered phases on the membrane of living plant cells and monitored their variations induced by cryptogein elicitation. We analyze these results in the context of plant defense signaling, discuss their meaning within the framework of the “membrane raft” hypothesis, and propose a new mechanism of signaling platform formation in response to elicitor treatment. PMID:24235133

  12. Impact of ionic liquids in aqueous solution on bacterial plasma membranes studied with molecular dynamics simulations.

    PubMed

    Lim, Geraldine S; Zidar, Jernej; Cheong, Daniel W; Jaenicke, Stephan; Klähn, Marco

    2014-09-01

    The impact of five different imidazolium-based ionic liquids (ILs) diluted in water on the properties of a bacterial plasma membrane is investigated using molecular dynamics (MD) simulations. Cations considered are 1-octyl-3-methylimidazolium (OMIM), 1-octyloxymethyl-3-methylimidazolium (OXMIM), and 1-tetradecyl-3-methylimidazolium (TDMIM), as well as the anions chloride and lactate. The atomistic model of the membrane bilayer is designed to reproduce the lipid composition of the plasma membrane of Gram-negative Escherichia coli. Spontaneous insertion of cations into the membrane is observed in all ILs. Substantially more insertions of OMIM than of OXMIM occur and the presence of chloride reduces cation insertions compared to lactate. In contrast, anions do not adsorb onto the membrane surface nor diffuse into the bilayer. Once inserted, cations are oriented in parallel to membrane lipids with cation alkyl tails embedded into the hydrophobic membrane core, while the imidazolium-ring remains mostly exposed to the solvent. Such inserted cations are strongly associated with one to two phospholipids in the membrane. The overall order of lipids decreased after OMIM and OXMIM insertions, while on the contrary the order of lipids in the vicinity of TDMIM increased. The short alkyl tails of OMIM and OXMIM generate voids in the bilayer that are filled by curling lipids. This cation induced lipid disorder also reduces the average membrane thickness. This effect is not observed after TDMIM insertions due to the similar length of cation alkyl chain and the fatty acids of the lipids. This lipid-mimicking behavior of inserted TDMIM indicates a high membrane affinity of this cation that could lead to an enhanced accumulation of cations in the membrane over time. Overall, the simulations reveal how cations are inserted into the bacterial membrane and how such insertions change its properties. Moreover, the different roles of cations and anions are highlighted and the fundamental

  13. Aquaporins: Their role in gastrointestinal malignancies.

    PubMed

    Nagaraju, Ganji Purnachandra; Basha, Riyaz; Rajitha, Balney; Alese, Olatunji Boladale; Alam, Afroz; Pattnaik, Subasini; El-Rayes, Bassel

    2016-04-01

    Aquaporins (AQPs) are small (~30 kDa monomers) integral membrane water transport proteins that allow water to flow through cell membranes in reaction to osmotic gradients in cells. In mammals, the family of AQPs has thirteen (AQP0-12) unique members that mediate critical biological functions. Since AQPs can impact cell proliferation, migration and angiogenesis, their role in various human cancers is well established. Recently, AQPs have been explored as potential diagnostic and therapeutic targets in gastrointestinal (GI) cancers. GI cancers encompass multiple sites including the colon, esophagus, stomach and pancreas. Research in the last three decades has revealed biological aspects and signaling pathways critical for the development of GI cancers. Since the majority of these cancers are very aggressive and rapidly metastasizes, identifying effective targets is crucial for treatment. Preclinical studies have utilized inhibitors of specific AQPs and knock down of AQP expression using siRNA. Although several studies have explored the role of AQPs in colorectal, esophageal, gastric, hepatocellular and pancreatic cancers, there is no comprehensive review compiling the available information on GI cancers as has been published for other malignancies such as ovarian cancer. Due to the similarities and association of various sites of GI cancers, it is helpful to consider these results collectively in order to better understand the role of specific AQPs in critical GI cancers. This review summarizes the current knowledge of the role of AQPs in GI malignancies with particular focus on diagnosis and therapeutic applications. PMID:26780474

  14. Expression of VAMP-2-like protein in kidney collecting duct intracellular vesicles. Colocalization with Aquaporin-2 water channels.

    PubMed

    Nielsen, S; Marples, D; Birn, H; Mohtashami, M; Dalby, N O; Trimble, M; Knepper, M

    1995-10-01

    Body water balance is controlled by vasopressin, which regulates Aquaporin-2 (AQP2) water channels in kidney collecting duct cells by vesicular trafficking between intracellular vesicles and the plasma membrane. To examine the molecular apparatus involved in vesicle trafficking and vasopressin regulation of AQP2 in collecting duct cells, we tested if targeting proteins expressed in the synaptic vesicles, namely vesicle-associated membrane proteins 1 and 2 (VAMP1 and 2), are expressed in kidney collecting duct. Immunoblotting revealed specific labeling of VAMP2 (18-kD band) but not VAMP1 in membrane fractions prepared from kidney inner medulla. Controls using preadsorbed antibody or preimmune serum were negative. Bands of identical molecular size were detected in immunoblots of brain membrane vesicles and purified synaptic vesicles. VAMP2 in kidney membranes was cleaved by tetanus toxin, revealing a tetanus toxin-sensitive VAMP homologue. Similarly, tetanus toxin cleaved VAMP2 in synaptic vesicles. In kidney inner medulla, VAMP2 was predominantly expressed in the membrane fraction enriched for intracellular vesicles, with little or no VAMP2 in the plasma membrane enriched fraction. This was confirmed by immunocytochemistry using semithin cryosections, which showed mainly vesicular labeling in collecting duct principal cells, with no labeling of intercalated cells. VAMP2 immunolabeling colocalized with AQP2 labeling in intracellular vesicles, as determined by immunoelectron microscopy after double immunolabeling of isolated vesicles. Quantitative analysis of 1,310 vesicles revealed a highly significant association of both AQP2 and VAMP2 in the same vesicles (P < 0.0001). Furthermore, the presence of AQP2 in vesicles immunoisolated with anti-VAMP2 antibodies was confirmed by immunoblotting. In conclusion, VAMP2, a component of the neuronal SNARE complex, is expressed in vesicles carrying AQP2, suggesting a role in vasopressin-regulated vesicle trafficking of AQP2

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

    PubMed

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

    2015-08-01

    For the first time, the effect of asymmetry of the membrane transport was studied for organic solvents and solutes upon their nanofiltration through the plasma-modified membranes based on poly(1-trimethylsilyl-1-propyne) (PTMSP). Plasma treatment is shown to provide a marked hydrophilization of the hydrophobic PTMSP surface (the contact angle of water decreases from 88 down to 20°) and leads to the development of a negative charge of -5.2 nC/cm(2). The XPS measurements prove the formation of the oxygen-containing groups (Si-O and C-O) due to the surface modification. The AFM images show that the small-scale surface roughness of the plasma-treated PTMSP sample is reduced but the large-scale surface heterogeneities become more pronounced. The modified membranes retain their hydrophilic surface properties even after the nanofiltration tests and 30-day storage under ambient conditions. The results of the filtration tests show that when the membrane is oriented so that its modified layer contacts the feed solution, the membrane permeability for linear alcohols (methanol-propanol) and acetone decreases nearly two times. When the modified membrane surface faces the permeate, the membrane is seen to regain its transport characteristics: the flux becomes equal to that of the unmodified PTMSP. The well-pronounced effect of the transport asymmetry is observed for the solution of the neutral dye Solvent Blue 35 in methanol, ethanol, and acetone. For example, the initial membrane shows the negative retention for the Solvent Blue 35 dye (-16%) upon its filtration from the ethanol solution whereas, for the modified PTMSP membrane, the retention increases up to 17%. Various effects contributing to the asymmetry of the membrane transport characteristics are discussed. PMID:25482845

  16. Cationic nanoparticles induce nanoscale disruption in living cell plasma membranes.

    PubMed

    Chen, Jiumei; Hessler, Jessica A; Putchakayala, Krishna; Panama, Brian K; Khan, Damian P; Hong, Seungpyo; Mullen, Douglas G; Dimaggio, Stassi C; Som, Abhigyan; Tew, Gregory N; Lopatin, Anatoli N; Baker, James R; Holl, Mark M Banaszak; Orr, Bradford G

    2009-08-13

    It has long been recognized that cationic nanoparticles induce cell membrane permeability. Recently, it has been found that cationic nanoparticles induce the formation and/or growth of nanoscale holes in supported lipid bilayers. In this paper, we show that noncytotoxic concentrations of cationic nanoparticles induce 30-2000 pA currents in 293A (human embryonic kidney) and KB (human epidermoid carcinoma) cells, consistent with a nanoscale defect such as a single hole or group of holes in the cell membrane ranging from 1 to 350 nm(2) in total area. Other forms of nanoscale defects, including the nanoparticle porating agents adsorbing onto or intercalating into the lipid bilayer, are also consistent; although the size of the defect must increase to account for any reduction in ion conduction, as compared to a water channel. An individual defect forming event takes 1-100 ms, while membrane resealing may occur over tens of seconds. Patch-clamp data provide direct evidence for the formation of nanoscale defects in living cell membranes. The cationic polymer data are compared and contrasted with patch-clamp data obtained for an amphiphilic phenylene ethynylene antimicrobial oligomer (AMO-3), a small molecule that is proposed to make well-defined 3.4 nm holes in lipid bilayers. Here, we observe data that are consistent with AMO-3 making approximately 3 nm holes in living cell membranes. PMID:19606833

  17. Membrane potential shapes regulation of dopamine transporter trafficking at the plasma membrane

    PubMed Central

    Richardson, Ben D.; Saha, Kaustuv; Krout, Danielle; Cabrera, Elizabeth; Felts, Bruce; Henry, L. Keith; Swant, Jarod; Zou, Mu-Fa; Newman, Amy Hauck; Khoshbouei, Habibeh

    2016-01-01

    The dopaminergic system is essential for cognitive processes, including reward, attention and motor control. In addition to DA release and availability of synaptic DA receptors, timing and magnitude of DA neurotransmission depend on extracellular DA-level regulation by the dopamine transporter (DAT), the membrane expression and trafficking of which are highly dynamic. Data presented here from real-time TIRF (TIRFM) and confocal microscopy coupled with surface biotinylation and electrophysiology suggest that changes in the membrane potential alone, a universal yet dynamic cellular property, rapidly alter trafficking of DAT to and from the surface membrane. Broadly, these findings suggest that cell-surface DAT levels are sensitive to membrane potential changes, which can rapidly drive DAT internalization from and insertion into the cell membrane, thus having an impact on the capacity for DAT to regulate extracellular DA levels. PMID:26804245

  18. Extraction of tumor-specific antigen from cells and plasma membranes of line-10 hepatoma.

    PubMed

    Leonard, E J; Richardson, A K; Hardy, A S; Rapp, H J

    1975-07-01

    Tumor-specific antigen was extracted with 3 M KCl from line-10 guinea pig hepatoma cells. The yield of antigenic activity, estimated by production of delayed cutaneous hypersensitivity reactions in line-10 immune guinea pigs, was 10-30% of the antigen present in intact cells. By ultracentrifugation criteria, the extracted antigen was soluble. Gel filtration, ion exchange chromatography, and salting-out studies showed that the antigen was heterogeneous in size and net charge. The possibility that 3 M KCl extracted a homogeneous population of molecules associating into polymers of various sizes at low ionic strength was ruled out by heterogeneity on Sephadex G-200 chromatography at high ionic strength. After osmotic lysis of sucrose-loaded line-10 cells, whole plasma membranes or large membrane fragments were obtained in a yield of about 20%. The isolation procedure did not cause detectable loss of membrane antigenic activity. The membranes had 33 skin test U/mg membrane protein, compared to the intact cell value of 1.7 skin test U/mg cell protein. Extracts of plasma membranes had 10-20% of the antigenic activity of the starting membrane material. In contrast to the wide variety of proteins liberated from intact cells, much of the protein extracted from the membranes was in the molecular weight range above 250,000. PMID:169367

  19. Polyphosphoinositides are present in plasma membranes isolated from fusogenic carrot cells

    SciTech Connect

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

    1987-10-01

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

  20. Transport of endocannabinoids across the plasma membrane and within the cell.

    PubMed

    Fowler, Christopher J

    2013-05-01

    Endocannabinoids are readily accumulated from the extracellular space by cells. Although their uptake properties have the appearance of a process of facilitated diffusion, it is by no means clear as to whether there is a plasma membrane transporter dedicated to this task. Intracellular carrier proteins that shuttle the endocannabinoid anandamide from the plasma membrane to its intracellular targets such as the metabolic enzyme, fatty acid amide hydrolase, have been identified. These include proteins with other primary functions, such as fatty-acid-binding proteins and heat shock protein 70, and possibly a fatty acid amide hydrolase-like anandamide transporter protein. Thus, anandamide uptake can be adequately described as a diffusion process across the plasma membrane followed by intracellular carrier-mediated transport to effector molecules, catabolic enzymes and sequestration sites, although it is recognized that different cells are likely to utilize different mechanisms of endocannabinoid transport depending upon the utility of the endocannabinoid for the cell in question. PMID:23441874

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

    PubMed Central

    Dittman, Jeremy S.; Kaplan, Joshua M.

    2006-01-01

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

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

    PubMed Central

    Liu, Zengyu; Persson, Staffan; Zhang, Yi

    2015-01-01

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

  3. Characterization of epidermal growth factor receptors on plasma membranes isolated from rat gastric mucosa

    SciTech Connect

    Hori, R.; Nomura, H.; Iwakawa, S.; Okumura, K. )

    1990-06-01

    The binding of human epidermal growth factor (hEGF), beta-urogastrone, to plasma membranes isolated from rat gastric mucosa was studied to characterize gastric EGF receptors. The binding of ({sup 125}I)hEGF was temperature dependent, reversible, and saturable. A single class of binding sites for EGF with a dissociation constant of 0.42 nM and maximal binding capacity of 42 fmol/mg protein was suggested. There was little change in the binding of ({sup 125}I)hEGF upon addition of peptide hormones (secretin, insulin), antiulcer drugs (cimetidine), or an ulcer-inducing reagent (aspirin). Cross-linking of ({sup 125}I)hEGF to gastric plasma membranes with the use of disuccinimidyl suberate resulted in the labeling of a protein of 150 kDa. These results indicate the presence of EGF receptors on plasma membranes of rat gastric mucosa.

  4. [Role of endoplasmic reticulum-plasma membrane junctions in intracellular calcium homeostasis and cardiovascular disease].

    PubMed

    Zhao, Ming; Jia, Hang-Huan; Xu, Man; Yu, Xiao-Jiang; Liu, Long-Zhu; Zang, Wei-Jin

    2016-08-25

    Calcium overload is one of the important mechanisms of cardiovascular disease. Endoplasmic reticulum is an important organelle which regulates intracellular calcium homeostasis by uptake, storage and mobilization of calcium. So it plays a critical role in regulation of intracellular calcium homeostasis. Endoplasmic reticulum, which is widely distributed in cytoplasm, has a large number of membrane junction sites. Recent studies have reported that these junction sites are distributed on plasma membrane and organelle membranes (mitochondria, lysosomes, Golgi apparatus, etc.), separately. They could form complexes to regulate calcium transport. In this review, we briefly outlined the recent research progresses of endoplasmic reticulum-plasma membrane junctions in intracellular calcium homeostasis and cardiovascular disease, which may offer a new strategy for prevention and treatment of cardiovascular disease. PMID:27546511

  5. Analytical characterization of plasma membrane-derived vesicles produced via osmotic and chemical vesiculation.

    PubMed

    Sarabipour, Sarvenaz; Chan, Robin B; Zhou, Bowen; Di Paolo, Gilbert; Hristova, Kalina

    2015-07-01

    Plasma membrane-derived vesicles are being used in biophysical and biochemical research as a simple, yet native-like model of the cellular membrane. Here we report on the characterization of vesicles produced via two different vesiculation methods from CHO and A431 cell lines. The first method is a recently developed method which utilizes chloride salts to induce osmotic vesiculation. The second is a well established chemical vesiculation method which uses DTT and formaldehyde. We show that both vesiculation methods produce vesicles which contain the lipid species previously reported in the plasma membrane of these cell lines. The two methods lead to small but statistically significant differences in two lipid species only; phosphatidylcholine (PC) and plasmalogen phosphatidylethanolamine (PEp). However, highly significant differences were observed in the degree of incorporation of a membrane receptor and in the degree of retention of soluble cytosolic proteins within the vesicles. PMID:25896659

  6. Plasma-induced Styrene Grafting onto the Surface of Polytetrafluoroethylene Powder for Proton Exchange Membrane Application

    NASA Astrophysics Data System (ADS)

    Lan, Yan; Cheng, Cheng; Zhang, Suzhen; Ni, Guohua; Chen, Longwei; Yang, Guangjie; Nagatsu, M.; Meng, Yuedong

    2011-10-01

    Low-temperature plasma treatment was adopted to graft styrene onto polytetrafluoroethylene (PTFE) powder, which is widely used in the fabrication of proton exchange membrane (PEM). The grafted PTFE powder was sulfonated in chlorosulfonic acid and fabricated into a membrane, which was used as inexpensive PEM material for a proton exchange membrane fuel cell (PEMFC). Fourier transform infrared spectroscopy attenuated total reflection spectroscopy (FTIR-ATR) and X-ray photoelectron spectroscopy (XPS) analysis were used to characterize the structure of the sulfonated PTFE powder. The results showed that all the PTFE powders were successfully grafted by nitrogen plasma and then sulfonated under such experimental conditions. A scanning electron microscopy (SEM) image indicated that the fabricated membrane exhibits flat morphology and homogenous structure. The ion exchange capacity (IEC) of this kind of PEM was also investigated.

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

    PubMed

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

    2016-07-01

    Plant cellulose microfibrils are synthesized by a process that propels the cellulose synthase complex (CSC) through the plane of the plasma membrane. How interactions between membranes and the CSC are regulated is currently unknown. Here, we demonstrate that all catalytic subunits of the CSC, known as cellulose synthase A (CESA) proteins, are S-acylated. Analysis of Arabidopsis CESA7 reveals four cysteines in variable region 2 (VR2) and two cysteines at the carboxy terminus (CT) as S-acylation sites. Mutating both the VR2 and CT cysteines permits CSC assembly and trafficking to the Golgi but prevents localization to the plasma membrane. Estimates suggest that a single CSC contains more than 100 S-acyl groups, which greatly increase the hydrophobic nature of the CSC and likely influence its immediate membrane environment. PMID:27387950

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

    PubMed Central

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

    2014-01-01

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

  9. Small unilamellar liposomes as a membrane model for cell inactivation by cold atmospheric plasma treatment

    NASA Astrophysics Data System (ADS)

    Maheux, S.; Frache, G.; Thomann, J. S.; Clément, F.; Penny, C.; Belmonte, T.; Duday, D.

    2016-09-01

    Cold atmospheric plasma is thought to be a promising tool for numerous biomedical applications due to its ability to generate a large diversity of reactive species in a controlled way. In some cases, it can also generate pulsed electric fields at the zone of treatment, which can induce processes such as electroporation in cell membranes. However, the interaction of these reactive species and the pulse electric field with cells in a physiological medium is very complex, and we still need a better understanding in order to be useful for future applications. A way to reach this goal is to work with model cell membranes such as liposomes, with the simplest physiological liquid and in a controlled atmosphere in order to limit the number of parallel reactions and processes. In this paper, where this approach has been chosen, 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) small unilamellar vesicles (SUV) have been synthesized in a phosphate buffered aqueous solution, and this solution has been treated by a nanosecond pulsed plasma jet under a pure nitrogen atmosphere. It is only the composition of the plasma gas that has been changed in order to generate different cocktails of reactive species. After the quantification of the main plasma reactive species in the phosphate buffered saline (PBS) solution, structural, surface charge state, and chemical modifications generated on the plasma treated liposomes, due to the interaction with the plasma reactive species, have been carefully characterized. These results allow us to further understand the effect of plasma reactive species on model cell membranes in physiological liquids. The permeation through the liposomal membrane and the reaction of plasma reactive species with molecules encapsulated inside the liposomes have also been evaluated. New processes of degradation are finally presented and discussed, which come from the specific conditions of plasma treatment under the pure nitrogen atmosphere.

  10. Interactions of normal and mutant vesicular stomatitis virus matrix proteins with the plasma membrane and nucleocapsids.

    PubMed Central

    Chong, L D; Rose, J K

    1994-01-01

    We demonstrated recently that a fraction of the matrix (M) protein of vesicular stomatitis virus (VSV) binds tightly to cellular membranes in vivo when expressed in the absence of other VSV proteins. This membrane-associated M protein was functional in binding purified VSV nucleocapsids in vitro. Here we show that the membrane-associated M protein is largely associated with a membrane fraction having the density of plasma membranes, indicating membrane specificity in the binding. In addition, we analyzed truncated forms of M protein to identify regions responsible for membrane association and nucleocapsid binding. Truncated M protein lacking the amino-terminal basic domain still associated with cellular membranes, although not as tightly as wild-type M protein, and could not bind nucleocapsids. In contrast, deletion of the carboxy-terminal 14 amino acids did not disrupt stable membrane association or nucleocapsid interaction. These results suggest that the amino terminus of M protein either interacts directly with membranes and nucleocapsids or stabilizes a conformation that is required for M protein to mediate both of these interactions. Images PMID:8254754

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

    PubMed Central

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

    2003-01-01

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

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

    SciTech Connect

    Steponkus, P.L.

    1991-01-01

    The principal goal of our program 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. Our immediate goals are (1) to provide an understanding of the mechanism by which freeze-induced dehydration affects the formation of aparticulate domains and lamellar-to-hexagonal{sub {parallel}} phase transitions in the plasma membrane of NA protoplasts, (2) to characterize the cellular and molecular mechanisms by which cold acclimation and cryoprotectants preclude or diminish these alterations in the plasma membrane of ACC protoplasts and (3) to elucidate the molecular basis for the lesion that limits the maximum freezing tolerance of cold-acclimated winter rye and which is believed to be the formation of domains of interdigitated lipids in the L{sub {beta}} phase. This past year our efforts have included (a) characterization of the ultrastructural changes in the plasma membrane that are associated with freezing injury of protoplasts isolated from cold-acclimated rye leaves; (b) determinations of the hydration characteristics of plasma membrane lipids and model lipid mixtures, including the thermal dependence of the hydration characteristics; (c) studies of dehydration-induced phase transitions and demixing in model systems of plasma membrane lipids; (d) differential scanning calorimetry studies to determine the amount of freezable/unfreezable water that is associated with lipids; and (e) preliminary cryo-SEM observations of in situ ice formation in rye leaves. 11 refs.

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

    PubMed Central

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

    1996-01-01

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

  14. Translocation of signalling proteins to the plasma membrane revealed by a new bioluminescent procedure

    PubMed Central

    2011-01-01

    Background Activation by extracellular ligands of G protein-coupled (GPCRs) and tyrosine kinase receptors (RTKs), results in the generation of second messengers that in turn control specific cell functions. Further, modulation/amplification or inhibition of the initial signalling events, depend on the recruitment onto the plasma membrane of soluble protein effectors. High throughput methodologies to monitor quantitatively second messenger production, have been developed over the last years and are largely used to screen chemical libraries for drug development. On the contrary, no such high throughput methods are yet available for the other aspect of GPCRs regulation, i.e. protein translocation to the plasma membrane, despite the enormous interest of this phenomenon for the modulation of receptor downstream functions. Indeed, to date, the experimental procedures available are either inadequate or complex and expensive. Results Here we describe the development of a novel conceptual approach to the study of cytosolic proteins translocation to the inner surface of the plasma membrane. The basis of the technique consists in: i) generating chimeras between the protein of interests and the calcium (Ca2+)-sensitive, luminescent photo-protein, aequorin and ii) taking advantage of the large Ca2+ concentration [Ca2+] difference between bulk cytosolic and the sub-plasma membrane rim. Conclusion This approach, that keeps unaffected the translocation properties of the signalling protein, can in principle be applied to any protein that, upon activation, moves from the cytosol to the plasma membrane. Thus, not only the modulation of GPCRs and RTKs can be investigated in this way, but that of all other proteins that can be recruited to the plasma membrane also independently of receptor activation. Moreover, its automated version, which can provide information about the kinetics and concentration-dependence of the process, is also applicable to high throughput screening of drugs

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

    SciTech Connect

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

    1987-05-01

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

  16. Glycaemia regulates the glucose transporter number in the plasma membrane of rat skeletal muscle.

    PubMed Central

    Dimitrakoudis, D; Ramlal, T; Rastogi, S; Vranic, M; Klip, A

    1992-01-01

    The number of glucose transporters was measured in isolated membranes from diabetic-rat skeletal muscle to determine the role of circulating blood glucose levels in the control of glucose uptake into skeletal muscle. Three experimental groups of animals were investigated in the post-absorptive state: normoglycaemic/normoinsulinaemic, hyperglycaemic/normoinsulinaemic and hyperglycaemic/normoinsulinaemic made normoglycaemic/normoinsulinaemic by phlorizin treatment. Hyperglycaemia caused a reversible decrease in total transporter number, as measured by cytochalasin B binding, in both plasma membranes and internal membranes of skeletal muscle. Changes in GLUT4 glucose transporter protein mirrored changes in cytochalasin B binding in plasma membranes. However, there was no recovery of GLUT4 levels in intracellular membranes with correction of glycaemia. GLUT4 mRNA levels decreased with hyperglycaemia and recovered only partially with correction of glycaemia. Conversely, GLUT1 glucose transporters were only detectable in the plasma membranes; the levels of this protein varied directly with glycaemia, i.e. in the opposite direction to GLUT4 glucose transporters. This study demonstrates that hyperglycaemia, in the absence of hypoinsulinaemia, is capable of down-regulating the glucose transport system in skeletal muscle, the major site of peripheral resistance to insulin-stimulated glucose transport in diabetes. Furthermore, correction of hyperglycaemia causes a complete restoration of the transport system in the basal state (determined by the transporter number in the plasma membrane), but possibly only an incomplete recovery of the transport system's ability to respond to insulin (since there is no recovery of GLUT4 levels in the intracellular membrane insulin-responsive transporter pool). Finally, the effect of hyperglycaemia is specific for glucose transporter isoforms, with GLUT1 and GLUT4 proteins varying respectively in parallel and opposite directions to levels of

  17. Roles of charged particles and reactive species on cell membrane permeabilization induced by atmospheric-pressure plasma irradiation

    NASA Astrophysics Data System (ADS)

    Sasaki, Shota; Kanzaki, Makoto; Hokari, Yutaro; Tominami, Kanako; Mokudai, Takayuki; Kanetaka, Hiroyasu; Kaneko, Toshiro

    2016-07-01

    As factors that influence cell membrane permeabilization during direct and indirect atmospheric-pressure plasma irradiation, charged particle influx, superoxide anion radicals (O2 ‑•), and hydrogen peroxide (H2O2) in plasma-irradiated solution were evaluated. These are the three strong candidate factors and might multiply contribute to cell membrane permeabilization. In particular, a shorter plasma diffusion distance leads to the enhancement of the direct effects such as charged particle influx and further increase cell membrane permeability. In addition, O2 ‑• dissipates over time (a life span of the order of minutes) in plasma-irradiated water, and the deactivation of a plasma-irradiated solution in term of cell membrane permeabilization occurs in a life span of the same order. These results could promote the understanding of the mechanism of plasma-induced cell membrane permeabilization.

  18. Increased water flux induced by an aquaporin-1/carbonic anhydrase II interaction

    PubMed Central

    Vilas, Gonzalo; Krishnan, Devishree; Loganathan, Sampath Kumar; Malhotra, Darpan; Liu, Lei; Beggs, Megan Rachele; Gena, Patrizia; Calamita, Giuseppe; Jung, Martin; Zimmermann, Richard; Tamma, Grazia; Casey, Joseph Roman; Alexander, Robert Todd

    2015-01-01

    Aquaporin-1 (AQP1) enables greatly enhanced water flux across plasma membranes. The cytosolic carboxy terminus of AQP1 has two acidic motifs homologous to known carbonic anhydrase II (CAII) binding sequences. CAII colocalizes with AQP1 in the renal proximal tubule. Expression of AQP1 with CAII in Xenopus oocytes or mammalian cells increased water flux relative to AQP1 expression alone. This required the amino-terminal sequence of CAII, a region that binds other transport proteins. Expression of catalytically inactive CAII failed to increase water flux through AQP1. Proximity ligation assays revealed close association of CAII and AQP1, an effect requiring the second acidic cluster of AQP1. This motif was also necessary for CAII to increase AQP1-mediated water flux. Red blood cell ghosts resealed with CAII demonstrated increased osmotic water permeability compared with ghosts resealed with albumin. Water flux across renal cortical membrane vesicles, measured by stopped-flow light scattering, was reduced in CAII-deficient mice compared with wild-type mice. These data are consistent with CAII increasing water conductance through AQP1 by a physical interaction between the two proteins. PMID:25609088

  19. Brain water mobility decreases after astrocytic aquaporin-4 inhibition using RNA interference

    PubMed Central

    Badaut, Jérôme; Ashwal, Stephen; Adami, Arash; Tone, Beatriz; Recker, Rebecca; Spagnoli, David; Ternon, Béatrice; Obenaus, Andre

    2011-01-01

    Neuroimaging with diffusion-weighted imaging is routinely used for clinical diagnosis/prognosis. Its quantitative parameter, the apparent diffusion coefficient (ADC), is thought to reflect water mobility in brain tissues. After injury, reduced ADC values are thought to be secondary to decreases in the extracellular space caused by cell swelling. However, the physiological mechanisms associated with such changes remain uncertain. Aquaporins (AQPs) facilitate water diffusion through the plasma membrane and provide a unique opportunity to examine the molecular mechanisms underlying water mobility. Because of this critical role and the recognition that brain AQP4 is distributed within astrocytic cell membranes, we hypothesized that AQP4 contributes to the regulation of water diffusion and variations in its expression would alter ADC values in normal brain. Using RNA interference in the rodent brain, we acutely knocked down AQP4 expression and observed that a 27% AQP4-specific silencing induced a 50% decrease in ADC values, without modification of tissue histology. Our results demonstrate that ADC values in normal brain are modulated by astrocytic AQP4. These findings have major clinical relevance as they suggest that imaging changes seen in acute neurologic disorders such as stroke and trauma are in part due to changes in tissue AQP4 levels. PMID:20877385

  20. AKAP220 manages apical actin networks that coordinate aquaporin-2 location and renal water reabsorption.

    PubMed

    Whiting, Jennifer L; Ogier, Leah; Forbush, Katherine A; Bucko, Paula; Gopalan, Janani; Seternes, Ole-Morten; Langeberg, Lorene K; Scott, John D

    2016-07-26

    Filtration through the kidney eliminates toxins, manages electrolyte balance, and controls water homeostasis. Reabsorption of water from the luminal fluid of the nephron occurs through aquaporin-2 (AQP2) water pores in principal cells that line the kidney-collecting duct. This vital process is impeded by formation of an "actin barrier" that obstructs the passive transit of AQP2 to the plasma membrane. Bidirectional control of AQP2 trafficking is managed by hormones and signaling enzymes. We have discovered that vasopressin-independent facets of this homeostatic mechanism are under the control of A-Kinase Anchoring Protein 220 (AKAP220; product of the Akap11 gene). CRISPR/Cas9 gene editing and imaging approaches show that loss of AKAP220 disrupts apical actin networks in organoid cultures. Similar defects are evident in tissue sections from AKAP220-KO mice. Biochemical analysis of AKAP220-null kidney extracts detected reduced levels of active RhoA GTPase, a well-known modulator of the actin cytoskeleton. Fluorescent imaging of kidney sections from these genetically modified mice revealed that RhoA and AQP2 accumulate at the apical surface of the collecting duct. Consequently, these animals are unable to appropriately dilute urine in response to overhydration. We propose that membrane-proximal signaling complexes constrained by AKAP220 impact the actin barrier dynamics and AQP2 trafficking to ensure water homeostasis. PMID:27402760

  1. Stress Regulates Aquaporin-8 Permeability to Impact Cell Growth and Survival

    PubMed Central

    Medraño-Fernandez, Iria; Bestetti, Stefano; Bertolotti, Milena; Bienert, Gerd P.; Bottino, Cinzia; Laforenza, Umberto; Rubartelli, Anna

    2016-01-01

    Abstract Aquaporin-8 (AQP8) allows the bidirectional transport of water and hydrogen peroxide across biological membranes. Depending on its concentration, H2O2 exerts opposite roles, amplifying growth factor signaling in physiological conditions, but causing severe cell damage when in excess. Thus, H2O2 permeability is likely to be tightly controlled in living cells. Aims: In this study, we investigated whether and how the transport of H2O2 through plasma membrane AQP8 is regulated, particularly during cell stress. Results: We show that diverse cellular stress conditions, including heat, hypoxia, and ER stress, reversibly inhibit the permeability of AQP8 to H2O2 and water. Preventing the accumulation of intracellular reactive oxygen species (ROS) during stress counteracts AQP8 blockade. Once inhibition is established, AQP8-dependent transport can be rescued by reducing agents. Neither H2O2 nor water transport is impaired in stressed cells expressing a mutant AQP8, in which cysteine 53 had been replaced by serine. Cells expressing this mutant are more resistant to stress-, drug-, and radiation-induced growth arrest and death. Innovation and Conclusion: The control of AQP8-mediated H2O2 transport provides a novel mechanism to regulate cell signaling and survival during stress. Antioxid. Redox Signal. 24, 1031–1044. PMID:26972385

  2. Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes.

    PubMed

    Castro-Gomes, Thiago; Corrotte, Matthias; Tam, Christina; Andrews, Norma W

    2016-01-01

    Eukaryotic cells rapidly repair wounds on their plasma membrane. Resealing is Ca(2+)-dependent, and involves exocytosis of lysosomes followed by massive endocytosis. Extracellular activity of the lysosomal enzyme acid sphingomyelinase was previously shown to promote endocytosis and wound removal. However, whether lysosomal proteases released during cell injury participate in resealing is unknown. Here we show that lysosomal proteases regulate plasma membrane repair. Extracellular proteolysis is detected shortly after cell wounding, and inhibition of this process blocks repair. Conversely, surface protein degradation facilitates plasma membrane resealing. The abundant lysosomal cysteine proteases cathepsin B and L, known to proteolytically remodel the extracellular matrix, are rapidly released upon cell injury and are required for efficient plasma membrane repair. In contrast, inhibition of aspartyl proteases or RNAi-mediated silencing of the lysosomal aspartyl protease cathepsin D enhances resealing, an effect associated with the accumulation of active acid sphingomyelinase on the cell surface. Thus, secreted lysosomal cysteine proteases may promote repair by facilitating membrane access of lysosomal acid sphingomyelinase, which promotes wound removal and is subsequently downregulated extracellularly by a process involving cathepsin D. PMID:27028538

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

    PubMed

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

    2015-07-15

    Thin-film composite membranes, primarily based on poly(amide) (PA) semipermeable materials, are nowadays the dominant technology used in pressure driven water desalination systems. Despite offering superior water permeation and salt selectivity, their surface properties, such as their charge and roughness, cannot be extensively tuned due to the intrinsic fabrication process of the membranes by interfacial polymerization. The alteration of these properties would lead to a better control of the materials surface zeta potential, which is critical to finely tune selectivity and enhance the membrane materials stability when exposed to complex industrial waste streams. Low pressure plasma was employed to introduce amine functionalities onto the PA surface of commercially available thin-film composite (TFC) membranes. Morphological changes after plasma polymerization were analyzed by SEM and AFM, and average surface roughness decreased by 29%. Amine enrichment provided isoelectric point changes from pH 3.7 to 5.2 for 5 to 15 min of plasma polymerization time. Synchrotron FTIR mappings of the amine-modified surface indicated the addition of a discrete 60 nm film to the PA layer. Furthermore, metal affinity was confirmed by the enhanced binding of silver to the modified surface, supported by an increased antimicrobial functionality with demonstrable elimination of E. coli growth. Essential salt rejection was shown minimally compromised for faster polymerization processes. Plasma polymerization is therefore a viable route to producing functional amine enriched thin-film composite PA membrane surfaces. PMID:26083007

  4. Deletion of the PDR16 gene influences the plasma membrane properties of the yeast Kluyveromyces lactis.

    PubMed

    Toth Hervay, Nora; Goffa, Eduard; Svrbicka, Alexandra; Simova, Zuzana; Griac, Peter; Jancikova, Iva; Gaskova, Dana; Morvova, Marcela; Sikurova, Libusa; Gbelska, Yvetta

    2015-04-01

    The plasma membrane is the first line of cell defense against changes in external environment, thus its integrity and functionality are of utmost importance. The plasma membrane properties depend on both its protein and lipid composition. The PDR16 gene is involved in the control of Kluyveromyces lactis susceptibility to drugs and alkali metal cations. It encodes the homologue of the major K. lactis phosphatidylinositol transfer protein Sec14p. Sec14p participates in protein secretion, regulation of lipid synthesis, and turnover in vivo. We report here that the plasma membrane of the Klpdr16Δ mutant is hyperpolarized and its fluidity is lower than that of the parental strain. In addition, protoplasts prepared from the Klpdr16Δ cells display decreased stability when subjected to hypo-osmotic conditions. These changes in membrane properties lead to an accumulation of radiolabeled fluconazole and lithium cations inside mutant cells. Our results point to the fact that the PDR16 gene of K. lactis (KlPDR16) influences the plasma membrane properties in K. lactis that lead to subsequent changes in susceptibility to a broad range of xenobiotics. PMID:25742422

  5. Disrupting Microtubules Network Immobilizes Amoeboid Chemotactic Receptor in the Plasma Membrane

    PubMed Central

    de Keijzer, S.; Galloway, J.; Harms, G.S.; Devreotes, P.N.; Iglesias, P.A.

    2011-01-01

    Signaling cascades are initiated in the plasma membrane via activation of one molecule by another. The interaction depends on the mutual availability of the molecules to each other and this is determined by their localization and lateral diffusion in the cell membrane. The cytoskeleton plays a very important role in this process by enhancing or restricting the possibility of the signaling partners to meet in the plasma membrane. In this study we explored the mode of diffusion of the cAMP receptor, cAR1, in the plasma membrane of Dictyostelium discoideum cells and how this is regulated by the cytoskeleton. Single-particle tracking of fluorescently labeled cAR1 using total internal reflection microscopy showed that 70% of the cAR1 molecules were mobile. These receptors showed directed motion and we demonstrate that this is not because of tracking along the actin cytoskeleton. Instead, destabilization of the microtubules abolished cAR1 mobility in the plasma membrane and this was confirmed by fluorescence recovery after photobleaching. As a result of microtubule stabilization, one of the first downstream signaling events, the jump of the PH domain of CRAC, was decreased. These results suggest a role for microtubules in cAR1 dynamics and in the ability of cAR1 molecules to interact with their signaling partners. PMID:21334306

  6. Identification of DNA-binding proteins on human umbilical vein endothelial cell plasma membrane.

    PubMed Central

    Chan, T M; Frampton, G; Cameron, J S

    1993-01-01

    The binding of anti-DNA antibodies to the endothelial cell is mediated through DNA, which forms a bridge between the immunoglobulin and the plasma membrane. We have shown that 32P-labelled DNA bound to the plasma membrane of human umbilical vein endothelial cells (HUVEC) by a saturable process, which could be competitively inhibited by non-radiolabelled DNA. In addition, DNA-binding was enhanced in HUVEC that had been treated with IL-1 alpha or tumour necrosis factor-alpha (TNF-alpha). DNA-binding proteins of mol. wt 46,000, 92,000, and 84,000 were identified by the binding of 32P-labelled DNA to plasma membrane proteins separated on SDS-PAGE. DNA-binding proteins of mol. wt 46,000 and 84,000 were also present in the cytosol and nucleus. Murine anti-DNA MoAb410 bound to a single band, at mol. wt 46,000, of plasma membrane protein, in the presence of DNA. Our results showed that DNA-binding proteins are present in different cellular fractions of endothelial cells. DNA-binding proteins on the cell membrane could participate in the in situ formation of immune deposits; and their presence in the cell nucleus suggests a potential role in the modulation of cell function. Images Fig. 3 Fig. 4 PMID:8419070

  7. Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes

    PubMed Central

    Castro-Gomes, Thiago; Corrotte, Matthias; Tam, Christina; Andrews, Norma W.

    2016-01-01

    Eukaryotic cells rapidly repair wounds on their plasma membrane. Resealing is Ca2+-dependent, and involves exocytosis of lysosomes followed by massive endocytosis. Extracellular activity of the lysosomal enzyme acid sphingomyelinase was previously shown to promote endocytosis and wound removal. However, whether lysosomal proteases released during cell injury participate in resealing is unknown. Here we show that lysosomal proteases regulate plasma membrane repair. Extracellular proteolysis is detected shortly after cell wounding, and inhibition of this process blocks repair. Conversely, surface protein degradation facilitates plasma membrane resealing. The abundant lysosomal cysteine proteases cathepsin B and L, known to proteolytically remodel the extracellular matrix, are rapidly released upon cell injury and are required for efficient plasma membrane repair. In contrast, inhibition of aspartyl proteases or RNAi-mediated silencing of the lysosomal aspartyl protease cathepsin D enhances resealing, an effect associated with the accumulation of active acid sphingomyelinase on the cell surface. Thus, secreted lysosomal cysteine proteases may promote repair by facilitating membrane access of lysosomal acid sphingomyelinase, which promotes wound removal and is subsequently downregulated extracellularly by a process involving cathepsin D. PMID:27028538

  8. The Plasma Membrane Proteins Prm1 and Fig1 Ascertain Fidelity of Membrane Fusion during Yeast Mating

    PubMed Central

    Engel, Alex; Walter, Peter

    2007-01-01

    As for most cell–cell fusion events, the molecular details of membrane fusion during yeast mating are poorly understood. The multipass membrane protein Prm1 is the only known component that acts at the step of bilayer fusion. In its absence, mutant mating pairs lyse or arrest in the mating reaction with tightly apposed plasma membranes. We show that deletion of FIG 1, which controls pheromone-induced Ca2+ influx, yields similar cell fusion defects. Although extracellular Ca2+ is not required for efficient cell fusion of wild-type cells, cell fusion in prm1 mutant mating pairs is dramatically reduced when Ca2+ is removed. This enhanced fusion defect is due to lysis. Time-lapse microscopy reveals that fusion and lysis events initiate with identical kinetics, suggesting that both outcomes result from engagement of the fusion machinery. The yeast synaptotagmin orthologue and Ca2+ binding protein Tcb3 has a role in reducing lysis of prm1 mutants, which opens the possibility that the observed role of Ca2+ is to engage a wound repair mechanism. Thus, our results suggest that Prm1 and Fig1 have a role in enhancing membrane fusion and maintaining its fidelity. Their absence results in frequent mating pair lysis, which is counteracted by Ca2+-dependent membrane repair. PMID:17151357

  9. Isolation and Characterization of Concanavalin A-labeled Plasma Membranes of Carrot Protoplasts 1

    PubMed Central

    Boss, Wendy F.; Ruesink, Albert W.

    1979-01-01

    The plasma membranes of protoplasts released from carrot suspension culture cells were labeled with [14C]acetyl-concanavalin A. After homogenization a single labeled membrane fraction was isolated in a continuous isopycnic Renografin gradient. The labeled membranes peaked at an apparent density of 1.14 grams per cubic centimeter between the Golgi fraction at a density of 1.11 grams per cubic centimeter as determined by latent IDPase activity and the mitochondria at a density of 1.16 grams per cubic centimeter as determined by the cytochrome c oxidase activity. This method provided a very discrete peak of putative plasma membrane. On discontinuous Renografin gradients a relatively pure fraction of labeled plasma membranes could be readily isolated at the 1.122 to 1.146 grams per cubic centimeter interface. The labeled fraction was enriched in both an ATPase (pH 6.5) and a glucan synthetase with a pH optimum of 6.5 whose activity was promoted by magnesium and cellobiose. Enzyme activities were not altered by the membrane label. PMID:16661082

  10. Radiation inactivation target size of rat adipocyte glucose transporters in the plasma membrane and intracellular pools

    SciTech Connect

    Jacobs, D.B.; Berenski, C.J.; Spangler, R.A.; Jung, C.Y.

    1987-06-15

    The in situ assembly states of the glucose transport carrier protein in the plasma membrane and in the intracellular (microsomal) storage pool of rat adipocytes were assessed by studying radiation-induced inactivation of the D-glucose-sensitive cytochalasin B binding activities. High energy radiation inactivated the glucose-sensitive cytochalasin B binding of each of these membrane preparations by reducing the total number of the binding sites without affecting the dissociation constant. The reduction in total number of binding sites was analyzed as a function of radiation dose based on target theory, from which a radiation-sensitive mass (target size) was calculated. When the plasma membranes of insulin-treated adipocytes were used, a target size of approximately 58,000 daltons was obtained. For adipocyte microsomal membranes, we obtained target sizes of approximately 112,000 and 109,000 daltons prior to and after insulin treatment, respectively. In the case of microsomal membranes, however, inactivation data showed anomalously low radiation sensitivities at low radiation doses, which may be interpreted as indicating the presence of a radiation-sensitive inhibitor. These results suggest that the adipocyte glucose transporter occurs as a monomer in the plasma membrane while existing in the intracellular reserve pool either as a homodimer or as a stoichiometric complex with a protein of an approximately equal size.

  11. Homeostasis of the apical plasma membrane during regulated exocytosis in the salivary glands of live rodents

    PubMed Central

    Masedunskas, Andrius; Sramkova, Monika; Weigert, Roberto

    2011-01-01

    In exocrine organs such as the salivary glands, fluids and proteins are secreted into ductal structures by distinct mechanisms that are tightly coupled. In the acinar cells, the major secretory units of the salivary glands, fluids are secreted into the acinar canaliculi through paracellular and intracellular transport, whereas proteins are stored in large granules that undergo exocytosis and fuse with the apical plasma membranes releasing their content into the canaliculi. Both secretory processes elicit a remodeling of the apical plasma membrane that has not been fully addressed in in vitro or ex vivo models. Recently, we have studied regulated exocytosis in the salivary glands of live rodents, focusing on the role that actin and myosin plays in this process. We observed that during exocytosis both secretory granules and canaliculi are subjected to the hydrostatic pressure generated by fluid secretion. Furthermore, the absorption of the membranes of the secretory granules contributes to the expansion and deformation of the canaliculi. Here we suggest that the homeostasis of the apical plasma membranes during exocytosis is maintained by various strategies that include: (1) membrane retrieval via compensatory endocytosis, (2) increase of the surface area via membrane folds and (3) recruitment of a functional actomyosin complex. Our observations underscore the important relationship between tissue architecture and cellular response, and highlight the potential of investigating biological processes in vivo by using intravital microscopy. PMID:22754613

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

    SciTech Connect

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

    1989-07-01

    Plasma membrane vesicles were isolated from zucchini (Cucurbita pepo) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-(7-{sup 3}H)IAA(({sup 3}H)N{sub 3}IAA), in a manner similar to the accumulation of ({sup 3}H)IAA. The association of the ({sup 3}H)N{sub 3}IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of ({sup 3}H)N{sub 3}IAA to plasma membrane vesicles prior to exposure to UV light and detected by subsequent NaDodSO{sub 4}/PAGE and fluorography. When the reaction temperature was lowered to {minus}196{degree}C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Collectively, these results suggest that the radiolabeled polypeptides are auxin receptors. The covalent nature of the label should facilitate purification and further characterization of the receptors.

  13. Spatiotemporal mapping of diffusion dynamics and organization in plasma membranes

    NASA Astrophysics Data System (ADS)

    Bag, Nirmalya; Ng, Xue Wen; Sankaran, Jagadish; Wohland, Thorsten

    2016-09-01

    Imaging fluorescence correlation spectroscopy (FCS) and the related FCS diffusion law have been applied in recent years to investigate the diffusion modes of lipids and proteins in membranes. These efforts have provided new insights into the membrane structure below the optical diffraction limit, new information on the existence of lipid domains, and on the influence of the cytoskeleton on membrane dynamics. However, there has been no systematic study to evaluate how domain size, domain density, and the probe partition coefficient affect the resulting imaging FCS diffusion law parameters. Here, we characterize the effects of these factors on the FCS diffusion law through simulations and experiments on lipid bilayers and live cells. By segmenting images into smaller 7  ×  7 pixel areas, we can evaluate the FCS diffusion law on areas smaller than 2 µm and thus provide detailed maps of information on the membrane structure and heterogeneity at this length scale. We support and extend this analysis by deriving a mathematical expression to calculate the mean squared displacement (MSDACF) from the autocorrelation function of imaging FCS, and demonstrate that the MSDACF plots depend on the existence of nanoscopic domains. Based on the results, we derive limits for the detection of domains depending on their size, density, and relative viscosity in comparison to the surroundings. Finally, we apply these measurements to bilayers and live cells using imaging total internal reflection FCS and single plane illumination microscopy FCS.

  14. Role of Aquaporin 0 in lens biomechanics.

    PubMed

    Sindhu Kumari, S; Gupta, Neha; Shiels, Alan; FitzGerald, Paul G; Menon, Anil G; Mathias, Richard T; Varadaraj, Kulandaiappan

    2015-07-10

    Maintenance of proper biomechanics of the eye lens is important for its structural integrity and for the process of accommodation to focus near and far objects. Several studies have shown that specialized cytoskeletal systems such as the beaded filament (BF) and spectrin-actin networks contribute to mammalian lens biomechanics; mutations or deletion in these proteins alters lens biomechanics. Aquaporin 0 (AQP0), which constitutes ∼45% of the total membrane proteins of lens fiber cells, has been shown to function as a water channel and a structural cell-to-cell adhesion (CTCA) protein. Our recent ex vivo study on AQP0 knockout (AQP0 KO) mouse lenses showed the CTCA function of AQP0 could be crucial for establishing the refractive index gradient. However, biomechanical studies on the role of AQP0 are lacking. The present investigation used wild type (WT), AQP5 KO (AQP5(-/-)), AQP0 KO (heterozygous KO: AQP0(+/-); homozygous KO: AQP0(-/-); all in C57BL/6J) and WT-FVB/N mouse lenses to learn more about the role of fiber cell AQPs in lens biomechanics. Electron microscopic images exhibited decreases in lens fiber cell compaction and increases in extracellular space due to deletion of even one allele of AQP0. Biomechanical assay revealed that loss of one or both alleles of AQP0 caused a significant reduction in the compressive load-bearing capacity of the lenses compared to WT lenses. Conversely, loss of AQP5 did not alter the lens load-bearing ability. Compressive load-bearing at the suture area of AQP0(+/-) lenses showed easy separation while WT lens suture remained intact. These data from KO mouse lenses in conjunction with previous studies on lens-specific BF proteins (CP49 and filensin) suggest that AQP0 and BF proteins could act co-operatively in establishing normal lens biomechanics. We hypothesize that AQP0, with its prolific expression at the fiber cell membrane, could provide anchorage for cytoskeletal structures like BFs and together they help to confer

  15. Interactions between Transmembrane Helices within Monomers of the Aquaporin AtPIP2;1 Play a Crucial Role in Tetramer Formation.

    PubMed

    Yoo, Yun-Joo; Lee, Hyun Kyung; Han, Wonhee; Kim, Dae Heon; Lee, Myoung Hui; Jeon, Jouhyun; Lee, Dong Wook; Lee, Junho; Lee, Yongjik; Lee, Juhun; Kim, Jin Seok; Cho, Yunje; Han, Jin-Kwan; Hwang, Inhwan

    2016-07-01

    Aquaporin (AQP) is a water channel protein found in various subcellular membranes of both prokaryotic and eukaryotic cells. The physiological functions of AQPs have been elucidated in many organisms. However, understanding their biogenesis remains elusive, particularly regarding how they assemble into tetramers. Here, we investigated the amino acid residues involved in the tetramer formation of the Arabidopsis plasma membrane AQP AtPIP2;1 using extensive amino acid substitution mutagenesis. The mutant proteins V41A/E44A, F51A/L52A, F87A/I91A, F92A/I93A, V95A/Y96A, and H216A/L217A, harboring alanine substitutions in the transmembrane (TM) helices of AtPIP2;1 polymerized into multiple oligomeric complexes with a variable number of subunits greater than four. Moreover, these mutant proteins failed to traffic to the plasma membrane, instead of accumulating in the endoplasmic reticulum (ER). Structure-based modeling revealed that these residues are largely involved in interactions between TM helices within monomers. These results suggest that inter-TM interactions occurring both within and between monomers play crucial roles in tetramer formation in the AtPIP2;1 complex. Moreover, the assembly of AtPIP2;1 tetramers is critical for their trafficking from the ER to the plasma membrane, as well as water permeability. PMID:27142778

  16. Identification and Expression Analysis of Aquaporins in the Potato Psyllid, Bactericera cockerelli

    PubMed Central

    Ibanez, Freddy; Hancock, Joseph; Tamborindeguy, Cecilia

    2014-01-01

    Aquaporin (AQPs) proteins transport water and uncharged low molecular-weight solutes across biological membranes. Six to 8 AQP genes have been identified in many insect species, but presently only three aquaporins have been characterized in phloem feeding insects. The objective of this study was to identify candidate AQPs in the potato psyllid, Bactericera cockerelli. Herein, we identified four candidate aquaporin cDNAs in B. cockerelli transcriptome. Phylogenetic analysis showed that candidate BcAQP2-like had high similarity to PRIP aquaporins; while candidates BcAQP4-like, BcAQP5-like and BcAQP9-like clustered within clade B. In particular, candidates BcAQP4-like and BcAQP5-like clustered with functionally validated insect aquaglyceroporin proteins. Expression analyses using RT-qPCR showed that all candidates were expressed in all life stages and tissues. Candidates BcAQP4-like and BcAQP5-like were highly expressed in bacteriocytes, while BcAQP9-like appeared to be expressed at high levels in whole body but not in the assayed tissues. This study is the first global attempt to identify putative aquaporins in a phloem feeding insect. PMID:25354208

  17. Membrane Protein Mobility and Orientation Preserved in Supported Bilayers Created Directly from Cell Plasma Membrane Blebs.

    PubMed

    Richards, Mark J; Hsia, Chih-Yun; Singh, Rohit R; Haider, Huma; Kumpf, Julia; Kawate, Toshimitsu; Daniel, Susan

    2016-03-29

    Membrane protein interactions with lipids are crucial for their native biological behavior, yet traditional characterization methods are often carried out on purified protein in the absence of lipids. We present a simple method to transfer membrane proteins expressed in mammalian cells to an assay-friendly, cushioned, supported lipid bilayer platform using cell blebs as an intermediate. Cell blebs, expressing either GPI-linked yellow fluorescent proteins or neon-green fused transmembrane P2X2 receptors, were induced to rupture on glass surfaces using PEGylated lipid vesicles, which resulted in planar supported membranes with over 50% mobility for multipass transmembrane proteins and over 90% for GPI-linked proteins. Fluorescent proteins were tracked, and their diffusion in supported bilayers characterized, using single molecule tracking and moment scaling spectrum (MSS) analysis. Diffusion was characterized for individual proteins as either free or confined, revealing details of the local lipid membrane heterogeneity surrounding the protein. A particularly useful result of our bilayer formation process is the protein orientation in the supported planar bilayer. For both the GPI-linked and transmembrane proteins used here, an enzymatic assay revealed that protein orientation in the planar bilayer results in the extracellular domains facing toward the bulk, and that the dominant mode of bleb rupture is via the "parachute" mechanism. Mobility, orientation, and preservation of the native lipid environment of the proteins using cell blebs offers advantages over proteoliposome reconstitution or disrupted cell membrane preparations, which necessarily result in significant scrambling of protein orientation and typically immobilized membrane proteins in SLBs. The bleb-based bilayer platform presented here is an important step toward integrating membrane proteomic studies on chip, especially for future studies aimed at understanding fundamental effects of lipid interactions

  18. The microviscosity of liver plasma membranes of rats fed with oleoylanilide.

    PubMed Central

    Pagani, R; Portoles, M T; Gavilanes, F G; Garcia-Barreno, P; Municio, A M

    1984-01-01

    Oleoylanilide was administered orally to groups of rats according to different patterns. Oleoylanilide was perfused at different concentrations through rat liver. Oleoylanilide was added to isolated hepatocytes. Oleoylanilide was added to plasma-membrane preparations. Membrane preparations were obtained after experiments performed in vivo and perfusion experiments and, by using 1,6-diphenylhexa-1,3,5-triene as fluorescence probe, the fluorescence polarization parameter was measured, from which the microviscosity (eta) was calculated. In all cases the microviscosity decreased markedly. Addition of oleoylanilide to hepatocyte preparations and to isolated membranes produced the same effect, increasing the fluidity of the membranes. These data suggest that oleoylanilide partitions into the membrane, disordering some lipid interactions. PMID:6712608

  19. Cross-tolerance of human placental plasma membranes of smokers to fluidizing effects of alcohol

    SciTech Connect

    Sastry, B.V.R.; Horst, M.A.; Naukam, R.J. )

    1991-03-11

    There is cross-tolerance between ethanol and several centrally acting drugs at the membrane level. In order to evaluate cross-tolerance between maternal smoking during pregnancy and alcohol, the authors have prepared plasma membranes of human term placentas from nonsmokers (NS, n=5) and smokers (S, 24 {plus minus} 8 cigarettes/day, n=5) and studied their microviscosities by steady state fluorescence polarization using trans-1,6-diphenyl-1,3,5-hexatriene as a fluorescent probe. These experiments gave the following results: (a) microviscosity was increased by maternal smoking; (b) alcohol decreased microviscosity of the membranes of smokers; (c) exogenous nicotine did not exert any significant effect on the membranes of smokers and nonsmokers. Therefore, the increase in the rigidity of placental plasma membranes is due to chronic smoking, and these membranes are tolerant to the fluidizing effects of alcohol. Cross-tolerance between smoking and ethanol suggests a common hydrophobic locus of the apparent adaptation at the membrane level.

  20. Mechanical Properties of the Plasma Membrane of Isolated Plant Protoplasts 1

    PubMed Central

    Wolfe, Joe; Steponkus, Peter 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. Images Fig. 2 PMID:16662817

  1. Membrane-based, sedimentation-assisted plasma separator for point-of-care applications.

    PubMed

    Liu, Changchun; Mauk, Michael; Gross, Robert; Bushman, Frederic D; Edelstein, Paul H; Collman, Ronald G; Bau, Haim H

    2013-11-01

    Often, high-sensitivity, point-of-care (POC) clinical tests, such as HIV viral load, require large volumes of plasma. Although centrifuges are ubiquitously used in clinical laboratories to separate plasma from whole blood, centrifugation is generally inappropriate for on-site testing. Suitable alternatives are not readily available to separate the relatively large volumes of plasma from milliliters of blood that may be needed to meet stringent limit-of-detection specifications for low-abundance target molecules. We report on a simple-to-use, low-cost, pump-free, membrane-based, sedimentation-assisted plasma separator capable of separating a relatively large volume of plasma from undiluted whole blood within minutes. This plasma separator consists of an asymmetric, porous, polysulfone membrane housed in a disposable chamber. The separation process takes advantage of both gravitational sedimentation of blood cells and size exclusion-based filtration. The plasma separator demonstrated a "blood in-plasma out" capability, consistently extracting 275 ± 33.5 μL of plasma from 1.8 mL of undiluted whole blood within less than 7 min. The device was used to separate plasma laden with HIV viruses from HIV virus-spiked whole blood with recovery efficiencies of 95.5% ± 3.5%, 88.0% ± 9.5%, and 81.5% ± 12.1% for viral loads of 35,000, 3500, and 350 copies/mL, respectively. The separation process is self-terminating to prevent excessive hemolysis. The HIV-laden plasma was then injected into our custom-made microfluidic chip for nucleic acid testing and was successfully subjected to reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP), demonstrating that the plasma is sufficiently pure to support high-efficiency nucleic acid amplification. PMID:24099566

  2. Mutational scanning reveals the determinants of protein insertion and association energetics in the plasma membrane

    PubMed Central

    Elazar, Assaf; Weinstein, Jonathan; Biran, Ido; Fridman, Yearit; Bibi, Eitan; Fleishman, Sarel Jacob

    2016-01-01

    Insertion of helix-forming segments into the membrane and their association determines the structure, function, and expression levels of all plasma membrane proteins. However, systematic and reliable quantification of membrane-protein energetics has been challenging. We developed a deep mutational scanning method to monitor the effects of hundreds of point mutations on helix insertion and self-association within the bacterial inner membrane. The assay quantifies insertion energetics for all natural amino acids at 27 positions across the membrane, revealing that the hydrophobicity of biological membranes is signi