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Sample records for oocyte plasma membrane

  1. 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.

  2. ATP crossing the cell plasma membrane generates an ionic current in xenopus oocytes.

    PubMed

    Bodas, E; Aleu, J; Pujol, G; Martin-Satué, M; Marsal, J; Solsona, C

    2000-07-07

    The presence of ATP within cells is well established. However, ATP also operates as an intercellular signal via specific purinoceptors. Furthermore, nonsecretory cells can release ATP under certain experimental conditions. To measure ATP release and membrane currents from a single cell simultaneously, we used Xenopus oocytes. We simultaneously recorded membrane currents and luminescence. Here, we show that ATP release can be triggered in Xenopus oocytes by hyperpolarizing pulses. ATP release (3.2 +/- 0.3 pmol/oocyte) generated a slow inward current (2.3 +/- 0.1 microA). During hyperpolarizing pulses, the permeability for ATP(4-) was more than 4000 times higher than that for Cl(-). The sensitivity to GdCl(3) (0. 2 mm) of hyperpolarization-induced ionic current, ATP release and E-ATPase activity suggests their dependence on stretch-activated ion channels. The pharmacological profile of the current inhibition coincides with the inhibition of ecto-ATPase activity. This enzyme is highly conserved among species, and in humans, it has been cloned and characterized as CD39. The translation, in Xenopus oocytes, of human CD39 mRNA encoding enhances the ATP-supported current, indicating that CD39 is directly or indirectly responsible for the electrodiffusion of ATP.

  3. Determination of ionic permeability coefficients of the plasma membrane of Xenopus laevis oocytes under voltage clamp.

    PubMed Central

    Costa, P F; Emilio, M G; Fernandes, P L; Ferreira, H G; Ferreira, K G

    1989-01-01

    1. A method of estimating absolute ionic permeability coefficients which does not depend on the use of impermeant substitutes is reported. 2. The method is based on a pump leak model of the Xenopus laevis oocyte membrane. The procedure consists of measuring, in the same experiment, the pump current and the currents generated under voltage clamp by the partial substitution of one or two ions at a time. For each experimental condition, the measured currents are substituted in a Goldman-Hodgkin-Katz type equation with two unknowns (the permeability coefficients). The set of equations thus generated enables the computation of all the ionic permeability coefficients. 3. The Xenopus oocyte membrane (stages IV and V, Dumont, 1972) has been found to be permeable to conventional ion substitutes such as N-methyl-D-glucamine (NMG), sulphate, isethionate and gluconate. 4. The values for sodium, potassium and chloride permeability coefficients obtained from sixty-eight pooled experiments were, respectively, 5.44, 17.41 and 1.49 x 10(-8) cm s-1. 5. The diffusional currents for sodium, potassium and chloride computed from the experiments referred to above were, respectively, -1.16, 0.69 and -0.038 microA cm-2. 6. A stoichiometry of the Na+-K+ pump exchange of 3/1.8 was computed. 7. The intracellular concentrations of sodium, potassium and chloride ions, as determined by ion-selective microelectrodes, were, respectively, 10.1 +/- 0.66 mM (n = 12), 109.5 +/- 3.3 mM (n = 13) and 37.7 +/- 1.18 mM (n = 19), corresponding to equilibrium potentials of 61, -95 and -28 mV. 8. Since chloride is not at equilibrium across the membrane, we propose that there is an inward uphill Cl- transport. PMID:2600847

  4. HCO3−/Cl− Exchange Inactivation and Reactivation during Mouse Oocyte Meiosis Correlates with MEK/MAPK-Regulated Ae2 Plasma Membrane Localization

    PubMed Central

    Zhou, Chenxi; Tiberi, Mario; Liang, Binhui; Alper, Seth L.; Baltz, Jay M.

    2009-01-01

    Background Germinal Vesicle (GV) stage mouse oocytes in first meiotic prophase exhibit highly active HCO3−/Cl− exchange—a class of transport nearly ubiquitously involved in regulation of intracellular pH and cell volume. During meiosis, however, oocyte HCO3−/Cl− exchange becomes inactivated during first metaphase (MI), remains inactive in second metaphase (MII), and is reactivated only after egg activation. Previous work using pharmacological manipulations had indicated that activity of the MEK/MAPK signaling pathway was negatively correlated with HCO3−/Cl− exchange activity during meiosis. However, the mechanism by which the exchanger is inactivated during meiotic progression had not been determined, nor had the role of MEK/MAPK been directly established. Methodology/Principal Findings Expression of a constitutively active form of MEK (MAP kinase kinase), which prevented the normal downregulation of MAPK after egg activation, also prevented reactivation of HCO3−/Cl− exchange. Conversely, suppression of endogenous MAPK activity with dominant negative MEK activated the normally quiescent HCO3−/Cl− exchange in mature MII eggs. A GFP-tagged form of the HCO3−/Cl− exchanger isoform Ae2 (Slc4a2) was strongly expressed at the GV oocyte plasma membrane, but membrane localization decreased markedly during meiotic progression. A similar pattern for endogenous Ae2 was confirmed by immunocytochemistry. The loss of membrane-localized Ae2 appeared selective, since membrane localization of a GFP-tagged human dopamine D1 receptor did not change during meiotic maturation. Conclusions Direct manipulation of MAPK activity indicated that GFP-tagged Ae2 localization depended upon MAPK activity. Inactivation of HCO3−/Cl− exchange during the meiotic cell cycle may therefore reflect the loss of Ae2 from the oocyte plasma membrane, downstream of MEK/MAPK signaling. This identifies a novel role for MEK/MAPK-mediated cytostatic factor (CSF) activity during

  5. Progesterone modulation of transmembrane helix-helix interactions between the α-subunit of Na/K-ATPase and phospholipid N-methyltransferase in the oocyte plasma membrane

    PubMed Central

    2010-01-01

    Background Progesterone binding to the surface of the amphibian oocyte initiates the meiotic divisions. Our previous studies with Rana pipiens oocytes indicate that progesterone binds to a plasma membrane site within the external loop between the M1 and M2 helices of the α-subunit of Na/K-ATPase, triggering a cascade of lipid second messengers and the release of the block at meiotic prophase. We have characterized this site, using a low affinity ouabain binding isoform of the α1-subunit. Results Preparations of isolated plasma membranes from Rana oocytes demonstrate that physiological levels of progesterone (or the non-metabolizable progestin R5020) successively activate phosphatidylethanolamine-N-methyltransferase (PE-NMT) and sphingomyelin synthase within seconds. Inhibition of PE-NMT blocks the progesterone induction of meiosis in intact oocytes, whereas its initial product, phosphatidylmonomethylethanolamine (PME), can itself initiate meiosis in the presence of the inhibitor. Published X-ray crystallographic data on Na/K-ATPase, computer-generated 3D projections, heptad repeat analysis and hydrophobic cluster analysis of the transmembrane helices predict that hydrophobic residues L, V, V, I, F and Y of helix M2 of the α1-subunit interact with F, L, G, L, L and F, respectively, of helix M3 of PE-NMT. Conclusion We propose that progesterone binding to the first external loop of the α1-subunit facilitates specific helix-helix interactions between integral membrane proteins to up-regulate PE-NMT, and, that successive interactions between two or more integral plasma membrane proteins induce the signaling cascades which result in completion of the meiotic divisions. PMID:20500835

  6. Does heat stress provoke the loss of a continuous layer of cortical granules beneath the plasma membrane during oocyte maturation?

    PubMed

    Andreu-Vázquez, C; López-Gatius, F; García-Ispierto, I; Maya-Soriano, M J; Hunter, R H F; López-Béjar, M

    2010-11-01

    The objective of the present study was to evaluate the influence of heat stress on bovine oocyte maturation. Both nuclear stage and distribution of cortical granules (CG) were simultaneously evaluated in each oocyte. Oocyte overmaturation under standard conditions of culture was also evaluated. For this purpose, logistic regression procedures were used to evaluate possible effects of factors such as heat stress, overmaturation, replicate, CG distribution and metaphase II (MII) morphology on oocyte maturation. Based on the odds ratio, oocytes on heat stressed (HSO) and overmaturated (OMO) oocyte group were, respectively, 14.5 and 5.4 times more likely to show anomalous MII morphology than those matured under control conditions (CO). The likelihood for an oocyte of showing the CG distribution pattern IV (aging oocyte) was 6.3 and 9.3 times higher for HSO and OMO groups, respectively, than for the CO group. The risk of undergoing anomalous oocyte maturation, considering both nuclear stage and distribution of CG was 17.1 and 18 times greater in oocytes cultured in HSO and OMO groups, respectively, than those in the CO group. In conclusion, heat stress proved to be valuable in aging oocytes. Heat stress advanced age for nuclear and cytoplasmic processes in a similar form to that of oocyte overmaturation.

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

    PubMed Central

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

    2002-01-01

    The Xenopus oocyte is a very powerful tool for studies of the structure and function of membrane proteins, e.g., messenger RNA extracted from the brain and injected into oocytes leads to the synthesis and membrane incorporation of many types of functional receptors and ion channels, and membrane vesicles from Torpedo electroplaques injected into oocytes fuse with the oocyte membrane and cause the appearance of functional Torpedo acetylcholine receptors and Cl− channels. This approach was developed further to transplant already assembled neurotransmitter receptors from human brain cells to the plasma membrane of Xenopus oocytes. Membranes isolated from the temporal neocortex of a patient, operated for intractable epilepsy, were injected into oocytes and, within a few hours, the oocyte membrane acquired functional neurotransmitter receptors to γ-aminobutyric acid, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, and glycine. These receptors were also expressed in the plasma membrane of oocytes injected with mRNA extracted from the temporal neocortex of the same patient. All of this makes the Xenopus oocyte a more useful model than it already is for studies of the structure and function of many human membrane proteins and opens the way to novel pathophysiological investigations of some human brain disorders. PMID:12237406

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

    NASA Astrophysics Data System (ADS)

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

    2002-10-01

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

  9. Evidence from simultaneous intracellular- and surface-pH transients that carbonic anhydrase IV enhances CO2 fluxes across Xenopus oocyte plasma membranes.

    PubMed

    Musa-Aziz, Raif; Occhipinti, Rossana; Boron, Walter F

    2014-11-01

    Human carbonic anhydrase IV (CA IV) is GPI-anchored to the outer membrane surface, catalyzing CO2/HCO3 (-) hydration-dehydration. We examined effects of heterologously expressed CA IV on intracellular-pH (pHi) and surface-pH (pHS) transients caused by exposing oocytes to CO2/HCO3 (-)/pH 7.50. CO2 influx causes a sustained pHi fall and a transient pHS rise; CO2 efflux does the opposite. Both during CO2 addition and removal, CA IV increases magnitudes of maximal rate of pHi change (dpHi/dt)max, and maximal pHS change (ΔpHS) and decreases time constants for pHi changes (τpHi ) and pHS relaxations (τpHS ). Decreases in time constants indicate that CA IV enhances CO2 fluxes. Extracellular acetazolamide blocks all CA IV effects, but not those of injected CA II. Injected acetazolamide partially reduces CA IV effects. Thus, extracellular CA is required for, and the equivalent of cytosol-accessible CA augments, the effects of CA IV. Increasing the concentration of the extracellular non-CO2/HCO3 (-) buffer (i.e., HEPES), in the presence of extracellular CA or at high [CO2], accelerates CO2 influx. Simultaneous measurements with two pHS electrodes, one on the oocyte meridian perpendicular to the axis of flow and one downstream from the direction of extracellular-solution flow, reveal that the downstream electrode has a larger (i.e., slower) τpHS , indicating [CO2] asymmetry over the oocyte surface. A reaction-diffusion mathematical model (third paper in series) accounts for the above general features, and supports the conclusion that extracellular CA, which replenishes entering CO2 or consumes exiting CO2 at the extracellular surface, enhances the gradient driving CO2 influx across the cell membrane.

  10. Microtransplantation of membranes from cultured cells to Xenopus oocytes: A method to study neurotransmitter receptors embedded in native lipids

    PubMed Central

    Palma, Eleonora; Trettel, Flavia; Fucile, Sergio; Renzi, Massimiliano; Miledi, Ricardo; Eusebi, Fabrizio

    2003-01-01

    The Xenopus oocyte is used as a convenient cell expression system to study the structure and function of heterogenic transmitter receptors and ion channels. Recently, we introduced a method to microtransplant already assembled neurotransmitter receptors from the human brain to the plasma membrane of Xenopus oocytes. The same approach was used here to transplant neurotransmitter receptors expressed from cultured cells to the oocytes. Membrane vesicles prepared from a human embryonic kidney cell line (HEK293) stably expressing the rat glutamate receptor 1 were injected into oocytes, and, within a few hours, the oocyte plasma membrane acquired α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors, which had the same properties as those expressed in the original HEK cells. Analogously, oocytes injected with membranes prepared from rat pituitary GH(4)C1 cells, stably expressing homomeric human neuronal α7 nicotinic acetylcholine receptors (α7-AcChoRs), incorporated in their plasma membrane AcChoRs that behaved as those expressed in GH(4)C1 cells. Similar results were obtained with HEK cells stably expressing heteromeric human neuronal α4β2-AcChoRs. All this makes the Xenopus oocyte a powerful tool for detailed investigations of receptors and other proteins expressed in the membrane of cultured cells. PMID:12595576

  11. Evidence from simultaneous intracellular- and surface-pH transients that carbonic anhydrase II enhances CO2 fluxes across Xenopus oocyte plasma membranes

    PubMed Central

    Occhipinti, Rossana; Boron, Walter F.

    2014-01-01

    The α-carbonic anhydrases (CAs) are zinc-containing enzymes that catalyze the interconversion of CO2 and HCO3−. Here, we focus on human CA II (CA II), a ubiquitous cytoplasmic enzyme. In the second paper in this series, we examine CA IV at the extracellular surface. After microinjecting recombinant CA II in a Tris solution (or just Tris) into oocytes, we expose oocytes to 1.5% CO2/10 mM HCO3−/pH 7.50 while using microelectrodes to monitor intracellular pH (pHi) and surface pH (pHS). CO2 influx causes the familiar sustained pHi fall as well as a transient pHS rise; CO2 efflux does the opposite. Both during CO2 addition and removal, CA II increases the magnitudes of the maximal rate of pHi change, (dpHi/dt)max, and the maximal change in pHS, ΔpHS. Preincubating oocytes with the inhibitor ethoxzolamide eliminates the effects of CA II. Compared with pHS, pHi begins to change only after a delay of ∼9 s and its relaxation has a larger (i.e., slower) time constant (τpHi > τpHS). Simultaneous measurements with two pHi electrodes, one superficial and one deep, suggest that impalement depth contributes to pHi delay and higher τpHi. Using higher CO2/HCO3− levels, i.e., 5%/33 mM HCO3− or 10%/66 mM HCO3−, increases (dpHi/dt)max and ΔpHS, though not in proportion to the increase in [CO2]. A reaction-diffusion mathematical model (described in the third paper in this series) accounts for the above general features and supports the conclusion that cytosolic CA—consuming entering CO2 or replenishing exiting CO2—increases CO2 fluxes across the cell membrane. PMID:24965587

  12. Incorporation of acetylcholine receptors and Cl- channels in Xenopus oocytes injected with Torpedo electroplaque membranes.

    PubMed Central

    Marsal, J; Tigyi, G; Miledi, R

    1995-01-01

    A method was developed to transplant assembled nicotinic acetylcholine receptors (AcChoRs) and Cl- channels from the electric organ of Torpedo to the membrane of Xenopus oocytes. Membrane vesicles from Torpedo electroplaques were injected into the oocytes and, within a few hours, the oocyte membrane acquired AcChoRs and Cl- channels. The mechanism of expression of these receptors and channels is very different from that which follows the injection of mRNA, since the appearance of receptors after membrane injection does not require de novo protein synthesis or N-glycosylation. This, and other controls, indicate that the foreign receptor-bearing membranes fuse with the oocyte membrane and cause the appearance of functional receptors and channels. All this makes the Xenopus oocyte an even more powerful tool for studies of the structure and function of membrane proteins. PMID:7761478

  13. Dynamics of intracellular phospholipid membrane organization during oocyte maturation and successful vitrification of immature oocytes retrieved by ovum pick-up in cattle.

    PubMed

    Aono, Akira; Nagatomo, Hiroaki; Takuma, Tetsuya; Nonaka, Rika; Ono, Yoshitaka; Wada, Yasuhiko; Abe, Yasuyuki; Takahashi, Masashi; Watanabe, Tomomasa; Kawahara, Manabu

    2013-05-01

    The objective was to determine if immature bovine oocytes with cumulus cells at the germinal vesicle (GV) stage could be vitrified by aluminum sheets (AS; pieces of sheet-like aluminum foil). Cleavage rates in fertilized oocytes previously vitrified by the AS procedure were higher than those vitrified by a nylon-mesh holder (NM) procedure (89.3 ± 2.1% vs. 65.0 ± 3.7%). Cleaved embryos derived from the AS but not from the NM procedures developed to blastocysts. Furthermore, to investigate the effects of vitrifying GV oocytes on cytoplasmic structure and on the ability to undergo cytoplasmic changes, the intracellular phospholipid membrane (IM) was stained with the lipophilic fluorescent dye, 3,3'-dioctadecyloxa-carbocyanine perchlorate. After vitrification by AS, the IM remained intact relative to that of oocytes vitrified by NM. During in vitro maturation, reorganization of the IM was also undamaged in oocytes vitrified by AS before oocyte maturation, and the IM within oocytes vitrified by the NM procedure was evidently impaired. Finally, vitrification (AS) was used for GV oocytes collected using the ovum pick-up method. A bull calf was born after in vitro production and subsequent embryo transfer. The vitrification techniques described herein should facilitate generation of viable in vitro production bovine blastocysts using oocytes recovered using the ovum pick-up method.

  14. Encapsulation of sex sorted boar semen: sperm membrane status and oocyte penetration parameters.

    PubMed

    Spinaci, Marcella; Chlapanidas, Theodora; Bucci, Diego; Vallorani, Claudia; Perteghella, Sara; Lucconi, Giulia; Communod, Ricardo; Vigo, Daniele; Galeati, Giovanna; Faustini, Massimo; Torre, Maria Luisa

    2013-03-01

    Although sorted semen is experimentally used for artificial, intrauterine, and intratubal insemination and in vitro fertilization, its commercial application in swine species is still far from a reality. This is because of the low sort rate and the large number of sperm required for routine artificial insemination in the pig, compared with other production animals, and the greater susceptibility of porcine spermatozoa to stress induced by the different sex sorting steps and the postsorting handling protocols. The encapsulation technology could overcome this limitation in vivo, protecting and allowing the slow release of low-dose sorted semen. The aim of this work was to evaluate the impact of the encapsulation process on viability, acrosome integrity, and on the in vitro fertilizing potential of sorted boar semen. Our results indicate that the encapsulation technique does not damage boar sorted semen; in fact, during a 72-hour storage, no differences were observed between liquid-stored sorted semen and encapsulated sorted semen in terms of plasma membrane (39.98 ± 14.38% vs. 44.32 ± 11.72%, respectively) and acrosome integrity (74.32 ± 12.17% vs. 66.07 ± 10.83%, respectively). Encapsulated sorted spermatozoa presented a lower penetration potential than nonencapsulated ones (47.02% vs. 24.57%, respectively, P < 0.0001), and a significant reduction of polyspermic fertilization (60.76% vs. 36.43%, respectively, polyspermic ova/total ova; P < 0.0001). However, no difference (P > 0.05) was observed in terms of total efficiency of fertilization expressed as normospermic oocytes/total oocytes (18.45% vs. 15.43% for sorted diluted and sorted encapsulated semen, respectively). The encapsulation could be an alternative method of storing of pig sex sorted spermatozoa and is potentially a promising technique in order to optimize the use of low dose of sexed spermatozoa in vivo.

  15. 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.

  16. Membrane-pipette interactions underlie delayed voltage activation of mechanosensitive channels in Xenopus oocytes.

    PubMed Central

    Gil, Z; Magleby, K L; Silberberg, S D

    1999-01-01

    To investigate the mechanism for the delayed activation by voltage of the predominant mechanosensitive (MS) channel in Xenopus oocytes, currents were recorded from on-cell and excised patches of membrane with the patch clamp technique and from intact oocytes with the two-electrode voltage clamp technique. MS channels could be activated by stretch in inside-out, on-cell, and outside-out patch configurations, using pipettes formed of either borosilicate or soft glass. In inside-out patches formed with borosilicate glass pipettes, depolarizing voltage steps activated MS channels in a cooperative manner after delays of seconds. This voltage-dependent activation was not observed for outside-out patches. Voltage-dependent activation was also not observed when the borosilicate pipettes were either replaced with soft glass pipettes or coated with soft glass. When depolarizing voltage steps were applied to the whole oocyte with a two-electrode voltage clamp, currents that could be attributed to MS channels were not observed. Yet the same depolarizing steps activated MS channels in on-cell patches formed with borosilicate pipettes on the same oocyte. These observations suggest that the delayed cooperative activation of MS channels by depolarization is not an intrinsic property of the channels, but requires interaction between the membrane and patch pipette. PMID:10354436

  17. Effect of magnetized extender on sperm membrane integrity and development of oocytes in vitro fertilized with liquid storage boar semen.

    PubMed

    Lee, Sang-Hee; Park, Choon-Keun

    2015-03-01

    The objective of this study was to evaluate the effect of a magnetized extender on sperm membrane damage and development of oocytes in vitro fertilized with liquid storage boar semen. Before semen dilution, extender was flowed through a neodymium magnet (0, 2000, 4000 and 6000G) for 5min and collected semen was preserved for 168h at 18°C. In results, plasma membrane integrity with live sperm was significantly higher in semen treated with extenders magnetized at 4000G than sperm treated with extenders magnetized at 0G during semen preservation for 120-168h (p<0.05). In addition, acrosomal membrane damage was significantly lower in semen treated with extenders magnetized at 4000 and 6000G compared to 0 and 2000G during semen preservation for 168h (p<0.05). And mitochondrial membrane damage with all sperm was significantly lower in semen treated with extenders magnetized at 2000G than other groups during semen preservation for 168h. The ability of semen to achieve successful in vitro fertilization was also not significantly different among the groups during preservation. However, when the semen was preserved for 168h, the blastocyst formation rates were significantly higher at 6000G compared to 0 and 2000G (p<0.05). In conclusion, these results suggest that highly magnetized semen extender could protect the sperm membrane from damage, and improve the ability of rates of in vitro blastocyst development and magnetized semen diluter is beneficial for long liquid preservation of boar semen.

  18. 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.

  19. Obox4-silencing-activated STAT3 and MPF/MAPK signaling accelerate nuclear membrane breakdown in mouse oocytes.

    PubMed

    Lee, Hyun-Seo; Kim, Kyeoung-Hwa; Kim, Eun-Young; Lee, Su-Yeon; Ko, Jung-Jae; Lee, Kyung-Ah

    2016-04-01

    Mouse oocytes begin to mature in vitro once liberated from ovarian follicles. Previously, we showed that oocyte-specific homeobox 4 (Obox4) is critical for maintaining the intact nuclear membrane of the germinal vesicle (GV) in oocytes and for completing meiosis at the metaphase I-II (MI-MII) transition. This study further examines the molecular mechanisms of OBOX4 in regulating GV nuclear membrane breakdown. Maturation-promoting factor (MPF) and MAPK are normally inactive in GV stage oocytes but were activated prematurely in arrested GV stage oocytes by 3-isobutyl-1-metyl-xanthine (IBMX) in vitro after Obox4 RNA interference (RNAi). Furthermore, signal transducer and activator of transcription 3 (STAT3) was significantly activated by Obox4 RNAi. We confirmed that this Obox4 RNAi-induced premature STAT3 and MPF/MAPK activation at the GV stage provoked subsequent GV breakdown (GVBD) despite the opposing force of high cAMP in the IBMX-supplemented medium to maintain intact GV. When cumulus-oocyte complexes were exposed to interferon α (IFNA), a STAT3 activator, oocytes matured and cumulus cells expanded to resume nuclear maturation in IBMX-supplemented medium, suggesting that STAT3 activation is sufficient for stimulating the continuation of meiosis. Using Stattic, a specific STAT3 inhibitor, we confirmed that GVBD involves STAT3 activation in Obox4-silenced oocytes. Based on these findings, we concluded that i) Obox4 is an important upstream regulator of MPF/MAPK and STAT3 signaling, and ii) Obox4 is a key regulator of the GV arrest mechanism in oocytes.

  20. Proteomic analysis of integral plasma membrane proteins.

    PubMed

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

    2004-04-01

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

  1. An improved method for real-time monitoring of membrane capacitance in Xenopus laevis oocytes.

    PubMed Central

    Schmitt, Bernhard M; Koepsell, Hermann

    2002-01-01

    Measurements of membrane capacitance (C(m)) in Xenopus laevis oocytes offer unique experimental possibilities but are difficult to perform with current methods. To improve C(m) measurements in the two-electrode voltage clamp (TEVC) mode, we developed a paired-ramp protocol and tested its performance in a model circuit (with tunable C(m), membrane resistance R(m), and series resistance R(s)) and in Xenopus oocytes. In the cell model and with R(s) = 0 Omega, inaccuracy of C(m) estimates was <1% under widely varying conditions (R(m) ranging from 100 to 2000 kOmega, and C(m) from 50 to 1000 nF). With R(s) > 0 Omega, C(m) was underestimated by a relative error epsilon closely approximated as epsilon approximate 2 x R(s)/(R(s) + R(m)), in keeping with the theoretical prediction. Thus, epsilon may be neglected under standard conditions or, under extreme conditions, corrected for if R(s) is known. Relative imprecision of C(m) estimates was small, independent of R(s), and inversely related to C(m) (<1.5% at 50 nF, <0.4% at 200 nF). Averaging allowed reliable detection of C(m) deviations from 200 nF of 0.1 nF, i.e., 0.05%. In Xenopus oocytes, we could resolve C(m) changes that were small (e.g., DeltaC(m) approximate 2 nF upon 100 muM 8-Br-cAMP), fast (e.g., DeltaC(m)/Deltat approximate 20nF/30s upon 1 muM phorbol myristate acetate (PMA)) or extended and complex (e.g., fast increase, followed by prolonged C(m) decrease upon 1 muM PMA). Rapidly alternating between paired ramps and a second, step protocol allowed quasi-simultaneous monitoring of additional electrical parameters such as R(m), slope conductance g(m), and reversal potential E(rev). Taken together, our method is suited to monitor C(m) in Xenopus oocytes conveniently, with high temporal resolution, accuracy and precision, and in parallel with other electrical parameters. Thus, it may be useful for the study of endo- and exocytosis and of membrane protein regulation and for electrophysiological high

  2. Progesterone Receptor Membrane Component 1 Mediates Progesterone-Induced Suppression of Oocyte Meiotic Prophase I and Primordial Folliculogenesis

    PubMed Central

    Guo, Meng; Zhang, Cheng; Wang, Yan; Feng, Lizhao; Wang, Zhengpin; Niu, Wanbo; Du, Xiaoyan; Tang, Wang; Li, Yuna; Wang, Chao; Chen, Zhenwen

    2016-01-01

    Well-timed progression of primordial folliculogenesis is essential for mammalian female fertility. Progesterone (P4) inhibits primordial follicle formation under physiological conditions; however, P4 receptor that mediates this effect and its underlying mechanisms are unclear. In this study, we used an in vitro organ culture system to show that progesterone receptor membrane component 1 (PGRMC1) mediated P4-induced inhibition of oocyte meiotic prophase I and primordial follicle formation. We found that membrane-impermeable BSA-conjugated P4 inhibited primordial follicle formation similar to that by P4. Interestingly, PGRMC1 and its partner serpine1 mRNA-binding protein 1 were highly expressed in oocytes in perinatal ovaries. Inhibition or RNA interference of PGRMC1 abolished the suppressive effect of P4 on follicle formation. Furthermore, P4-PGRMC1 interaction blocked oocyte meiotic progression and decreased intra-oocyte cyclic AMP (cAMP) levels in perinatal ovaries. cAMP analog dibutyryl cAMP reversed P4–PGRMC1 interaction-induced inhibition of meiotic progression and follicle formation. Thus, our results indicated that PGRMC1 mediated P4-induced suppression of oocyte meiotic progression and primordial folliculogenesis by decreasing intra-oocyte cAMP levels. PMID:27848973

  3. Xenopus laevis Oocytes as a Model System for Studying the Interaction Between Asbestos Fibres and Cell Membranes.

    PubMed

    Bernareggi, Annalisa; Ren, Elisa; Borelli, Violetta; Vita, Francesca; Constanti, Andrew; Zabucchi, Giuliano

    2015-06-01

    The mode of interaction of asbestos fibres with cell membranes is still debatable. One reason is the lack of a suitable and convenient cellular model to investigate the causes of asbestos toxicity. We studied the interaction of asbestos fibres with Xenopus laevis oocytes, using electrophysiological and morphological methods. Oocytes are large single cells, with a limited ability to endocytose molecular ligands; we therefore considered these cells to be a good model for investigating the nature of asbestos/membrane interactions. Electrophysiological recordings were performed to compare the passive electrical membrane properties, and those induced by applying positive or negative voltage steps, in untreated oocytes and those exposed to asbestos fibre suspensions. Ultrastructural analysis visualized in detail, any morphological changes of the surface membrane caused by the fibre treatment. Our results demonstrate that Amosite and Crocidolite-type asbestos fibres significantly modify the properties of the membrane, starting soon after exposure. Cells were routinely depolarized, their input resistance decreased, and the slow outward currents evoked by step depolarizations were dramatically enhanced. Reducing the availability of surface iron contained in the structure of the fibres with cation chelators, abolished these effects. Ultrastructural analysis of the fibre-exposed oocytes showed no evidence of phagocytic events. Our results demonstrate that asbestos fibres modify the oocyte membrane, and we propose that these cells represent a viable model for studying the asbestos/cell membrane interaction. Our findings also open the possibly for finding specific competitors capable of hindering the asbestos-cell membrane interaction as a means of tackling the long-standing asbestos toxicity problem.

  4. Age-Associated Lipidome Changes in Metaphase II Mouse Oocytes.

    PubMed

    Mok, Hyuck Jun; Shin, Hyejin; Lee, Jae Won; Lee, Geun-Kyung; Suh, Chang Suk; Kim, Kwang Pyo; Lim, Hyunjung Jade

    2016-01-01

    The quality of mammalian oocytes declines with age, which negatively affects fertilization and developmental potential. The aging process often accompanies damages to macromolecules such as proteins, DNA, and lipids. To investigate if aged oocytes display an altered lipidome compared to young oocytes, we performed a global lipidomic analysis between oocytes from 4-week-old and 42 to 50-week-old mice. Increased oxidative stress is often considered as one of the main causes of cellular aging. Thus, we set up a group of 4-week-old oocytes treated with hydrogen peroxide (H2O2), a commonly used oxidative stressor, to compare if similar lipid species are altered between aged and oxidative-stressed oocytes. Between young and aged oocytes, we identified 26 decreased and 6 increased lipids in aged oocytes; and between young and H2O2-treated oocytes, we identified 35 decreased and 26 increased lipids in H2O2-treated oocytes. The decreased lipid species in these two comparisons were overlapped, whereas the increased lipid species were distinct. Multiple phospholipid classes, phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylserine (PS), and lysophosphatidylserine (LPS) significantly decreased both in H2O2-treated and aged oocytes, suggesting that the integrity of plasma membrane is similarly affected under these conditions. In contrast, a dramatic increase in diacylglycerol (DG) was only noted in H2O2-treated oocytes, indicating that the acute effect of H2O2-caused oxidative stress is distinct from aging-associated lipidome alteration. In H2O2-treated oocytes, the expression of lysophosphatidylcholine acyltransferase 1 increased along with increases in phosphatidylcholine. Overall, our data reveal that several classes of phospholipids are affected in aged oocytes, suggesting that the integrity of plasma membrane is associated with maintaining fertilization and developmental potential of mouse oocytes.

  5. Age-Associated Lipidome Changes in Metaphase II Mouse Oocytes

    PubMed Central

    Lee, Jae Won; Lee, Geun-Kyung; Suh, Chang Suk; Kim, Kwang Pyo; Lim, Hyunjung Jade

    2016-01-01

    The quality of mammalian oocytes declines with age, which negatively affects fertilization and developmental potential. The aging process often accompanies damages to macromolecules such as proteins, DNA, and lipids. To investigate if aged oocytes display an altered lipidome compared to young oocytes, we performed a global lipidomic analysis between oocytes from 4-week-old and 42 to 50-week-old mice. Increased oxidative stress is often considered as one of the main causes of cellular aging. Thus, we set up a group of 4-week-old oocytes treated with hydrogen peroxide (H2O2), a commonly used oxidative stressor, to compare if similar lipid species are altered between aged and oxidative-stressed oocytes. Between young and aged oocytes, we identified 26 decreased and 6 increased lipids in aged oocytes; and between young and H2O2-treated oocytes, we identified 35 decreased and 26 increased lipids in H2O2-treated oocytes. The decreased lipid species in these two comparisons were overlapped, whereas the increased lipid species were distinct. Multiple phospholipid classes, phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylserine (PS), and lysophosphatidylserine (LPS) significantly decreased both in H2O2-treated and aged oocytes, suggesting that the integrity of plasma membrane is similarly affected under these conditions. In contrast, a dramatic increase in diacylglycerol (DG) was only noted in H2O2-treated oocytes, indicating that the acute effect of H2O2-caused oxidative stress is distinct from aging-associated lipidome alteration. In H2O2-treated oocytes, the expression of lysophosphatidylcholine acyltransferase 1 increased along with increases in phosphatidylcholine. Overall, our data reveal that several classes of phospholipids are affected in aged oocytes, suggesting that the integrity of plasma membrane is associated with maintaining fertilization and developmental potential of mouse oocytes. PMID:26881843

  6. ISOLATION OF RAT LIVER PLASMA MEMBRANES

    PubMed Central

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

    1970-01-01

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

  7. 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.

  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. 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

  10. 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.

  11. Effect of blood plasma collected after adrenocorticotropic hormone administration during the preovulatory period in the sow on oocyte in vitro maturation.

    PubMed

    González, R; Sjunnesson, Y C B

    2013-10-01

    Reproduction may be affected by stressful events changing the female endocrine or metabolic profile. An altered environment during oocyte development could influence the delicate process of oocyte maturation. Here, the effect of simulated stress by media supplementation with blood plasma from sows after adrenocorticotropic hormone (ACTH) administration during the preovulatory period was assessed. Oocytes were matured for 46 hours in the presence of plasma from ACTH-treated sows, or plasma from NaCl-treated control sows, or medium without plasma (BSA group). The plasma used had been collected at 36 and 12 hours (±2 hours) before ovulation (for the first 24 hours + last 22 hours of maturation, respectively). Subsequent fertilization and embryo development were evaluated. Actin cytoskeleton and mitochondrial patterns were studied by confocal microscopy both in the oocytes and the resulting blastocysts. Nuclear maturation did not differ between treatments. Subtle differences were observed in the actin microfilaments in oocytes; however, mitochondrial patterns were associated with the treatment (P < 0.001). These differences in mitochondrial patterns were not reflected by in vitro outcomes, which were similar in all groups. In conclusion, an altered hormonal environment provided by a brief exposure to plasma from ACTH-treated sows during in vitro oocyte maturation could induce alterations in actin cytoskeleton and mitochondrial patterns in oocytes. However, these changes might not hamper the subsequent in vitro embryo development.

  12. Determination of oocyte membrane permeability coefficients and their application to cryopreservation in a rabbit model.

    PubMed

    Liu, Jun; Mullen, Steve; Meng, Qinggang; Critser, John; Dinnyes, Andras

    2009-10-01

    Having an effective means to cryopreserve human oocytes would offer more flexibility in healthcare services for infertility patients, and obviate cryopreservation of preimplantation embryos. It is essential to establish good animal models for human oocyte cryopreservation and the rabbit is a good candidate. Attempts to improve oocyte cryopreservation are often empirical, with results often being irreproducible. Cryopreservation protocols may be optimized by modeling the changes in oocyte volume and the associated damages incurred during the addition and dilution of cryoprotective agents (CPA). The objectives of the current study were to determine cryobiological properties of rabbit oocytes, including the isotonic volume, osmotically inactive cell fraction (V(b)), hydraulic conductivity (L(p)), permeability (P(s)) to dimethylsulfoxide (Me(2)SO), ethylene glycol (EG), and glycerol (GLY) and to examine the correlation between cell volume excursions and viability. This has led to the development of the accumulative osmotic damage (AOD) model associated with the processes of CPA addition/dilution. Mature rabbit oocytes were perfused with 15% (V/V) CPA medium (dissolved in 1x PBS). The osmotic responses of the oocytes were videotaped. A two-parameter model was fit to the experimental data to determine the values of L(p) and P(s). Oocyte volumes reached upon equilibration with 285, 600, 900, and 1200 mOsm (milliosmolal) solutions of non-permeating compounds were plotted in a Boyle van't Hoff plot. The average radius of rabbit oocytes in an isotonic solution was determined to be 55.7+/-1.2 microm (n=16). The rabbit oocyte exhibited an "ideal" osmotic response in the range from iso-osmolity to 1200 mOsm. The V(b) was determined to be 20% of the isotonic value with r(2)=0.97. The values of L(p) were determined to be 0.79+/-0.26, 0.82+/-0.22, and 0.64+/-0.16 microm min(-1)atm(-1) and the P(s) values were determined to be 2.9+/-1.3, 2.7+/-1.3, and 0.27+/-0.18x10(-3) cm min(-1

  13. Store-activated Ca2+ inflow in Xenopus laevis oocytes: inhibition by primaquine and evaluation of the role of membrane fusion.

    PubMed Central

    Gregory, R B; Barritt, G J

    1996-01-01

    The role of membrane fusion in the activation of store-activated Ca2+ channels (SACCs) in the plasma membrane of Xenopus laevis oocytes was investigated with primaquine, an inhibitor of vesicle trafficking, reagents that disrupt the cytoskeleton, and reagents that activate or inhibit the functions of monomeric and trimeric GTP-binding regulatory proteins. Ca2+ inflow was assessed by measuring the rate of increase in the fluorescence of the intracellular Ca2+ chelator fluo-3 after the addition of extracellular Ca2+ to oocytes previously incubated in the absence of added Ca2+. Primaquine inhibited the 3-deoxy-3-fluoro Ins(1,4,5)P3 (Ins(1,4,5)P3F)-stimulated increase in Ca2+O,-induced fluo-3 fluorescence with no detectable effect on the release of Ca2+ from intracellular stores. The effect of primaquine was observed within 1.5 min, showed similarity to the inhibition induced by Gd3+, was reversible, and was observed when primaquine was added either before or after activation of the SACCs. The degree of inhibition of Ca2+ inflow by primaquine was halved when the extracellular concentration of Ca2+ was increased from 3.1 to 12.5 mM. Primaquine also inhibited Ca2+ inflow through cholera toxin-activated divalent cation channels and Drosophila Trpl channels (expressed in oocytes after injection of trp1 cRNA). These results indicate that primaquine inhibits open SACCs, possibly by directly inhibiting Ca2+ flow through the channel pore. Colchicine plus cytochalasin B, Brefeldin A, the peptide Arf-1 (2-17) (introduced by microinjection), lovastatin or pertussis toxin did not inhibit the Ins(1,4,5)P3F stimulated increase in fluo-3 fluorescence. In contrast, guanosine 5'-[gamma-thio]triphosphate (GTP[S]), guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) and A1F4-, but not guanosine 5'-[beta-thio]diphosphate, inhibited the Ins(1,4,5)P3F-stimulated increase in fluo-3 fluorescence. Co-administration of GTP did not prevent the inhibition by GTP[S] of FA1F4-. Staurosporine

  14. Detection of oocyte perivitelline membrane-bound sperm: a tool for avian collection management.

    PubMed

    Croyle, Kaitlin E; Durrant, Barbara S; Jensen, Thomas

    2015-01-01

    The success and sustainability of an avian breeding programme depend on managing productive and unproductive pairs. Given that each breeding season can be of immeasurable importance, it is critical to resolve pair fertility issues quickly. Such problems are traditionally diagnosed through behavioural observations, egg lay history and hatch rates, with a decision to re-pair generally taking one or more breeding seasons. In pairs producing incubated eggs that show little or no signs of embryonic development, determining fertility is difficult. Incorporating a technique to assess sperm presence on the oocyte could, in conjunction with behaviour and other data, facilitate a more timely re-pair decision. Detection of perivitelline membrane-bound (PVM-bound) sperm verifies successful copulation, sperm production and sperm functionality. Alternatively, a lack of detectable sperm, at least in freshly laid eggs, suggests no mating, lack of sperm production/function or sperm-oviduct incompatibility. This study demonstrated PVM-bound sperm detection by Hoechst staining in fresh to 24-day-incubated exotic eggs from 39 species representing 13 orders. However, a rapid and significant time-dependent loss of detectable PVM-bound sperm was observed following incubation of chicken eggs. The PCR detection of sperm in seven species, including two bacterially infected eggs, demonstrated that this method was not as reliable as visual detection using Hoechst staining. The absence of amplicons in visually positive PVMs was presumably due to large PVM size and low sperm count, resulting in DNA concentrations too low for standard PCR detection. In summary, this study demonstrated the feasibility and limitations of using PVM-bound sperm detection as a management tool for exotic avian species. We verified that sperm presence or absence on fluorescence microscopy can aid in the differentiation of fertile from infertile eggs to assist breeding managers in making prompt decisions for pair

  15. Detection of oocyte perivitelline membrane-bound sperm: a tool for avian collection management

    PubMed Central

    Croyle, Kaitlin E.; Durrant, Barbara S.; Jensen, Thomas

    2015-01-01

    The success and sustainability of an avian breeding programme depend on managing productive and unproductive pairs. Given that each breeding season can be of immeasurable importance, it is critical to resolve pair fertility issues quickly. Such problems are traditionally diagnosed through behavioural observations, egg lay history and hatch rates, with a decision to re-pair generally taking one or more breeding seasons. In pairs producing incubated eggs that show little or no signs of embryonic development, determining fertility is difficult. Incorporating a technique to assess sperm presence on the oocyte could, in conjunction with behaviour and other data, facilitate a more timely re-pair decision. Detection of perivitelline membrane-bound (PVM-bound) sperm verifies successful copulation, sperm production and sperm functionality. Alternatively, a lack of detectable sperm, at least in freshly laid eggs, suggests no mating, lack of sperm production/function or sperm–oviduct incompatibility. This study demonstrated PVM-bound sperm detection by Hoechst staining in fresh to 24-day-incubated exotic eggs from 39 species representing 13 orders. However, a rapid and significant time-dependent loss of detectable PVM-bound sperm was observed following incubation of chicken eggs. The PCR detection of sperm in seven species, including two bacterially infected eggs, demonstrated that this method was not as reliable as visual detection using Hoechst staining. The absence of amplicons in visually positive PVMs was presumably due to large PVM size and low sperm count, resulting in DNA concentrations too low for standard PCR detection. In summary, this study demonstrated the feasibility and limitations of using PVM-bound sperm detection as a management tool for exotic avian species. We verified that sperm presence or absence on fluorescence microscopy can aid in the differentiation of fertile from infertile eggs to assist breeding managers in making prompt decisions for pair

  16. Plasma membrane regulates Ras signaling networks

    PubMed Central

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

    2015-01-01

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

  17. Plasma membrane regulates Ras signaling networks.

    PubMed

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

    2015-01-01

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

  18. Characterization of the platelet-activating factor acetylhydrolase from human plasma by heterologous expression in Xenopus laevis oocytes.

    PubMed Central

    Yamada, Y; Stafforini, D M; Imaizumi, T; Zimmerman, G A; McIntyre, T M; Prescott, S M

    1994-01-01

    Platelet-activating factor (PAF) has been implicated as a mediator of inflammation and atherosclerosis. A specific degradative enzyme found in plasma, PAF acetylhydrolase, plays important roles in various pathophysiological events induced by PAF. Human macrophages and Hep G2 cells secrete PAF acetylhydrolase with characteristics identical to the plasma activity. Other investigators reported that apolipoprotein B may possess phospholipase A2 activity, which suggested that apolipoprotein B might be a zymogen for PAF acetylhydrolase. However, while macrophages express PAF acetylhydrolase activity, we did not detect cDNAs for apolipoprotein B in a cDNA library from these cells, indicating that macrophages do not express this protein. In contrast, Hep G2 cells had high levels of cDNA for apolipoprotein B, as expected. We next injected Xenopus laevis oocytes with poly(A)+ RNA extracted from cultured human macrophages and Hep G2 cells. Twenty-five to 50% of Xenopus oocytes injected with poly(A)+ RNA from macrophages or Hep G2 cells secreted a PAF acetylhydrolase activity (1.0-7.8 nmol/ml per h) that also utilized a synthetic oxidized phospholipid as substrate. The activity secreted by poly(A)+ RNA-injected oocytes associated with lipoproteins and transferred between the particles in a pH-dependent manner, much like the plasma activity. These experiments establish that the properties of the enzyme released from poly(A)+ RNA-injected oocytes are identical to those of the plasma form of PAF acetylhydrolase and that the activity detected is not the expression of a domain in apolipoprotein B. Images PMID:7937948

  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. Dysferlin is essential for endocytosis in the sea star oocyte.

    PubMed

    Oulhen, Nathalie; Onorato, Thomas M; Ramos, Isabela; Wessel, Gary M

    2014-04-01

    Dysferlin is a calcium-binding transmembrane protein involved in membrane fusion and membrane repair. In humans, mutations in the dysferlin gene are associated with muscular dystrophy. In this study, we isolated plasma membrane-enriched fractions from full-grown immature oocytes of the sea star, and identified dysferlin by mass spectrometry analysis. The full-length dysferlin sequence is highly conserved between human and the sea star. We learned that in the sea star Patiria miniata, dysferlin RNA and protein are expressed from oogenesis to gastrulation. Interestingly, the protein is highly enriched in the plasma membrane of oocytes. Injection of a morpholino against dysferlin leads to a decrease of endocytosis in oocytes, and to a developmental arrest during gastrulation. These results suggest that dysferlin is critical for normal endocytosis during oogenesis and for embryogenesis in the sea star and that this animal may be a useful model for studying the relationship of dysferlin structure as it relates to its function.

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

    PubMed

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

    2016-02-29

    The ability of nanoparticles (NPs) to be promptly uptaken by the cells makes them both dangerous and useful to human health. It was recently postulated that some NPs might cross the plasma membrane also by a non-endocytotic pathway gaining access to the cytoplasm. To this aim, after having filled mature Xenopus oocytes with Calcein, whose fluorescence is strongly quenched by divalent metal ions, we have exposed them to different cobalt NPs quantifying quenching as evidence of the increase of the concentration of Co(2+) released by the NPs that entered into the cytoplasm. We demonstrated that cobalt oxide NPs, but not cobalt nor cobalt oxide NPs that were surrounded by a protein corona, can indeed cross plasma membranes.

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

    PubMed Central

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

    2016-01-01

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

  3. Distribution of prepubertal and adult goat oocyte cortical granules during meiotic maturation and fertilisation: ultrastructural and cytochemical study.

    PubMed

    Velilla, E; Izquierdo, D; Rodríguez-González, E; López-Béjar, M; Vidal, F; Paramio, M T

    2004-08-01

    The aim of this study was evaluate cortical granule (CG) distribution during in vitro maturation (IVM) and fertilisation of prepubertal goat oocytes compared to CG distribution of ovulated and in vitro fertilised oocytes from adult goats. Oocytes from prepubertal goats were recovered from a slaughterhouse and were matured in M199 with hormones and serum for 27 hr. Ovulated oocytes were collected from gonadotrophin treated Murciana goats. Frozen-thawed spermatozoa were selected by centrifugation in percoll gradient and were capacitated in DMH with 20% steer serum for 1 hr. Ovulated and IVM-oocytes were inseminated in DMH medium with steer serum and calcium lactate for 20 hr. Oocytes and presumptive zygotes were stained with FITC-LCA (Lens culinaris agglutinin labelled with fluorescein isothiocyanate) and observed under a confocal laser scanning microscope. Ultrastructure morphology of oocytes and presumptive zygotes were analysed by transmission electron microscopy (TEM). Prepubertal goat oocytes at germinal vesicle stage show a homogeneous CG distribution in the cytoplasm. IVM-oocytes at Metaphase II (MII) and ovulated oocytes presented CGs located in the cortex with the formation of a monolayer beneath to the plasma membrane. At 20 hr postinsemination (hpi), zygotes from IVM-oocytes showed a complete CG exocytosis whereas zygotes from ovulated oocytes presented aggregates of CGs located at the cortical region. Images by TEM detected that CGs were more electrodense and compacts in oocytes from prepubertal than from adult goats.

  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. Cellular membrane collapse by atmospheric-pressure plasma jet

    NASA Astrophysics Data System (ADS)

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

    2014-01-01

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

  6. Preparation of the cortical reaction: maturation-dependent migration of SNARE proteins, clathrin, and complexin to the porcine oocyte's surface blocks membrane traffic until fertilization.

    PubMed

    Tsai, Pei-Shiue; van Haeften, Theo; Gadella, Bart M

    2011-02-01

    The cortical reaction is a calcium-dependent exocytotic process in which the content of secretory granules is released into the perivitellin space immediately after fertilization, which serves to prevent polyspermic fertilization. In this study, we investigated the involvement and the organization of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins in the docking and fusion of the cortical granule membrane with the oolemma in porcine oocytes. During meiotic maturation, secretory vesicles that were labeled with a granule-specific binding lectin, peanut agglutinin (PNA), migrated toward the oocyte's surface. This surface-orientated redistribution behavior was also observed for the oocyte-specific SNARE proteins SNAP23 and VAMP1 that colocalized with the PNA-labeled structures in the cortex area just under the oolemma and with the exclusive localization area of complexin (a trans-SNARE complex-stabilizing protein). The coming together of these proteins serves to prevent the spontaneous secretion of the docked cortical granules and to prepare the oocyte's surface for the cortical reaction, which should probably be immediately compensated for by a clathrin-mediated endocytosis. In vitro fertilization resulted in the secretion of the cortical granule content and the concomitant release of complexin and clathrin into the oocyte's cytosol, and this is considered to stimulate the observed endocytosis of SNARE-containing membrane vesicles.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-08-21

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

  9. The mitochondria-plasma membrane contact site.

    PubMed

    Westermann, Benedikt

    2015-08-01

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

  10. The product of the gene GEF1 of Saccharomyces cerevisiae transports Cl- across the plasma membrane.

    PubMed

    López-Rodríguez, Angélica; Trejo, Alfonso Cárabez; Coyne, Leanne; Halliwell, Robert F; Miledi, Ricardo; Martínez-Torres, Ataúlfo

    2007-12-01

    Expression of GEF1 in Xenopus laevis oocytes and HEK-293 cells gave rise to a Cl- channel that remained permanently open and was blocked by nitro-2-(3-phenyl-propylamino) benzoic acid and niflumic acid. NPPB induced petite-like colonies, resembling the GEF1 knock-out. The fluorescent halide indicator SPQ was quenched in a wild-type strain, in contrast to both a GEF1 knock-out strain and yeast grown in the presence of NPPB. Immunogold and electron microscopy located Gef1p in the plasma membrane, vacuole, endoplasmic reticulum and Golgi apparatus. Eleven substitutions in five residues forming the ion channel of GEF1 were introduced; some of them (S186A, I188N, Y459D, Y459F, Y459V, I467A, I467N and F468N) did not rescue the pet phenotype, whereas F468A, A558F and A558Y formed normal colonies. All the pet mutants showed reduced O2 consumption, small mitochondria and mostly disrupted organelles. Finally, electron microscopy revealed that the plasma membrane of the mutants develop multiple foldings and highly ordered cylindrical protein-membrane complexes. All the experiments above suggest that Gef1p transports Cl- through the plasma membrane and reveal the importance of critical amino acids for the proper function of the protein as suggested by structural models. However, the mechanism of activation of the channel has yet to be defined.

  11. Mouse Oocytes Acquire Mechanisms that Permit Independent Cell Volume Regulation at the End of Oogenesis.

    PubMed

    Richard, Samantha; Tartia, Alina P; Boison, Detlev; Baltz, Jay M

    2016-09-02

    Mouse embryos employ a unique mechanism of cell volume regulation in which glycine is imported via the GLYT1 transporter to regulate intracellular osmotic pressure. Independent cell volume regulation normally becomes active in the oocyte after ovulation is triggered. This involves two steps: the first is the release of the strong adhesion between the oocyte and zona pellucida (ZP) while the second is the activation of GLYT1. In fully-grown oocytes, release of adhesion and GLYT1 activation also occur spontaneously in oocytes removed from the follicle. It is unknown, however, whether the capacity to release oocyte-ZP adhesion or activate GLYT1 first arises in the oocyte after ovulation is triggered or instead growing oocytes already possess these capabilities but they are suppressed in the follicle. Here, we assessed when during oogenesis oocyte-ZP adhesion can be released and when GLYT1 can be activated, with adhesion assessed by an osmotic assay and GLYT1 activity determined by [(3) H]-glycine uptake. Oocyte-ZP adhesion could not be released by growing oocytes until they were nearly fully grown. Similarly, the amount of GLYT1 activity that can be elicited in oocytes increased sharply at the end of oogenesis. The SLC6A9 protein that is responsible for GLYT1 activity and Slc6a9 transcripts are present in growing oocytes and increased over the course of oogenesis. Furthermore, SLC6A9 becomes localized to the oocyte plasma membrane as the oocyte grows. Thus, oocytes acquire the ability to regulate their cell volume by releasing adhesion to the ZP and activating GLYT1 as they approach the end of oogenesis. This article is protected by copyright. All rights reserved.

  12. Molecular and Cellular Mechanisms of Sperm-Oocyte Interactions Opinions Relative to in Vitro Fertilization (IVF)

    PubMed Central

    Anifandis, George; Messini, Christina; Dafopoulos, Konstantinos; Sotiriou, Sotiris; Messinis, Ioannis

    2014-01-01

    One of the biggest prerequisites for pregnancy is the fertilization step, where a human haploid spermatozoon interacts and penetrates one haploid oocyte in order to produce the diploid zygote. Although fertilization is defined by the presence of two pronuclei and the extraction of the second polar body the process itself requires preparation of both gametes for fertilization to take place at a specific time. These preparations include a number of consecutive biochemical and molecular events with the help of specific molecules and with the consequential interaction between the two gametes. These events take place at three different levels and in a precise order, where the moving spermatozoon penetrates (a) the outer vestments of the oocyte, known as the cumulus cell layer; (b) the zona pellucida (ZP); where exocytosis of the acrosome contents take place and (c) direct interaction of the spermatozoon with the plasma membrane of the oocyte, which involves a firm adhesion of the head of the spermatozoon with the oocyte plasma membrane that culminates with the fusion of both sperm and oocyte membranes (Part I). After the above interactions, a cascade of molecular signal transductions is initiated which results in oocyte activation. Soon after the entry of the first spermatozoon into the oocyte and oocyte activation, the oocyte’s coat (the ZP) and the oocyte’s plasma membrane seem to change quickly in order to initiate a fast block to a second spermatozoon (Part II). Sometimes, two spermatozoa fuse with one oocyte, an incidence of 1%–2%, resulting in polyploid fetuses that account for up to 10%–20% of spontaneously aborted human conceptuses. The present review aims to focus on the first part of the human sperm and oocyte interactions, emphasizing the latest molecular and cellular mechanisms controlling this process. PMID:25054321

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

    PubMed

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

    1996-06-01

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

  14. Calcium ion currents mediating oocyte maturation events

    PubMed Central

    Tosti, Elisabetta

    2006-01-01

    During maturation, the last phase of oogenesis, the oocyte undergoes several changes which prepare it to be ovulated and fertilized. Immature oocytes are arrested in the first meiotic process prophase, that is morphologically identified by a germinal vesicle. The removal of the first meiotic block marks the initiation of maturation. Although a large number of molecules are involved in complex sequences of events, there is evidence that a calcium increase plays a pivotal role in meiosis re-initiation. It is well established that, during this process, calcium is released from the intracellular stores, whereas less is known on the role of external calcium entering the cell through the plasma membrane ion channels. This review is focused on the functional role of calcium currents during oocyte maturation in all the species, from invertebrates to mammals. The emerging role of specific L-type calcium channels will be discussed. PMID:16684344

  15. A plasma membrane template for macropinocytic cups

    PubMed Central

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

    2016-01-01

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

  16. A Trial to Cryopreserve Immature Medaka (Oryzias latipes) Oocytes after Enhancing Their Permeability by Exogenous Expression of Aquaporin 3

    PubMed Central

    VALDEZ JR, Delgado M.; TSUCHIYA, Ryoma; SEKI, Shinsuke; SAIDA, Naoya; NIIMI, Saori; KOSHIMOTO, Chihiro; MATSUKAWA, Kazutsugu; KASAI, Magosaburo; EDASHIGE, Keisuke

    2013-01-01

    Abstract Fish oocytes have not been cryopreserved successfully, probably because it is difficult to prevent intracellular ice from forming. Previously, we have shown in medaka that immature oocytes are more suitable for cryopreservation than mature oocytes or embryos, in terms of permeability. We have also shown in immature medaka oocytes that the exogenous expression of aquaporin 3 (AQP3), a water/cryoprotectant channel, promotes the movement of water and cryoprotectants through the plasma membrane. In the present study, we attempted to cryopreserve immature medaka oocytes expressing AQP3. We first examined effects of hypertonic stress and the chemical toxicity of cryoprotectants on the survival of the AQP3-expressing oocytes. Exposure to hypertonic solutions containing sucrose decreased the survival of oocytes, but the expression of AQP3 did not affect sensitivity to hypertonic stress. Also, AQP3 expression did not markedly increase sensitivity to the toxicity of cryoprotectants. Of the four cryoprotectants tested, propylene glycol was the least toxic. Using a propylene glycol-based solution, therefore, we tried to cryopreserve immature oocytes by vitrification. During cooling with liquid nitrogen, all intact oocytes became opaque, but many AQP3-expressing oocytes remained transparent. This indicates that the expression of AQP3 is effective in preventing intracellular ice from forming during cooling. During warming, however, all the AQP3-expressing oocytes became opaque, indicating that intracellular ice formed. Therefore, the dehydration and permeation by propylene glycol were still insufficient. Further studies are necessary to realize the cryopreservation of fish oocytes. PMID:23337101

  17. Lectin-Magnetic Beads for Plasma Membrane Isolation.

    PubMed

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

    2015-07-01

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

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

    PubMed

    Elovson, J

    1980-06-25

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

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

    PubMed

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

    2011-04-01

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

  20. Light-induced modification of plant plasma membrane ion transport.

    PubMed

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

    2010-09-01

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

  1. 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

  2. The plasma membrane calcium ATPase and disease.

    PubMed

    Tempel, B L; Shilling, D J

    2007-01-01

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

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

    DTIC Science & Technology

    1993-08-16

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

  4. Plasma membrane reorganization induced by chemical transformation in cultura

    SciTech Connect

    Packard, B.S.

    1984-04-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

    Johnsen, S; Stokke, T; Prydz, H

    1974-11-01

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

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

    PubMed Central

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

    2017-01-01

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

  8. ISOLATION OF PLASMA MEMBRANE FRAGMENTS FROM HELA CELLS

    PubMed Central

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

    1969-01-01

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

  9. Calcium currents correlate with oocyte maturation during the reproductive cycle in Octopus vulgaris.

    PubMed

    Cuomo, Annunziata; Di Cristo, Carlo; Paolucci, Marina; Di Cosmo, Anna; Tosti, Elisabetta

    2005-03-01

    Using the whole-cell voltage clamp technique, we have studied the Ca2+ currents and the steady-state conductance during different oocyte growth stages and during the reproductive cycle of the female of Octopus vulgaris. Evidence is presented that L-type Ca2+ currents are high in small pre-vitellogenic oocytes (80-150 microm diameter) and significantly lower in early vitellogenic oocytes (180-300 microm diameter). Similarly, a significant decrease of the steady-state conductance occurred from the pre to early- vitellogenic oocytes. Octopus oocytes showed larger Ca2+ currents in the reproductive rather than non-reproductive periods. These data indicates that ion and L-type Ca2+ currents play a role in oocyte growth and cytoplasmic maturation, and possibly in preparing the plasma membrane to the interaction with the spermatozoon. By using fluorescent microscopy, we show that oocytes from 80 to 400 microm diameter have the large germinal vesicle characteristic of the immature oocytes. In subsequent stages of growth (up to 1000 microm diameter) the nucleus is no more visible and the metaphase spindle appears. These data demonstrate that Octopus vulgaris oocytes are arrested in the first meiotic prophase up to the early-vitellogenic stage and resume meiosis at this stage up to a second block presumably in metaphase I. We discuss a possible role for progesterone as the hormonal stimulus for the first prophase-metaphase meiotic transition.

  10. The crucial role of zona pellucida in cryopreservation of oocytes by vitrification.

    PubMed

    Choi, Jung Kyu; Yue, Tao; Huang, Haishui; Zhao, Gang; Zhang, Mingjun; He, Xiaoming

    2015-10-01

    Mammalian oocytes have a proteinaceous hydrogel-like outer shell known as the zona pellucida (ZP) that semi-encloses their plasma membrane and cytoplasm. In this study, we cryopreserved mouse oocytes either with or without ZP by vitrification. Our results show that the presence of an intact ZP could significantly improve the post-vitrification survival of oocytes to 92.1% from 13.3% for oocytes without ZP. Moreover, there was no significant difference in embryonic development between fresh and cryopreserved oocytes with ZP after in vitro fertilization (IVF). Further atomic force microscopy (AFM) analysis showed that the intact oocytes with ZP have an elastic modulus that is more than 85 times higher than that of oocytes without ZP. This may partially explain the important role of ZP in protecting the oocytes by resisting the mechanical stress due to possible ice formation during cryopreservation by vitrification. Collectively, this study reveals a new biophysical role of ZP during vitrification of oocytes and suggests microencapsulation of the many mammalian cells without a ZP in ZP-like hydrogel is an effective strategy to improve their survival post cryopreservation by vitrification.

  11. The Crucial Role of Zona Pellucida in Cryopreservation of Oocytes by Vitrification

    PubMed Central

    Choi, Jung Kyu; Yue, Tao; Huang, Haishui; Zhao, Gang; Zhang, Mingjun; He, Xiaoming

    2015-01-01

    Mammalian oocytes have a proteinaceous hydrogel-like outer shell known as the zona pellucida (ZP) that semi-encloses their plasma membrane and cytoplasm. In this study, we cryopreserved mouse oocytes either with or without ZP by vitrification. Our results show that the presence of an intact ZP could significantly improve the post-vitrification survival of oocytes to 92.1% from 13.3% for oocytes without ZP. Moreover, there was no significant difference in embryonic development between fresh and cryopreserved oocytes with ZP after in vitro fertilization (IVF). Further atomic force microscopy (AFM) analysis showed that the intact oocytes with ZP have an elastic modulus that is more than 85 times higher than that of oocytes without ZP. This may partially explain the important role of ZP in protecting the oocytes by resisting the mechanical stress due to possible ice formation during cryopreservation by vitrification. Collectively, this study reveals a new biophysical role of ZP during vitrification of oocytes and suggests microencapsulation of the many mammalian cells without a ZP in ZP-like hydrogel is an effective strategy to improve their survival post cryopreservation by vitrification. PMID:26297946

  12. Functional expression in frog oocytes of human ρ1 receptors produced in Saccharomyces cerevisiae

    PubMed Central

    Martínez-Martínez, Alejandro; Reyes-Ruiz, Jorge Mauricio; Martínez-Torres, Ataúlfo; Miledi, Ricardo

    2004-01-01

    The yeast Saccharomyces cerevisiae was engineered to express the ρ1 subunit of the human γ-aminobutyric acid ρ1 (GABAρ1) receptor. RNA that was isolated from several transformed yeast strains produced fully functional GABA receptors in Xenopus oocytes. The GABA currents elicited in the oocytes were fast, nondesensitizing chloride currents; and the order of agonist potency was GABA > β-alanine > glycine. Moreover, the receptors were resistant to bicuculline, strongly antagonized by (1,2,5,6 tetrahydropyridine-4-yl)methylphosphinic acid, and modulated by zinc and lanthanum. Thus, the GABA receptors expressed by the yeast mRNA retained all of the principal characteristics of receptors expressed by cRNA or native retina mRNAs. Western blot assays showed immunoreactivity in yeast plasma membrane preparations, and a ρ1-GFP fusion gene showed mostly intracellular distribution with a faint fluorescence toward the plasma membrane. In situ immunodetection of ρ1 in yeast demonstrated that some receptors reach the plasma membrane. Furthermore, microtransplantation of yeast plasma membranes to frog oocytes resulted in the incorporation of a small number of functional yeast ρ1 receptors into the oocyte plasma membrane. These results show that yeast may be useful to produce complete functional ionotropic receptors suitable for structural analysis. PMID:14704273

  13. Analysis of vitelline envelope synthesis and composition during early oocyte development in gilthead seabream (Sparus aurata).

    PubMed

    Modig, Carina; Raldúa, Demetrio; Cerdà, Joan; Olsson, Per-Erik

    2008-08-01

    The oocyte vitelline envelope (VE) of gilthead seabream is composed of four known zona pellucida (ZP) proteins, ZPBa, ZPBb, ZPC, and ZPX. We have previously shown that the gilthead seabream ZP proteins are differentially transcribed in liver and ovary, with the expression in liver being under estrogenic control. However, although mRNA was found in both liver and ovary, only low ZPBa protein levels were detected in liver and plasma. Using isoform-specific ZP antibodies we show that ZPBa and ZPX translation products are present in the cytosol of stage I and II oocytes. In addition, the zpBa and zpX mRNAs were detected in early developing oocytes. During oocyte growth (vitellogenesis), the VE increased in thickness (>10 microm), and we show that the four ZP isoforms are present in different regions of the VE. ZPX was detected closest to the oocyte plasma membrane while the intermediate region was composed of ZPBa, ZPBb, and ZPC. At the outer layer, only ZPC was detected. When oocytes reach the fully grown stage they resume meiosis and hydration. As the oocyte expands, thinning to 4 microm, the VE acquire a striped and compact appearance at the electron microscopy level. This study provides further evidence for the oocyte origin of some ZP proteins in the gilthead seabream and suggests that the ZP proteins are differentially distributed within the VE.

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

    PubMed Central

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

    2015-01-01

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

  15. 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.

  16. 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

  17. A study relating the composition of follicular fluid and blood plasma from individual Holstein dairy cows to the in vitro developmental competence of pooled abattoir-derived oocytes.

    PubMed

    Sutton-McDowall, Melanie L; Yelland, Robert; MacMillan, Keith L; Robker, Rebecca L; Thompson, Jeremy G

    2014-07-01

    The fertility of high-performance (high milk yield) dairy breeds such as the Holstein within the Australian dairy herd has been on the decline for the past two decades. The 12-month calving interval for pasture-based farming practices results in oocyte maturation coinciding with peak lactation, periods of negative energy balance, and energy partitioning for lactation, causing energy deficiency in some organ systems, including the reproductive system. Oocyte developmental competence (the ability to undergo successful fertilization, embryo development, and establishment of pregnancy) is intrinsically linked with the composition of follicular fluid (FF). The aim of this study was to determine if there was a relationship between the fat and carbohydrate levels in plasma and FF and the ability to support in vitro oocyte maturation (IVM). Plasma and FF were collected in vivo from eight Holstein cows between 52 and 151 days post-partum. Plasma glucose trended (P = 0.072) higher and triglyceride levels were significantly higher than in FF (P < 0.05), but there were no relationships between FF and plasma composition. Glucose FF concentration was negatively related to follicular lactate and nonesterified fatty acid (NEFA) levels and days post-partum. Conversely, FF triglyceride concentrations were positively related to FF NEFA levels and negatively related to milk fat and protein composition. Abattoir-derived cumulus-oocyte complexes were cultured in either 50% FF (FF-IVM) or 50% plasma (plasma-IVM), with on-time embryo development then assessed. Although there were no differences between animals, the blastocyst rates after FF-IVM were negatively related to plasma glucose and days post-partum and positively related to body condition score and plasma NEFA levels. In comparison to the previous studies, total NEFA levels in FF were not related to animal parameters and did not influence oocyte developmental competence in vitro. Results from this study suggest that days

  18. Plasma Membrane Ca-ATPase of Radish Seedlings 1

    PubMed Central

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

    1992-01-01

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

  19. 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

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

    PubMed

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

    2005-10-28

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

  1. Effects of cryopreservation on meiotic spindles of oocytes and its dynamics after thawing: clinical implications in oocyte freezing--a review article.

    PubMed

    Chen, S U; Lien, Y R; Chao, K H; Ho, H N; Yang, Y S; Lee, T Y

    2003-04-28

    Embryo freezing has been a successful practice, but oocyte cryopreservation formerly achieved poorer results. This was mainly due to low rates of survival, fertilization, and development. The major dissimilarities for oocytes to embryos are the character of the plasma membrane, the presence of cortical granules, at the metaphase of meiosis II with the spindle system. In addition, the oocytes must be fertilized by sperm at the appropriate time. To improve the survival rate, a refined slow freezing method with increased sucrose concentration would dehydrate oocytes more sufficiently. Vitrification is another approach to prevent ice crystal formation. Intracytoplasmic sperm injection is used to overcome possible zona hardening from the release of cortical granules. The microtubules of meiotic spindles are vulnerable to the thermal changes and would depolymerize. Cryopreserved oocytes exhibited serious disturbances of the microtubules immediately after thawing. Fertilization of oocytes with disorganized spindles could lead to chromosomal aneuploidy, digyny, and arrest of cleavage. After incubation, the microtubules would repolymerize in a time-dependent way. Normal fertilization and development of cryopreserved oocytes improved after appropriate incubation and timing of insemination, compatible with recovery of the spindles. With the improvement of survival, fertilization, and cleavage, oocyte cryopreservation would gain an imperative role.

  2. There Is No Simple Model of the Plasma Membrane Organization

    PubMed Central

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

    2016-01-01

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

  3. Plasma Membrane Repair in Health and Disease

    PubMed Central

    Demonbreun, Alexis R.; McNally, Elizabeth M.

    2016-01-01

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

  4. 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

  5. The egg membrane microdomain-associated uroplakin III-Src system becomes functional during oocyte maturation and is required for bidirectional gamete signaling at fertilization in Xenopus laevis.

    PubMed

    Mahbub Hasan, A K M; Hashimoto, Aki; Maekawa, Yuka; Matsumoto, Takashi; Kushima, Shota; Ijiri, Takashi W; Fukami, Yasuo; Sato, Ken-ichi

    2014-04-01

    In Xenopus laevis, sperm-egg interaction promotes partial proteolysis and/or tyrosine phosphorylation of uroplakin III (UPIII) and the tyrosine kinase Src, which both localize to the cholesterol-enriched egg membrane microdomains (MDs). Here we show that sperm promote proteolysis and/or tyrosine phosphorylation of UPIII and Src in MDs isolated from ovulated and unfertilized eggs (UF-MDs). An antibody against the extracellular domain of UPIII interferes with these events. Inhibition of fertilization by anti-UPIII antibody is rescued by co-incubation with UF-MDs. This suggests that, like MDs in intact eggs, the isolated UF-MDs are capable of interacting with sperm, an interaction that does not interfere with normal fertilization but rather augments the ability of sperm to fertilize eggs pretreated with anti-UPIII antibody. This unexpected effect of UF-MDs on sperm requires UPIII function in UF-MDs and protein kinase activity in sperm. MDs isolated from progesterone-treated mature oocytes, but not ovarian immature oocytes, are similarly functional as UF-MDs. The anti-UPIII extracellular domain antibody binds more effectively to the surface of mature than immature ovarian oocytes. We propose that the structural and functional competency of the UPIII-Src signaling system in MDs is strictly regulated during oocyte maturation and subsequently in sperm-mediated egg activation and fertilization. The fertilization-related signaling properties seen in UF-MDs can be partially reconstituted in MDs of human embryonic kidney 293 cells (293-MDs) expressing UPIII, Src and uroplakin Ib. However, 293-MDs expressing a proteolysis-resistant mutant of UPIII are less functional, suggesting that the availability of UPIII to protease action is important for MD function.

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2011-04-06

    Membrane proteins and membrane lipids are frequently organized in submicron-sized domains within cellular membranes. Factors thought to be responsible for domain formation include lipid-lipid interactions, lipid-protein interactions and protein-protein interactions. However, it is unclear whether the domain structure is regulated by other factors such as divalent cations. Here, we have examined in native plasma membranes and intact cells the role of the second messenger Ca(2+) in membrane protein organization. We find that Ca(2+) at low micromolar concentrations directly redistributes a structurally diverse array of membrane proteins via electrostatic effects. Redistribution results in a more clustered pattern, can be rapid and triggered by Ca(2+) influx through voltage-gated calcium channels and is reversible. In summary, the data demonstrate that the second messenger Ca(2+) strongly influences the organization of membrane proteins, thus adding a novel and unexpected factor that may control the domain structure of biological membranes.

  8. Plant plasma membrane proteomics for improving cold tolerance.

    PubMed

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

    2013-01-01

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

  9. Plasma Membrane Ca-ATPase of Radish Seedlings 1

    PubMed Central

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

    1992-01-01

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

  10. Plasma membrane appearance of phosphatidylethanolamine in stimulated macrophages

    SciTech Connect

    Sandra, A.; Cai, J. )

    1991-07-01

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

  11. Effect of BCD Plasma on a Bacteria Cell Membrane

    NASA Astrophysics Data System (ADS)

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

    2013-07-01

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

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

    PubMed

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

    2009-12-01

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

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

    PubMed

    Mellgren, Ronald L

    2008-04-24

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

  14. Ras Diffusion Is Sensitive to Plasma Membrane Viscosity

    PubMed Central

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

    2005-01-01

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

  15. Ras diffusion is sensitive to plasma membrane viscosity.

    PubMed

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

    2005-08-01

    The cell surface contains a variety of barriers and obstacles that slow the lateral diffusion of glycosylphosphatidylinositol (GPI)-anchored and transmembrane proteins below the theoretical limit imposed by membrane viscosity. How the diffusion of proteins residing exclusively on the inner leaflet of the plasma membrane is regulated has been largely unexplored. We show here that the diffusion of the small GTPase Ras is sensitive to the viscosity of the plasma membrane. Using confocal fluorescence recovery after photobleaching, we examined the diffusion of green fluorescent protein (GFP)-tagged HRas, NRas, and KRas in COS-7 cells loaded with or depleted of cholesterol, a well-known modulator of membrane bilayer viscosity. In cells loaded with excess cholesterol, the diffusional mobilities of GFP-HRas, GFP-NRas, and GFP-KRas were significantly reduced, paralleling the behavior of the viscosity-sensitive lipid probes DiIC(16) and DiIC(18). However, the effects of cholesterol depletion on protein and lipid diffusion in cell membranes were highly dependent on the depletion method used. Cholesterol depletion with methyl-beta-cyclodextrin slowed Ras diffusion by a viscosity-independent mechanism, whereas overnight cholesterol depletion slightly increased both protein and lipid diffusion. The ability of Ras to sense membrane viscosity may represent a general feature of proteins residing on the cytoplasmic face of the plasma membrane.

  16. 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.

  17. Localization of ubiquitin C-terminal hydrolase L1 in mouse ova and its function in the plasma membrane to block polyspermy.

    PubMed

    Sekiguchi, Satoshi; Kwon, Jungkee; Yoshida, Etsuko; Hamasaki, Hiroko; Ichinose, Shizuko; Hideshima, Makoto; Kuraoka, Mutsuki; Takahashi, Akio; Ishii, Yoshiyuki; Kyuwa, Shigeru; Wada, Keiji; Yoshikawa, Yasuhiro

    2006-11-01

    Protein degradation is essential for oogenesis and embryogenesis. The ubiquitin-proteasome system regulates many cellular processes via the rapid degradation of specific proteins. Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is exclusively expressed in neurons, testis, ovary, and placenta, each of which has unique biological activities. However, the functional role of UCH-L1 in mouse oocytes remains unknown. Here, we report the expression pattern of UCH-L1 and its isozyme UCH-L3 in mouse ovaries and embryos. Using immunocytochemistry, UCH-L1 was selectively detected on the plasma membrane, whereas UCH-L3 was mainly detected in the cytoplasm, suggesting that these isozymes have distinct functions in mouse eggs. To further investigate the functional role of UCH-L1 in mouse eggs, we analyzed the fertilization rate of UCH-L1-deficient ova of gad female mice. Female gad mice had a significantly increased rate of polyspermy in in vitro fertilization assays, although the rate of fertilization did not differ significantly from wild-type mice. In addition, the litter size of gad female mice was significantly reduced compared with wild-type mice. These results may identify UCH-L1 as a candidate for a sperm-oocyte interactive binding or fusion protein on the plasma membrane that functions during the block to polyspermy in mouse oocytes.

  18. 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

  19. 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.

  20. Requirements for F-BAR proteins TOCA-1 and TOCA-2 in actin dynamics and membrane trafficking during Caenorhabditis elegans oocyte growth and embryonic epidermal morphogenesis.

    PubMed

    Giuliani, Chiara; Troglio, Flavia; Bai, Zhiyong; Patel, Falshruti B; Zucconi, Adriana; Malabarba, Maria Grazia; Disanza, Andrea; Stradal, Theresia B; Cassata, Giuseppe; Confalonieri, Stefano; Hardin, Jeffrey D; Soto, Martha C; Grant, Barth D; Scita, Giorgio

    2009-10-01

    The TOCA family of F-BAR-containing proteins bind to and remodel lipid bilayers via their conserved F-BAR domains, and regulate actin dynamics via their N-Wasp binding SH3 domains. Thus, these proteins are predicted to play a pivotal role in coordinating membrane traffic with actin dynamics during cell migration and tissue morphogenesis. By combining genetic analysis in Caenorhabditis elegans with cellular biochemical experiments in mammalian cells, we showed that: i) loss of CeTOCA proteins reduced the efficiency of Clathrin-mediated endocytosis (CME) in oocytes. Genetic interference with CeTOCAs interacting proteins WSP-1 and WVE-1, and other components of the WVE-1 complex, produced a similar effect. Oocyte endocytosis defects correlated well with reduced egg production in these mutants. ii) CeTOCA proteins localize to cell-cell junctions and are required for proper embryonic morphogenesis, to position hypodermal cells and to organize junctional actin and the junction-associated protein AJM-1. iii) Double mutant analysis indicated that the toca genes act in the same pathway as the nematode homologue of N-WASP/WASP, wsp-1. Furthermore, mammalian TOCA-1 and C. elegans CeTOCAs physically associated with N-WASP and WSP-1 directly, or WAVE2 indirectly via ABI-1. Thus, we propose that TOCA proteins control tissues morphogenesis by coordinating Clathrin-dependent membrane trafficking with WAVE and N-WASP-dependent actin-dynamics.

  1. Requirements for F-BAR Proteins TOCA-1 and TOCA-2 in Actin Dynamics and Membrane Trafficking during Caenorhabditis elegans Oocyte Growth and Embryonic Epidermal Morphogenesis

    PubMed Central

    Patel, Falshruti B.; Zucconi, Adriana; Malabarba, Maria Grazia; Disanza, Andrea; Stradal, Theresia B.; Cassata, Giuseppe; Confalonieri, Stefano; Hardin, Jeffrey D.; Soto, Martha C.; Grant, Barth D.; Scita, Giorgio

    2009-01-01

    The TOCA family of F-BAR–containing proteins bind to and remodel lipid bilayers via their conserved F-BAR domains, and regulate actin dynamics via their N-Wasp binding SH3 domains. Thus, these proteins are predicted to play a pivotal role in coordinating membrane traffic with actin dynamics during cell migration and tissue morphogenesis. By combining genetic analysis in Caenorhabditis elegans with cellular biochemical experiments in mammalian cells, we showed that: i) loss of CeTOCA proteins reduced the efficiency of Clathrin-mediated endocytosis (CME) in oocytes. Genetic interference with CeTOCAs interacting proteins WSP-1 and WVE-1, and other components of the WVE-1 complex, produced a similar effect. Oocyte endocytosis defects correlated well with reduced egg production in these mutants. ii) CeTOCA proteins localize to cell–cell junctions and are required for proper embryonic morphogenesis, to position hypodermal cells and to organize junctional actin and the junction-associated protein AJM-1. iii) Double mutant analysis indicated that the toca genes act in the same pathway as the nematode homologue of N-WASP/WASP, wsp-1. Furthermore, mammalian TOCA-1 and C. elegans CeTOCAs physically associated with N-WASP and WSP-1 directly, or WAVE2 indirectly via ABI-1. Thus, we propose that TOCA proteins control tissues morphogenesis by coordinating Clathrin-dependent membrane trafficking with WAVE and N-WASP–dependent actin-dynamics. PMID:19798448

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

    PubMed

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

    2017-03-13

    In order to obtain a durable cost-effective membrane for membrane distillation (MD) process, flat sheet polyethersulfone (PES) membranes were modified by an atmospheric pressure nonequilibrium plasma generated using a dielectric barrier discharge in a mixture of argon and hexamethyldisiloxane as the organosilicon precursor. The surface properties of the plasma-modified membranes were characterized by water contact angle (CA), liquid entry pressure, X-ray photoelectron spectroscopy, scanning electron microscopy, and atomic force microscopy. The water CA of the membrane was increased from 64° to 104° by depositing a Si(CH3)-rich thin layer. While the pristine PES membrane was not applicable in the MD process, the modified PES membrane could be applied for the first time in an air gap membrane distillation setup for the removal of benzene as a volatile organic compound from water. The experimental design using central composite design and response surface methodology was applied to study the effects of feed temperature, concentration, and flow rate as well as their binary interactions on the overall permeate flux and separation factor. The separation factor and permeation flux of the modified PES membrane at optimum conditions were comparable with those of commercial polytetrafluoroethylene membrane.

  3. Evolutionary plasticity of plasma membrane interaction in DREPP family proteins.

    PubMed

    Vosolsobě, Stanislav; Petrášek, Jan; Schwarzerová, Kateřina

    2017-05-01

    The plant-specific DREPP protein family comprises proteins that were shown to regulate the actin and microtubular cytoskeleton in a calcium-dependent manner. Our phylogenetic analysis showed that DREPPs first appeared in ferns and that DREPPs have a rapid and plastic evolutionary history in plants. Arabidopsis DREPP paralogues called AtMDP25/PCaP1 and AtMAP18/PCaP2 are N-myristoylated, which has been reported as a key factor in plasma membrane localization. Here we show that N-myristoylation is neither conserved nor ancestral for the DREPP family. Instead, by using confocal microscopy and a new method for quantitative evaluation of protein membrane localization, we show that DREPPs rely on two mechanisms ensuring their plasma membrane localization. These include N-myristoylation and electrostatic interaction of a polybasic amino acid cluster. We propose that various plasma membrane association mechanisms resulting from the evolutionary plasticity of DREPPs are important for refining plasma membrane interaction of these signalling proteins under various conditions and in various cells.

  4. Large Plasma Membrane Disruptions Are Rapidly Resealed by Ca2+-dependent Vesicle–Vesicle Fusion Events

    PubMed Central

    Terasaki, Mark; Miyake, Katsuya; McNeil, Paul L.

    1997-01-01

    A microneedle puncture of the fibroblast or sea urchin egg surface rapidly evokes a localized exocytotic reaction that may be required for the rapid resealing that follows this breach in plasma membrane integrity (Steinhardt, R.A,. G. Bi, and J.M. Alderton. 1994. Science (Wash. DC). 263:390–393). How this exocytotic reaction facilitates the resealing process is unknown. We found that starfish oocytes and sea urchin eggs rapidly reseal much larger disruptions than those produced with a microneedle. When an ∼40 by 10 μm surface patch was torn off, entry of fluorescein stachyose (FS; 1,000 mol wt) or fluorescein dextran (FDx; 10,000 mol wt) from extracellular sea water (SW) was not detected by confocal microscopy. Moreover, only a brief (∼5–10 s) rise in cytosolic Ca2+ was detected at the wound site. Several lines of evidence indicate that intracellular membranes are the primary source of the membrane recruited for this massive resealing event. When we injected FS-containing SW deep into the cells, a vesicle formed immediately, entrapping within its confines most of the FS. DiI staining and EM confirmed that the barrier delimiting injected SW was a membrane bilayer. The threshold for vesicle formation was ∼3 mM Ca2+ (SW is ∼10 mM Ca2+). The capacity of intracellular membranes for sealing off SW was further demonstrated by extruding egg cytoplasm from a micropipet into SW. A boundary immediately formed around such cytoplasm, entrapping FDx or FS dissolved in it. This entrapment did not occur in Ca2+-free SW (CFSW). When egg cytoplasm stratified by centrifugation was exposed to SW, only the yolk platelet–rich domain formed a membrane, suggesting that the yolk platelet is a critical element in this response and that the ER is not required. We propose that plasma membrane disruption evokes Ca2+ regulated vesicle–vesicle (including endocytic compartments but possibly excluding ER) fusion reactions. The function in resealing of this cytoplasmic fusion

  5. Incapacity of Response to Disulfide-Reducing Agent in Triton X-100-Treated Oocytes of Starfish, Asterina pectinifera

    NASA Astrophysics Data System (ADS)

    Mita, Masatoshi

    2005-04-01

    Resumption of meiosis in starfish oocytes is induced by the natural maturation-inducing hormone, 1-methyladenine (1-MeAde). Oocyte maturation is also induced by the disulfide-reducing agent, dithiothreitol (DTT). Previous studies have shown that 1-MeAde controls meiosis by interacting with its receptors, which are located exclusively on oocyte plasma membrane. However, little is known about the mechanism of oocyte maturation induced by DTT. Thus, this study examined whether DTT interacts with 1-MeAde receptors to induce oocyte maturation. When oocytes were treated with Triton X-100, they failed to respond to 1-MeAde and DTT. Although the Triton X-100-treated oocytes recovered the capacity to respond to 1-MeAde during incubation in seawater, they remained unresponsive to DTT during seawater incubations. These results suggest that DTT does not interact with 1-MeAde receptors to induce oocyte maturation in starfish. It is possible that a protein essential for mediating DTT-induced maturation is eliminated from the oocytes surface following Triton X-100 treatment.

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

    PubMed

    Luo, W j; Chang, A

    2000-02-01

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

  7. Therapeutic plasmapheresis using membrane plasma separation.

    PubMed

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

    2012-08-01

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

  8. Remodeling of the postsynaptic plasma membrane during neural development

    PubMed Central

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

    2016-01-01

    Neuronal synapses are the fundamental units of neural signal transduction and must maintain exquisite signal fidelity while also accommodating the plasticity that underlies learning and development. To achieve these goals, the molecular composition and spatial organization of synaptic terminals must be tightly regulated; however, little is known about the regulation of lipid composition and organization in synaptic membranes. Here we quantify the comprehensive lipidome of rat synaptic membranes during postnatal development and observe dramatic developmental lipidomic remodeling during the first 60 postnatal days, including progressive accumulation of cholesterol, plasmalogens, and sphingolipids. Further analysis of membranes associated with isolated postsynaptic densities (PSDs) suggests the PSD-associated postsynaptic plasma membrane (PSD-PM) as one specific location of synaptic remodeling. We analyze the biophysical consequences of developmental remodeling in reconstituted synaptic membranes and observe remarkably stable microdomains, with the stability of domains increasing with developmental age. We rationalize the developmental accumulation of microdomain-forming lipids in synapses by proposing a mechanism by which palmitoylation of the immobilized scaffold protein PSD-95 nucleates domains at the postsynaptic plasma membrane. These results reveal developmental changes in lipid composition and palmitoylation that facilitate the formation of postsynaptic membrane microdomains, which may serve key roles in the function of the neuronal synapse. PMID:27535429

  9. 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.

  10. Magnetic apatite for structural insights on the plasma membrane.

    PubMed

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

    2015-01-21

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

  11. 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.

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

    PubMed

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

    2009-06-01

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

  13. 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

  14. Requirement for Coenzyme Q in Plasma Membrane Electron Transport

    NASA Astrophysics Data System (ADS)

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

    1992-12-01

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

  15. 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

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

    PubMed

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

    1993-02-24

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

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

    PubMed

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

    2002-08-14

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

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

    PubMed

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

    2010-05-03

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

  19. 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

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

    PubMed

    Figard, Lauren; Sokac, Anna Marie

    2014-01-01

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

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

    PubMed Central

    Gao, Xinxin

    2008-01-01

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

  2. Effect of polyunsaturated fatty acid supplementation on biophysical parameters and chilling sensitivity of ewe oocytes.

    PubMed

    Zeron, Yoel; Sklan, David; Arav, Amir

    2002-02-01

    Fat supplementation in the diet influences reproductive performance of lactating ruminants. Changes in the fat supply alter fatty acid composition and this can affect physical properties of cell membranes. This study examined the effect of rumen bypass polyunsaturated fatty acid (PUFA) supplementation on oocyte quality, chilling sensitivity, and lipid phase transition in oocytes of the sheep. Ewes were fed a diet supplemented with calcium soaps of fish oil for 13 weeks. More follicles and oocytes were found in the ovaries of ewes supplemented with PUFA than in control ewes. The number of high-quality oocytes was higher in ewes fed PUFA than in control ewes (74.3 and 57.0%, P < 0.05, respectively). Evaluation of phospholipid fatty acid composition indicated that PUFA were present in small proportions in oocytes, and eicosapentaenoic acid and docosahexaenoic acid were absent. Supplementation with PUFA increased the proportion of long chain unsaturated fatty acid in the plasma and cumulus cells phospholipids by 7.4 and 12.7%, respectively (P < 0.05). Integrity of oocyte membranes following chilling (16 degrees C, 15 min) was improved by PUFA supplementation increasing from 62.5 to 90.0% (P < 0.05). Due to changes in the oocyte's fatty acid profile, physical properties of the membrane were changed and the midpoint temperature of lipid transition reduced by 11 degrees C. These results suggest that supplementation of rumen bypass PUFA to ruminant diets can change fatty acid composition of follicle components and influence parameters such as number and quality of oocytes and their chilling resistance.

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

    PubMed

    Elandaloussi, Laurence M; Smith, Peter J

    2006-01-01

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

  4. Oocyte Activation and Fertilisation: Crucial Contributors from the Sperm and Oocyte.

    PubMed

    Yeste, Marc; Jones, Celine; Amdani, Siti Nornadhirah; Coward, Kevin

    2017-01-01

    This chapter intends to summarise the importance of sperm- and oocyte-derived factors in the processes of sperm-oocyte binding and oocyte activation. First, we describe the initial interaction between sperm and the zona pellucida, with particular regard to acrosome exocytosis. We then describe how sperm and oocyte membranes fuse, with special reference to the discovery of the sperm protein IZUMO1 and its interaction with the oocyte membrane receptor JUNO. We then focus specifically upon oocyte activation, the fundamental process by which the oocyte is alleviated from metaphase II arrest by a sperm-soluble factor. The identity of this sperm factor has been the source of much debate recently, although mounting evidence, from several different laboratories, provides strong support for phospholipase C ζ (PLCζ), a sperm-specific phospholipase. Herein, we discuss the evidence in support of PLCζ and evaluate the potential role of other candidate proteins, such as post-acrosomal WW-binding domain protein (PAWP/WBP2NL). Since the cascade of downstream events triggered by the sperm-borne oocyte activation factor heavily relies upon specialised cellular machinery within the oocyte, we also discuss the critical role of oocyte-borne factors, such as the inositol trisphosphate receptor (IP3R), protein kinase C (PKC), store-operated calcium entry (SOCE) and calcium/calmodulin-dependent protein kinase II (CaMKII), during the process of oocyte activation. In order to place the implications of these various factors and processes into a clinical context, we proceed to describe their potential association with oocyte activation failure and discuss how clinical techniques such as the in vitro maturation of oocytes may affect oocyte activation ability. Finally, we contemplate the role of artificial oocyte activating agents in the clinical rescue of oocyte activation deficiency and discuss options for more endogenous alternatives.

  5. 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

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

    PubMed

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

    2011-07-01

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

  7. From The Cover: Microtransplantation of functional receptors and channels from the Alzheimer's brain to frog oocytes

    NASA Astrophysics Data System (ADS)

    Miledi, R.; Dueñas, Z.; Martinez-Torres, A.; Kawas, C. H.; Eusebi, F.

    2004-02-01

    About a decade ago, cell membranes from the electric organ of Torpedo and from the rat brain were transplanted to frog oocytes, which thus acquired functional Torpedo and rat neurotransmitter receptors. Nevertheless, the great potential that this method has for studying human diseases has remained virtually untapped. Here, we show that cell membranes from the postmortem brains of humans that suffered Alzheimer's disease can be microtransplanted to the plasma membrane of Xenopus oocytes. We show also that these postmortem membranes carry neurotransmitter receptors and voltage-operated channels that are still functional, even after they have been kept frozen for many years. This method provides a new and powerful approach to study directly the functional characteristics and structure of receptors, channels, and other membrane proteins of the Alzheimer's brain. This knowledge may help in understanding the basis of Alzheimer's disease and also help in developing new treatments. -aminobutyric acid receptors | sodium channels | calcium channels | postmortem brain

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

    PubMed Central

    Costello, Donald P.

    1949-01-01

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

  9. Plasma-membrane calcium pumps and hereditary deafness.

    PubMed

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

    2007-11-01

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

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

    NASA Astrophysics Data System (ADS)

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

    1990-05-01

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

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

    PubMed

    Briskin, D P; Poole, R J

    1983-03-01

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

  12. 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.

  13. Plasma Membrane Lipids and Their Role in Fungal Virulence

    PubMed Central

    Del Poeta, Maurizio

    2016-01-01

    There has been considerable evidence in recent years suggesting that plasma membrane lipids are important regulators of fungal pathogenicity. Various glycolipids have been shown to impart virulent properties in several fungal species, while others have been shown to play a role in host defense. In addition to their role as virulence factors, lipids also contribute to other virulence mechanisms such as drug resistance, biofilm formation, and release of extracellular vesicles. In addition, lipids also affect the mechanical properties of the plasma membrane through the formation of packed microdomains composed mainly of sphingolipids and sterols. Changes in the composition of lipid microdomains has been shown to disrupt the localization of virulence factors and affect fungal pathogenicity. This review gathers evidence on the various roles of plasma membrane lipids in fungal virulence and how lipids might contribute to the different processes that occur during infection and treatment. Insight into the role of lipids in fungal virulence can lead to an improved understanding of the process of fungal pathogenesis and the development of new lipid-mediated therapeutic strategies. PMID:26703191

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

    PubMed

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

    2008-06-17

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

  15. 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

  16. 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

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

    PubMed

    Eggeling, Christian

    2015-01-01

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

  18. Plasma membrane wounding and repair in pulmonary diseases.

    PubMed

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

    2017-03-01

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

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

    PubMed

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

    2006-05-01

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

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

    PubMed

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

    2004-02-15

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

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

    PubMed Central

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

    1993-01-01

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

  2. Subcellular compartmentalization of maize storage proteins in Xenopus oocytes injected with zein messenger RNAs

    PubMed Central

    1981-01-01

    Maize storage proteins synthesized in oocytes were compartmentalized in membrane vesicles because they were resistant to hydrolysis by protease, unless detergent was present. The site of storage protein deposition within the oocyte was determined by subcellular fractionation. Optimal separation of oocyte membranes and organelles was obtained when EDTA and high concentrations of NaCl were included in the homogenization and gradient buffers. Under these conditions, fractions in sucrose gradients containing a heterogeneous mixture of smooth membranes (presumably endoplasmic reticulum, Golgi apparatus, and plasma membrane, density = 1.10-1.12 g/cm3), mitochondria (densities = 1.14 and 1.16 g/cm3), yolk platelets (density = 1.21 g/cm3), and a dense matrix material (density = 1.22 g/cm3) could be separated. Some zein proteins were recovered in the mixed membrane fraction, but the majority occurred in vesicles sedimenting with yolk platelets and granular material at a density of approximately 1.22 g/cm3. When metrizamide was included in the gradient to increase the density, little of the dense matrix material was isolated, and vesicles containing zein proteins were separated from other oocyte components. These vesicles were similar to protein bodies in maize endosperm because they were of identical density and contained the same group of polypeptides. PMID:7251653

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

    PubMed

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

    2013-08-01

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

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

    PubMed Central

    1981-01-01

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

  5. 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

  6. 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.

  7. 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.

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

    PubMed Central

    1984-01-01

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

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

    PubMed

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

    2006-03-01

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

  10. 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.

  11. Plant Endoplasmic Reticulum-Plasma Membrane Contact Sites.

    PubMed

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

    2017-04-01

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

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

    PubMed Central

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

    1973-01-01

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

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

    PubMed

    Fowler, Velia M

    2013-01-01

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

  14. 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.

  15. Coupling of inositol phospholipid hydrolysis to peptide hormone receptors expressed from adrenal and pituitary mRNA in Xenopus laevis oocytes

    SciTech Connect

    McIntosh, R.P.; Catt, K.J.

    1987-12-01

    The expression of several neurotransmitter and drug receptors from injected exogenous mRNA in Xenopus laevis oocytes has been demonstrated by electrophysiological measurements of ion channel activation. The expression of specific receptors for peptide hormones in such a translation system would facilitate studies on the structure and regulation of cell-surface receptors as well as their coupling to membrane transduction mechanisms. The expression of receptors for calcium-mobilizing hormones in Xenopus oocytes was sought by analysis of phospholipid turnover in hormone-stimulated oocytes. For this purpose, Xenopus oocytes were injected with mRNA extracted from bovine adrenal and pituitary glands and incubated with myo-(/sup 3/H)inositol to label plasma-membrane phosphatidylinositol phosphates. The expression of functionally active receptors for angiotensin II (AII) and thyrotropin-releasing hormone (TRH) was demonstrated by the stimulation of (/sup 3/H)inositol phosphate production by AII and TRH in the mRNA-injected, (/sup 3/H)inositol-prelabeled oocytes. The ability of AII and TRH to act by way of newly synthesized receptors from mammalian endocrine tissues to stimulate phosphatidylinositol polyphosphate hydrolysis in Xenopus oocytes suggests a generalized and conserved mechanism of receptor coupling to the transduction mechanism responsible for activation of phospholipase C in the plasma membrane.

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

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

  18. Effect of the expression of aquaporins 1 and 3 in mouse oocytes and morulae on the nucleation temperature for intracellular ice formation

    PubMed Central

    Seki, Shinsuke; Edashige, Keisuke; Wada, Sakiko; Mazur, Peter

    2013-01-01

    The occurrence of intracellular ice formation (IIF) is the most important factor determining whether or not cells survive a cryopreservation procedure. What is not clear is the mechanism or route by which an external ice crystal can traverse the plasma membrane and cause the heterogeneous nucleation of the supercooled solution within the cell. We have hypothesized that one route is through preexisting pores in aquaporin (AQP) proteins that span the plasma membranes of many cell types. Since the plasma membrane of mature mouse oocytes expresses little AQP, we compared the ice nucleation temperature of native oocytes with that of oocytes induced to express AQP1 and AQP3. The oocytes were suspended in 1.0 M ethylene glycol in PBS for 15 minutes, cooled in a Linkam cryostage to –7.0 °C, induced to freeze externally, and finally cooled at 20 °C/min to –70 °C. IIF that occurred during the 20 °C/min cooling is manifested by abrupt black flashing. The mean IIF temperatures for native oocytes, for oocytes sham injected with water, for oocytes expressing AQP1, and for those expressing AQP3 were –34, –40, –35, and –25 °C, respectively. The fact that the ice nucleation temperature of oocytes expressing AQP3 was 10° to 15° C higher than the others is consistent with our hypothesis. AQP3 pores can supposedly be closed by low pH or by treatment with double-stranded AQP3 RNA. However, when morulae were subjected to such treatments, the IIF temperature still remained high. A possible explanation is suggested. PMID:21734033

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

    PubMed Central

    Kraft, Mary L.

    2017-01-01

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

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

    PubMed

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

    2010-10-26

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

  1. 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.

  2. 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.

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

    PubMed Central

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

    2014-01-01

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

  4. 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

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

    PubMed

    Goldfinger, Lawrence E; Michael, James V

    2017-02-07

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

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

    PubMed

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

    2012-03-01

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

  7. 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

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

    PubMed

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

    2013-12-13

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

  9. Phosphatidic acid phosphatase and phospholipdase A activities in plasma membranes from fusing muscle cells.

    PubMed

    Kent, C; Vagelos, P R

    1976-06-17

    Plasma membrane from fusing embryonic muscle cells were assayed for phospholipase A activity to determine if this enzyme plays a role in cell fusion. The membranes were assayed under a variety of conditions with phosphatidylcholine as the substrate and no phospholipase A activity was found. The plasma membranes did contain a phosphatidic acid phosphatase which was optimally active in the presence of Triton X-100 and glycerol. The enzyme activity was constant from pH 5.2 to 7.0, and did not require divalent cations. Over 97% of the phosphatidic acid phosphatase activity was in the particulate fraction. The subcellular distribution of the phosphatidic acid phosphatase was the same as the distributions of the plasma membrane markers, (Na+ + k+)-ATPase and the acetylcholine receptor, which indicates that this phosphatase is located exclusively in the plasma membranes. There was no detectable difference in the phosphatidic acid phosphatase activities of plasma membranes from fusing and non-fusing cells.

  10. Voltage- and Tension-Dependent Lipid Mobility in the Outer Hair Cell Plasma Membrane

    NASA Astrophysics Data System (ADS)

    Oghalai, John S.; Zhao, Hong-Bo; Kutz, J. Walter; Brownell, William E.

    2000-01-01

    The mechanism responsible for electromotility of outer hair cells in the ear is unknown but is thought to reside within the plasma membrane. Lipid lateral diffusion in the outer hair cell plasma membrane is a sigmoidal function of transmembrane potential and bathing media osmolality. Cell depolarization or hyposmotic challenge shorten the cell and reduce membrane fluidity by half. Changing the membrane tension with amphipathic drugs results in similar reductions. These dynamic changes in membrane fluidity represent the modulation of membrane tension by lipid-protein interactions. The voltage dependence may be associated with the force-generating motors that contribute to the exquisite sensitivity of mammalian hearing.

  11. Size of lethality target in mouse immature oocytes determined with accelerated heavy ions.

    PubMed

    Straume, T; Dobson, R L; Kwan, T C

    1989-01-01

    Mouse immature oocytes were irradiated in vivo with highly charged, heavy ions from the Bevalac accelerator at the Lawrence Berkeley Laboratory. The particles used were 670-MeV/nucleon Si14+, 570-MeV/nucleon Ar18+, and 450-MeV/nucleon Fe26+. The cross-sectional area of the lethality target in these extremely radiosensitive cells was determined from fluence-response curves and information on energy deposition by delta rays. Results indicate a target cross-section larger than that of the nucleus, one which closely approximates the cross-sectional area of the entire oocyte. For 450-MeV/nucleon Fe26+ particles, the predicted target cross-sectional area is 120 +/- 16 microns2, comparing well with the microscopically determined cross-sectional area of 111 +/- 12 microns2 for these cells. The present results are in agreement with our previous target studies which implicate the oocyte plasma membrane.

  12. 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

  13. Plasma membrane calcium ATPase (PMCA4): A housekeeper for RT-PCR relative quantification of polytopic membrane proteins

    PubMed Central

    Calcagno, Anna Maria; Chewning, Katherine J; Wu, Chung-Pu; Ambudkar, Suresh V

    2006-01-01

    Background Although relative quantification of real-time RT-PCR data can provide valuable information, one limitation remains the selection of an appropriate reference gene. No one gene has emerged as a universal reference gene and much debate surrounds some of the more commonly used reference genes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). At this time, no gene encoding for a plasma membrane protein serves as a reference gene, and relative quantification of plasma membrane proteins is performed with genes encoding soluble proteins, which differ greatly in quantity and in targeting and trafficking from plasma membrane proteins. In this work, our aim was to identify a housekeeping gene, ideally one that codes for a plasma membrane protein, whose expression remains the same regardless of drug treatment and across a wide range of tissues to be used for relative quantification of real-time RT-PCR data for ATP binding cassette (ABC) plasma membrane transporters. Results In studies evaluating the expression levels of two commonly used reference genes coding for soluble proteins and two genes coding for membrane proteins, one plasma membrane protein, plasma membrane calcium-ATPase 4 (PMCA4), was comparable to the two reference genes already in use. In addition, PMCA4 expression shows little variation across eight drug-treated cell lines and was found to be superior to GAPDH and HPRT1, commonly used reference genes. Finally, we show PMCA4 used as a reference gene for normalizing ABC transporter expression in a drug-resistant lung carcinoma cell line. Conclusion We have found that PMCA4 is a good housekeeping gene for normalization of gene expression for polytopic membrane proteins including transporters and receptors. PMID:16978418

  14. A Comparative Spin-Label Study of Isolated Plasma Membranes and Plasma Membranes of Whole Cells and Protoplasts from Cold-Hardened and Nonhardened Winter Rye

    PubMed Central

    Windle, John J.

    1988-01-01

    Lipid-lipid and lipid-protein interactions in the plasma membranes of whole cells and protoplasts and an isolated plasma membrane fraction from winter rye (Secale cereale L. cv Puma) have been studied by spin labeling. Spectra were recorded between −40°C and 40°C using the freely diffusing spin-label, 16-doxyl stearic acid, as a midbilayer membrane probe. The probe was reduced by the whole cells and protoplasts and reoxidized by external potassium ferricyanide. The reoxidized probe was assumed to be localized in the plasma membrane. The spectra consisted of the superposition of a narrow and a broad component indicating that both fluid and immobilized lipids were present in the plasma membrane. The two components were separated by digital subtraction of the immobilized component. Temperature profiles of the membranes were developed using the percentage of immobilized lipid present at each temperature and the separation between the outermost hyperfine lines for the fluid lipid component. Lipid immobilization was attributed to lipid-protein interactions, lipid-cell wall interactions, and temperature-induced lipid phase transitions to the gel-state. Temperature profiles were compared for both cold-hardened and nonhardened protoplasts, plasma membranes, and plasma membrane lipids, respectively. Although cold-hardening extended the range of lipid fluidity by 5°C, it had no effect on lipid-protein interactions or activation energies of lipid mobility. Differences were found, however, between the temperature profiles for the different samples, suggesting that alterations in the plasma membrane occurred as a consequence of the isolation methods used. PMID:16666471

  15. Phospholipid transfer activities in toad oocytes and developing embryos. [Bufo arenarum

    SciTech Connect

    Rusinol, A.; Salomon, R.A.; Bloj, B.

    1987-01-01

    The role of lipid transfer proteins during plasma membrane biogenesis was explored. Developing amphibia embryos were used because during their growth an active plasma membrane biosynthesis occurs together with negligible mitochondrial and endoplasmic reticulum proliferation. Sonicated vesicles, containing /sup 14/C-labeled phospholipids and /sup 3/H-labeled triolein, as donor particles and cross-linked erythrocyte ghosts as acceptor particles were used to measure phospholipid transfer activities in unfertilized oocytes and in developing embryos of the toad Bufo arenarum. Phosphatidylcholine transfer activity in pH 5.1 supernatant of unfertilized oocytes was 8-fold higher than the activity found in female toad liver supernatant, but dropped steadily after fertilization. After 20 hr of development, at the stage of late blastula, the phosphatidylcholine transfer activity had dropped 4-fold. Unfertilized oocyte supernatant exhibited phosphatidylinositol and phosphatidylethanolamine transfer activity also, but at the late blastula stage the former had dropped 18-fold and the latter was no longer detectable under our assay conditions. Our results show that fertilization does not trigger a phospholipid transport process catalyzed by lipid transfer proteins. Moreover, they imply that 75% of the phosphatidylcholine transfer activity and more than 95% of the phosphatidylinositol and phosphatidylethanolamine transfer activities present in pH 5.1 supernatants of unfertilized oocytes may not be essential for toad embryo development. Our findings do not rule out, however, that a phosphatidylcholine-specific lipid transfer protein could be required for embryo early growth.

  16. Expression and localization of urokinase-type plasminogen activator receptor in bovine cumulus-oocyte complexes.

    PubMed

    García, Daniela C; Miceli, Dora C; Rizo, Gabriela; García, Elina V; Valdecantos, Pablo A; Roldán-Olarte, Mariela

    2016-04-01

    Urokinase-type plasminogen activator (uPA) is a serine protease involved in extracellular matrix remodeling through plasmin generation. uPA usually binds to its receptor, uPAR, which is anchored to the plasma membrane through a glycosylphosphatidylinositol anchor. uPA/uPAR binding increases proteolytic activity in the neighborhood of the cells containing uPAR and activates intracellular signaling pathways involved in extracellular matrix remodeling, cell migration and proliferation. The aim of this work was to study the expression of uPA, uPAR and plasminogen activator inhibitor-1 (PAI-1) in immature and in vitro matured bovine cumulus-oocyte complexes (COCs). uPA is only expressed in the cumulus cells of immature and in vitro matured COCs, while uPAR and PAI-1 are expressed in both the cumulus cells and the immature and in vitro matured oocytes. In addition, uPAR protein was localized by confocal microscopy in the plasma membrane of oocytes and cumulus cells of immature COCs. Results from this research led us to hypothesize that the uPA/uPAR interaction could cause the local production of uPA-mediated plasmin over oocyte and cumulus cell surface; plasmin formation could also be regulated by PAI-1.

  17. A Gs-linked receptor maintains meiotic arrest in mouse oocytes, but luteinizing hormone does not cause meiotic resumption by terminating receptor-Gs signaling

    PubMed Central

    Norris, Rachael P.; Freudzon, Leon; Freudzon, Marina; Hand, Arthur R.; Mehlmann, Lisa M.; Jaffe, Laurinda A.

    2008-01-01

    The maintenance of meiotic prophase arrest in fully grown vertebrate oocytes depends on the activity of a Gs G-protein that activates adenylyl cyclase and elevates cAMP, and in the mouse oocyte, Gs is activated by a constitutively active orphan receptor, GPR3. To determine whether the action of luteinizing hormone (LH) on the mouse ovarian follicle causes meiotic resumption by inhibiting GPR3-Gs signaling, we examined the effect of LH on the localization of Gαs. Gs activation in response to stimulation of an exogenously expressed β2-adrenergic receptor causes Gαs to move from the oocyte plasma membrane into the cytoplasm, whereas Gs inactivation in response to inhibition of the β2-adrenergic receptor causes Gαs to move back to the plasma membrane. However, LH does not cause a change in Gαs localization, indicating that LH does not act by terminating receptor-Gs signaling. PMID:17850783

  18. Glycosidases in the plasma membrane of Ceratitis capitata spermatozoa.

    PubMed

    Intra, Jari; De Caro, Daniela; Perotti, Maria-Elisa; Pasini, Maria Enrica

    2011-02-01

    Fruit flies in the family Tephritidae are rated among the world's most destructive agricultural pests. The Mediterranean fruit fly Ceratitis capitata is emerging as a model organism to study the fertilization in Insects. Three integral proteins with glycosidase activity are present in the plasma membrane of spermatozoa. The glycosidases have been purified and characterized. We have demonstrated the presence of three enzymes, a β-N-acetylhexosaminidase, an α-mannosidase and an α-l-fucosidase. The molecular mass of the native enzymes estimated by gel filtration was 160 kDa for β-N-acetylhexosaminidase, 310 kDa for α-mannosidase and 140 kDa for α-l-fucosidase. SDS-PAGE showed that β-N-acetylhexosaminidase is a dimer of a single protein of 73 kDa, α-mannosidase consists of six subunits with different molecular weights and α-l-fucosidase is a dimer made up by two different monomers. Characterization of the purified enzymes included glycosylation pattern, pI, optimal pH, substrate preference, kinetic properties and thermal stability. Soluble forms similar to the sperm associated glycosidases are present. Polyclonal antibodies raised against synthetic peptides designed from the predicted products of the Drosophila melanogaster genes encoding β-N-acetylhexosaminidase and α-l-fucosidase were used. Immunofluorescence labelling of spermatozoa showed that the enzymes are present in the sperm plasma membrane overlying the acrosome and the tail. This work represents the first report on the characterization in C. capitata of sperm proteins that are potentially involved in primary gamete recognition.

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

    PubMed Central

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

    2015-01-01

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

  20. β-Glucan Synthetases of Plasma Membrane and Golgi Apparatus from Onion Stem 1

    PubMed Central

    Van Der Woude, William J.; Lembi, Carole A.; Morré, D. James; Kindinger, Jaunita I.; Ordin, Lawrence

    1974-01-01

    Biosynthesis of glucans occurred in cell-free fractions isolated from onion stem (Allium cepa L.) enriched in either dictyosomes or plasma membranes. β-1,3- and β-1, 4-Glucans were synthesized in differing proportions and at different rates as the concentration of uridine diphosphoglucose or the proportion of dictyosomes or plasma membrane varied. At low (1.5 μm) UDP-glucose concentrations synthesis of alkali-insoluble glucan was correlated with abundance of dicytosomes; most of the substrate utilized by plasma membrane was for glycolipid synthesis. At high (1 mm) UDP-glucose concentration, the synthesis of alkali-insoluble glucans correlated with the abundance of plasma membrane. Substrate enhancement of β-1, 4-glucan synthesis in dictyosome fractions was less than proportional to increases in substrate concentration. In contrast, β-1, 4-glucan synthesis by plasma membrane was more than proportionately increased. At high substrate concentrations the synthesis of β-1, 3-glucans predominated in both dictyosome and plasma membrane fractions. The results show that the capacity to synthesize glucans resides in both Golgi apparatus and plasma membranes of onion stem, but that the plasma membrane has the greatest capacity for synthesis of alkali-insoluble glucans at high UDP-glucose concentrations. Images PMID:16658884

  1. 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...

  2. Rapid Response of the Yeast Plasma Membrane Proteome to Salt Stress*

    PubMed Central

    Szopinska, Aleksandra; Degand, Hervé; Hochstenbach, Jean-François; Nader, Joseph; Morsomme, Pierre

    2011-01-01

    The plasma membrane separates the cell from the external environment and plays an important role in the stress response of the cell. In this study, we compared plasma membrane proteome modifications of yeast cells exposed to mild (0.4 m NaCl) or high (1 m NaCl) salt stress for 10, 30, or 90 min. Plasma membrane-enriched fractions were isolated, purified, and subjected to iTRAQ labeling for quantitative analysis. In total, 88–109 plasma membrane proteins were identified and quantified. The quantitative analysis revealed significant changes in the abundance of several plasma membrane proteins. Mild salt stress caused an increase in abundance of 12 plasma membrane proteins, including known salt-responsive proteins, as well as new targets. Interestingly, 20 plasma membrane proteins, including the P-type H+-ATPase Pma1, ABC transporters, glucose and amino acid transporters, t-SNAREs, and proteins involved in cell wall biogenesis showed a significant and rapid decrease in abundance in response to both 0.4 m and 1 m NaCl. We propose that rapid protein internalization occurs as a response to hyper-osmotic and/or ionic shock, which might affect plasma membrane morphology and ionic homeostasis. This rapid response might help the cell to survive until the transcriptional response takes place. PMID:21825281

  3. Photodynamic activity of substituted zinc trisulfophthalocyanines: role of plasma membrane damage.

    PubMed

    Cauchon, Nicole; Nader, Moni; Bkaily, Ghassan; van Lier, Johan E; Hunting, Darel

    2006-01-01

    We recently reported that variations in cellular phototoxicity among a series of alkynyl-substituted zinc trisulfophthalocyanines (ZnPcS3Cn) correlates with their hydrophobicity, with the most amphiphilic derivatives showing the highest cell uptake and phototoxicity. In this study we address the role of the plasma membrane in the photodynamic response as it relates to the overall hydrophobicity of the photosensitizer. The membrane tracker dye 1-[4(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), which is incorporated into plasma membranes by endocytosis, was used to establish plasma membrane uptake by EMT-6 cells of the ZnPcS3C, by colocalization, and TMA-DPH membrane uptake rates after photodynamic therapy were used to quantify membrane damage. TMA-DPH colocalization patterns show plasma membrane uptake of the photosensitizers after short 1 h incubation periods. TMA-DPH plasma membrane uptake rates after illumination of the photosensitizer-treated cells show a parabolic relationship with photosensitizer hydrophobicity that correlates well with the phototoxicity of the ZnPcS3C,. After a 1 h incubation period, overall phototoxicity correlates closely with the postillumination rate of TMA-DPH incorporation into the cell membrane, suggesting a major role of plasma membrane damage in the overall PDT effect. In contrast, after a 24 h incubation, phototoxicity shows a stronger but imperfect correlation with total cellular photosensitizer uptake rather than TMA-DPH membrane uptake, suggesting a partial shift in the cellular damage responsible for photosensitization from the plasma membrane to intracellular targets. We conclude that plasma membrane localization of the amphiphilic ZnPcS3C6-C9 is a major factor in their overall photodynamic activity.

  4. 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.

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

    PubMed Central

    Wen, Peter J.; Grenklo, Staffan; Arpino, Gianvito; Tan, Xinyu; Liao, Hsien-Shun; Heureaux, Johanna; Peng, Shi-Yong; Chiang, Hsueh-Cheng; Hamid, Edaeni; Zhao, Wei-Dong; Shin, Wonchul; Näreoja, Tuomas; Evergren, Emma; Jin, Yinghui; Karlsson, Roger; Ebert, Steven N.; Jin, Albert; Liu, Allen P.; Shupliakov, Oleg; Wu, Ling-Gang

    2016-01-01

    Vesicle fusion is executed via formation of an Ω-shaped structure (Ω-profile), followed by closure (kiss-and-run) or merging of the Ω-profile into the plasma membrane (full fusion). Although Ω-profile closure limits release but recycles vesicles economically, Ω-profile merging facilitates release but couples to classical endocytosis for recycling. Despite its crucial role in determining exocytosis/endocytosis modes, how Ω-profile merging is mediated is poorly understood in endocrine cells and neurons containing small ∼30–300 nm vesicles. Here, using confocal and super-resolution STED imaging, force measurements, pharmacology and gene knockout, we show that dynamic assembly of filamentous actin, involving ATP hydrolysis, N-WASP and formin, mediates Ω-profile merging by providing sufficient plasma membrane tension to shrink the Ω-profile in neuroendocrine chromaffin cells containing ∼300 nm vesicles. Actin-directed compounds also induce Ω-profile accumulation at lamprey synaptic active zones, suggesting that actin may mediate Ω-profile merging at synapses. These results uncover molecular and biophysical mechanisms underlying Ω-profile merging. PMID:27576662

  6. Yeast mutants affecting possible quality control of plasma membrane proteins.

    PubMed

    Li, Y; Kane, T; Tipper, C; Spatrick, P; Jenness, D D

    1999-05-01

    Mutations gef1, stp22, STP26, and STP27 in Saccharomyces cerevisiae were identified as suppressors of the temperature-sensitive alpha-factor receptor (mutation ste2-3) and arginine permease (mutation can1(ts)). These suppressors inhibited the elimination of misfolded receptors (synthesized at 34 degrees C) as well as damaged surface receptors (shifted from 22 to 34 degrees C). The stp22 mutation (allelic to vps23 [M. Babst and S. Emr, personal communication] and the STP26 mutation also caused missorting of carboxypeptidase Y, and ste2-3 was suppressed by mutations vps1, vps8, vps10, and vps28 but not by mutation vps3. In the stp22 mutant, both the mutant and the wild-type receptors (tagged with green fluorescent protein [GFP]) accumulated within an endosome-like compartment and were excluded from the vacuole. GFP-tagged Stp22p also accumulated in this compartment. Upon reaching the vacuole, cytoplasmic domains of both mutant and wild-type receptors appeared within the vacuolar lumen. Stp22p and Gef1p are similar to tumor susceptibility protein TSG101 and voltage-gated chloride channel, respectively. These results identify potential elements of plasma membrane quality control and indicate that cytoplasmic domains of membrane proteins are translocated into the vacuolar lumen.

  7. Intrarenal localization of the plasma membrane ATP channel pannexin1.

    PubMed

    Hanner, Fiona; Lam, Lisa; Nguyen, Mien T X; Yu, Alan; Peti-Peterdi, János

    2012-11-15

    In the renal tubules, ATP released from epithelial cells stimulates purinergic receptors, regulating salt and water reabsorption. However, the mechanisms by which ATP is released into the tubular lumen are multifaceted. Pannexin1 (Panx1) is a newly identified. ubiquitously expressed protein that forms connexin-like channels in the plasma membrane, which have been demonstrated to function as a mechanosensitive ATP conduit. Here, we report on the localization of Panx1 in the mouse kidney. Using immunofluorescence, strong Panx1 expression was observed in renal tubules, including proximal tubules, thin descending limbs, and collecting ducts, along their apical cell membranes. In the renal vasculature, Panx1 expression was localized to vascular smooth muscle cells in renal arteries, including the afferent and efferent arterioles. Additionally, we tested whether Panx1 channels expressed in renal epithelial cells facilitate luminal ATP release by measuring the ATP content of urine samples freshly collected from wild-type and Panx1(-/-) mice. Urinary ATP levels were reduced by 30% in Panx1(-/-) compared with wild-type mice. These results suggest that Panx1 channels in the kidney may regulate ATP release and via purinergic signaling may participate in the control of renal epithelial fluid and electrolyte transport and vascular functions.

  8. Intrarenal localization of the plasma membrane ATP channel pannexin1

    PubMed Central

    Hanner, Fiona; Lam, Lisa; Nguyen, Mien T. X.; Yu, Alan

    2012-01-01

    In the renal tubules, ATP released from epithelial cells stimulates purinergic receptors, regulating salt and water reabsorption. However, the mechanisms by which ATP is released into the tubular lumen are multifaceted. Pannexin1 (Panx1) is a newly identified. ubiquitously expressed protein that forms connexin-like channels in the plasma membrane, which have been demonstrated to function as a mechanosensitive ATP conduit. Here, we report on the localization of Panx1 in the mouse kidney. Using immunofluorescence, strong Panx1 expression was observed in renal tubules, including proximal tubules, thin descending limbs, and collecting ducts, along their apical cell membranes. In the renal vasculature, Panx1 expression was localized to vascular smooth muscle cells in renal arteries, including the afferent and efferent arterioles. Additionally, we tested whether Panx1 channels expressed in renal epithelial cells facilitate luminal ATP release by measuring the ATP content of urine samples freshly collected from wild-type and Panx1−/− mice. Urinary ATP levels were reduced by 30% in Panx1−/− compared with wild-type mice. These results suggest that Panx1 channels in the kidney may regulate ATP release and via purinergic signaling may participate in the control of renal epithelial fluid and electrolyte transport and vascular functions. PMID:22952282

  9. 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

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

    PubMed Central

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

    2013-01-01

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

  11. 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.

  12. Enhancement of RNA Polymerase Activity by a Factor Released by Auxin from Plasma Membrane*

    PubMed Central

    Hardin, James W.; Cherry, Joe H.; Morré, D. James; Lembi, Carole A.

    1972-01-01

    Using recently developed techniques for solubilization of RNA polymerase from soybean chromatin and isolation of plasma membrane fractions from plants we can show the presence of a transcriptional factor specifically released from the membranes by auxin, 2,4-dichlorophenoxyacetic acid. The nonauxin, 3,5-dichlorophenoxyacetic acid, does not release the factor, but subsequent exposure of the membranes to auxin results in its release. Factor activity could not be demonstrated in fractions devoid of plasma membranes. The presence of a regulatory factor for RNA polymerase associated with plant plasma membrane and specifically released by auxin provides a mechanism whereby both rapid growth responses and delayed nuclear changes could be derived from a common auxin receptor site associated with plasma membrane. Images PMID:4508307

  13. Effect of calmodulin antagonists on calcium pump of ram spermatozoa plasma membrane.

    PubMed

    Breitbart, H; Rubinstein, S

    1988-01-01

    Plasma membranes isolated from ram spermatozoa contain calmodulin, which represents approximately 0.03% of the total sperm calmodulin and 0.025% of the membrane protein. When membranes were isolated in the presence of ethylene glycol (beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), the amount of calmodulin associated with the plasma membranes was reduced by only 20%. The ATP-dependent calcium transport activity of the isolated plasma membranes is not enhanced by adding calmodulin and not inhibited by the calmodulin antagonists trifluoperazinc (TFP), compound 48/80, or calmidazolium. In fact, there is an enhancement of calcium uptake by the calmodulin antagonists and this enhancement can be blocked by the Ca2+-channel blocker D-600. It is suggested that the ATP-dependent calcium transport activity in the plasma membrane of ram spermatozoa is not regulated by calmodulin.

  14. Electrogenic Transport of Protons Driven by the Plasma Membrane ATPase in Membrane Vesicles from Radish 1

    PubMed Central

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

    1985-01-01

    Mg:ATP-dependent H+ pumping has been studied in microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings by monitoring both intravesicular acidification and the building up of an inside positive membrane potential difference (Δ ψ). ΔpH was measured as the decrease of absorbance of Acridine orange and Δ ψ as the shift of absorbance of bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol. Both Mg:ATP-dependent Δ pH and Δ ψ generation are completely inhibited by vanadate and insensitive to oligomycin; moreover, Δ pH generation is not inhibited by NO3−. These findings indicate that this membrane preparation is virtually devoid of mitochondrial and tonoplast H+-ATPases. Both intravesicular acidification and Δ ψ generation are influenced by anions: Δ pH increases and Δ ψ decreases following the sequence SO42−, Cl−, Br−, NO3−. ATP-dependent H+ pumping strictly requires Mg2+. It is very specific for ATP (apparent Km 0.76 millimolar) compared to GTP, UTP, CTP, ITP. Δ pH generation is inhibited by CuSO4 and diethylstilbestrol as well as vanadate. Δ pH generation is specificially stimulated by K+ (+ 80%) and to a lesser extent by Na+ and choline (+28% and +14%, respectively). The characteristics of H+ pumping in these microsomal vesicles closely resemble those described for the plasma membrane ATPase partially purified from several plant materials. PMID:16664008

  15. Red wine activates plasma membrane redox system in human erythrocytes.

    PubMed

    Tedesco, Idolo; Moccia, Stefania; Volpe, Silvestro; Alfieri, Giovanna; Strollo, Daniela; Bilotto, Stefania; Spagnuolo, Carmela; Di Renzo, Massimo; Aquino, Rita P; Russo, Gian Luigi

    2016-01-01

    In the present study, we report that polyphenols present in red wine obtained by a controlled microvinification process are able to protect human erythrocytes from oxidative stress and to activate Plasma Membrane Redox System (PMRS). Human plasma obtained from healthy subjects was incubated in the presence of whole red wine at a concentration corresponding to 9.13-73 μg/ml gallic acid equivalents to verify the capacity to protect against hypochlorous acid (HOCl)-induced plasma oxidation and to minimize chloramine formation. Red wine reduced hemolysis and chloramine formation induced by HOCl of 40 and 35%, respectively. PMRS present on human erythrocytes transfers electrons from intracellular molecules to extracellular electron acceptors. We demonstrated that whole red wine activated PMRS activity in human erythrocytes isolated from donors in a dose-dependent manner with a maximum at about 70-100 μg/ml gallic acid equivalents. We also showed that red wine increased glutathione (GSH) levels and erythrocytic antioxidant capacity, measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) quenching assay. Furthermore, we reported that GSH played a crucial role in regulating PMRS activity in erythrocytes. In fact, the effect of iodoacetamide, an alkylating agent that induces depletion of intracellular GSH, was completely counteracted by red wine. Bioactive compounds present in red wine, such as gallic acid, resveratrol, catechin, and quercetin were unable to activate PMRS when tested at the concentrations normally present in aged red wines. On the contrary, the increase of PMRS activity was associated with the anthocyanin fraction, suggesting the capacity of this class of compounds to positively modulate PMRS enzymatic activity.

  16. Graft polymerization and plasma treatment of polymer membranes for fouling reduction: a review.

    PubMed

    Kochkodan, Victor M; Sharma, Virender K

    2012-01-01

    This article presents a review of recent developments in surface modification of polymer membranes via graft polymerization and plasma treatment for reduction of fouling with organic compounds and microorganisms in pressure driven membrane processes. The factors affecting membrane fouling, such as membrane hydrophilicity, charge and surface roughness are discussed. The recent studies in which the reduction of organic fouling and biofouling by the modification of the membrane surface via ultraviolet/redox initiated surface grafting of hydrophilic polymers and low temperature plasma treatment are reviewed.

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

    PubMed

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

    2012-07-30

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

  18. 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

  19. 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

  20. Previtellogenic oocyte growth in salmon: relationships among body growth, plasma insulin-like growth factor-1, estradiol-17beta, follicle-stimulating hormone and expression of ovarian genes for insulin-like growth factors, steroidogenic-acute regulatory protein and receptors for gonadotropins, growth hormone, and somatolactin.

    PubMed

    Campbell, B; Dickey, J; Beckman, B; Young, G; Pierce, A; Fukada, H; Swanson, P

    2006-07-01

    Body growth during critical periods is known to be an important factor in determining the age of maturity and fecundity in fish. However, the endocrine mechanisms controlling oogenesis in fish and the effects of growth on this process are poorly understood. In this study interactions between the growth and reproductive systems were examined by monitoring changes in various components of the FSH-ovary axis, plasma insulin-like growth factor 1 (Igf1), and ovarian gene expression in relation to body and previtellogenic oocyte growth in coho salmon. Samples were collected from females during two hypothesized critical periods when growth influences maturation in this species. Body growth during the fall-spring months was strongly related to the degree of oocyte development, with larger fish possessing more advanced oocytes than smaller, slower growing fish. The accumulation of cortical alveoli in the oocytes was associated with increases in plasma and pituitary FSH, plasma estradiol-17beta, and ovarian steroidogenic acute regulatory protein (star) gene expression, whereas ovarian transcripts for growth hormone receptor and somatolactin receptor decreased. As oocytes accumulated lipid droplets, a general increase occurred in plasma Igf1 and components of the FSH-ovary axis, including plasma FSH, estradiol-17beta, and ovarian mRNAs for gonadotropin receptors, star, igf1, and igf2. A consistent positive relationship between plasma Igf1, estradiol-17beta, and pituitary FSH during growth in the spring suggests that these factors are important links in the mechanism by which body growth influences the rate of oocyte development.

  1. Calcium and actin in the saga of awakening oocytes

    SciTech Connect

    Santella, Luigia Limatola, Nunzia; Chun, Jong T.

    2015-04-24

    The interaction of the spermatozoon with the egg at fertilization remains one of the most fascinating mysteries of life. Much of our scientific knowledge on fertilization comes from studies on sea urchin and starfish, which provide plenty of gametes. Large and transparent, these eggs have served as excellent model systems for studying egg activation and embryo development in seawater, a plain natural medium. Starfish oocytes allow the study of the cortical, cytoplasmic and nuclear changes during the meiotic maturation process, which can also be triggered in vitro by hormonal stimulation. These morphological and biochemical changes ensure successful fertilization of the eggs at the first metaphase. On the other hand, sea urchin eggs are fertilized after the completion of meiosis, and are particularly suitable for the study of sperm–egg interaction, early events of egg activation, and embryonic development, as a large number of mature eggs can be fertilized synchronously. Starfish and sea urchin eggs undergo abrupt changes in the cytoskeleton and ion fluxes in response to the fertilizing spermatozoon. The plasma membrane and cortex of an egg thus represent “excitable media” that quickly respond to the stimulus with the Ca{sup 2+} swings and structural changes. In this article, we review some of the key findings on the rapid dynamic rearrangements of the actin cytoskeleton in the oocyte/egg cortex upon hormonal or sperm stimulation and their roles in the modulation of the Ca{sup 2+} signals and in the control of monospermic fertilization. - Highlights: • Besides microtubules, microfilaments may anchor the nucleus to oocyte surface. • The cortical Ca{sup 2+} flash and wave at fertilization mirror electrical membrane change. • Artificial egg activation lacks microvilli extension in the perivitelline space. • Calcium is necessary but not sufficient for cortical granules exocytosis. • Actin cytoskeleton modulates Ca{sup 2+} release at oocyte maturation

  2. The Role of Microfilaments in Early Meiotic Maturation of Mouse Oocytes

    NASA Astrophysics Data System (ADS)

    Calarco, Patricia G.

    2005-04-01

    Mouse oocyte microfilaments (MF) were perturbed by depolymerization (cytochalasin B) or stabilization (jasplakinolide) and correlated meiotic defects examined by confocal microscopy. MF, microtubules, and mitochondria were vitally stained; centrosomes ([gamma]-tubulin), after fixation. MF depolymerization by cytochalasin in culture medium did not affect central migration of centrosomes, mitochondria, or nuclear breakdown (GVBD); some MF signal was localized around the germinal vesicle (GV). In maturation-blocking medium (containing IBMX), central movement was curtailed and cortical MF aggregations made the plasma membrane wavy. Occasional long MF suggested that not all MF were depolymerized. MF stabilization by jasplakinolide led to MF aggregations throughout the cytoplasm. GVBD occurred (unless IBMX was present) but no spindle formed. Over time, most oocytes constricted creating a dumbbell shape with MF concentrated under one-half of the oocyte cortex and on either side of the constriction. In IBMX medium, the MF-containing half of the dumbbell over time sequestered the GV, MF, mitochondria, and one to two large cortical centrosomes; the non-MF half appeared empty. Cumulus processes contacted the oocyte surface (detected by microtubule content) and mirrored MF distribution. Results demonstrated that MF play an essential role in meiosis, primarily through cortically mediated events, including centrosome localization, spindle (or GV) movement to the periphery, activation of (polar body) constriction, and establishment of oocyte polarity. The presence of a cortical “organizing pole” is hypothesized.

  3. Sperm-Egg Interaction: Evidence for Boar Sperm Plasma Membrane Receptors for Porcine Zona Pellucida

    NASA Astrophysics Data System (ADS)

    Peterson, Rudolph N.; Russell, Lonnie; Bundman, Donna; Freund, Matthew

    1980-01-01

    Freshly ejaculated, noncapacitated boar sperm bind rapidly and in large numbers to pig egg zona pellucida in vitro. In the present study, the number of sperm bound decreased sharply when sperm motility was lowered by energy poisons or by reducing the temperature. Highly motile sperm from humans, guinea pigs, and rats, added at concentrations ten times higher than control sperm, did not bind to the porcine zona. At the same high concentration, a small number of hamster and bull sperm bound to the zona. Binding of boar sperm to the zona pellucida was blocked almost completely by diluted whole antiserum to sperm plasma membranes and by univalent (Fab) antibody to these membranes. When antibody to sperm plasma membrane was first absorbed with plasma membrane vesicles, sperm binding was not inhibited. These results provide direct evidence for the existence of sperm plasma membrane receptors for the zona pellucida of the pig.

  4. Accumulation of raft lipids in T-cell plasma membrane domains engaged in TCR signalling

    PubMed Central

    Zech, Tobias; Ejsing, Christer S; Gaus, Katharina; de Wet, Ben; Shevchenko, Andrej; Simons, Kai; Harder, Thomas

    2009-01-01

    Activating stimuli for T lymphocytes are transmitted through plasma membrane domains that form at T-cell antigen receptor (TCR) signalling foci. Here, we determined the molecular lipid composition of immunoisolated TCR activation domains. We observed that they accumulate cholesterol, sphingomyelin and saturated phosphatidylcholine species as compared with control plasma membrane fragments. This provides, for the first time, direct evidence that TCR activation domains comprise a distinct molecular lipid composition reminiscent of liquid-ordered raft phases in model membranes. Interestingly, TCR activation domains were also enriched in plasmenyl phosphatidylethanolamine and phosphatidylserine. Modulating the T-cell lipidome with polyunsaturated fatty acids impaired the plasma membrane condensation at TCR signalling foci and resulted in a perturbed molecular lipid composition. These results correlate the accumulation of specific molecular lipid species with the specific plasma membrane condensation at sites of TCR activation and with early TCR activation responses. PMID:19177148

  5. Expression of a P-selectin ligand in zona pellucida of porcine oocytes and P-selectin on acrosomal membrane of porcine sperm cells. Potential implications for their involvement in sperm-egg interactions.

    PubMed

    Geng, J G; Raub, T J; Baker, C A; Sawada, G A; Ma, L; Elhammer, A P

    1997-05-05

    The selectin family of cell adhesion molecules mediates initial leukocyte adhesion to vascular endothelial cells at sites of inflammation. O-glycan structural similarities between oligosaccharides from human leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) and from zona pellucida glycoproteins of porcine oocytes indicate the possible existence of a P-selectin ligand in the zona pellucida. Here, using biochemical as well as morphological approaches, we demonstrate that a P-selectin ligand is expressed in the porcine zona pellucida. In addition, a search for a specific receptor for this ligand leads to the identification of P-selectin on the acrosomal membrane of porcine sperm cells. In vitro binding of porcine acrosome-reacted sperm cells to oocytes was found to be Ca2+ dependent and inhibitable with either P-selectin, P-selectin receptor-globulin, or leukocyte adhesion blocking antibodies against P-selectin and PSGL-1. Moreover, porcine sperm cells were found to be capable of binding to human promyeloid cell line HL-60. Taken together, our findings implicate a potential role for the oocyte P-selectin ligand and the sperm P-selectin in porcine sperm-egg interactions.

  6. In vitro fusion of lung lamellar bodies and plasma membrane is augmented by lung synexin.

    PubMed

    Chander, A; Wu, R D

    1991-11-05

    Lamellar bodies of lung epithelial type II cells undergo fusion with plasma membrane prior to exocytosis of surfactant into the alveolar lumen. Since synexin from adrenal glands promotes aggregation and fusion of chromaffin granules, we purified synexin-like proteins from bovine lung cytosolic fraction, and evaluated their effect on the fusion of isolated lamellar bodies and plasma membrane fractions. Synexin activity, which co-purified with an approx. 47 kDa protein (pI 6.8), was assessed by following calcium-dependent aggregation of liposomes prepared from a mixture of phosphatidylcholine:phosphatidylserine (PC:PS, 3:1, mol/mol). Lung synexin caused aggregation of liposomes approximating lung surfactant lipid-like composition, isolated lamellar bodies, or isolated plasma membrane fraction. Lung synexin promoted fusion only in the presence of calcium. It augmented fusion between lamellar bodies and plasma membranes, lamellar bodies and liposomes, or between two populations of liposomes. However, selectivity with regard to synexin-mediated fusion was observed as synexin did not promote fusion between plasma membrane and liposomes, or between liposomes of surfactant lipid-like composition and other liposomes. These observations support a role for lung synexin in membrane fusion between the plasma membrane and lamellar bodies during exocytosis of lung surfactant, and suggest that such fusion is dependent on composition of interacting membranes.

  7. Tailoring the properties of asymmetric cellulose acetate membranes by gas plasma etching.

    PubMed

    Olde Riekerink, M B; Engbers, G H M; Wessling, M; Feijen, J

    2002-01-15

    Cellulose triacetate (CTA) ultrafilters and cellulose acetate blend (CAB) desalination membranes were treated with a radiofrequency gas plasma (tetrafluoromethane (CF(4)) or carbon dioxide (CO(2)), 47-49 W, 0.04-0.08 mbar). Treatment times were varied between 15 s and 120 min. The plasma-treated top layer of the membranes was characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, and contact angle measurements to obtain information about surface structure, chemistry, and wettability, respectively. The membrane properties (e.g., permeability, selectivity, fouling) were studied by waterflux measurements, molecular weight cutoff measurements, and fouling experiments with bovine serum albumin. CO(2) plasma treatment resulted in gradual etching of the membrane's dense top layer. Permeation and selectivity changed significantly for treatment times of 0-15 min for CTA and 5-60 min for CAB membranes. Moreover, CTA membranes were hydrophilized during CO(2) plasma treatment whereas CF(4) plasma treatment led to hydrophobic surfaces due to strong fluorination of the top layer. This study shows that gas plasma etching can tailor the properties of asymmetric cellulose acetate membranes by simultaneously modifying the chemistry and structure of the top layer. The low fouling properties of CTA membranes were thereby largely maintained.

  8. Plasma Membrane H+-ATPase in Maize Roots Induced for NO3- Uptake.

    PubMed Central

    Santi, S.; Locci, G.; Pinton, R.; Cesco, S.; Varanini, Z.

    1995-01-01

    Plasma membrane H+-ATPase was studied in maize (Zea mays L.) roots induced for NO3- uptake. Membrane vesicles were isolated by means of Suc density gradient from roots exposed for 24 h either to 1.5 mM NO3- or 1.5 mM SO4-. The two populations of vesicles had similar composition as shown by diagnostic inhibitors of membrane-associated ATPases. However, both ATP-dependent intravesicular H+ accumulation and ATP hydrolysis were considerably enhanced (60-100%) in vesicles isolated from NO3--induced roots. Km for Mg:ATP and pH dependency were not influenced by NO3- treatment of the roots. ATP hydrolysis in plasma membrane vesicles for both control and NO3--induced roots was not affected by 10 to 150 mM NO3- or Cl-. On the other hand, kinetics of NO3-- or Cl--stimulated ATP-dependent intravesicular H+ accumulation were modified in plasma membrane vesicles isolated from NO3-- induced roots. Immunoassays carried out with polyclonal antibodies against plasma membrane H+-ATPase revealed an increased steady-state level of the enzyme in plasma membrane vesicles isolated from NO3--induced roots. Results are consistent with the idea of an involvement of plasma membrane H+-ATPase in the overall response of roots to NO3-. PMID:12228668

  9. Plasma Membrane H+-ATPase in Maize Roots Induced for NO3- Uptake.

    PubMed

    Santi, S.; Locci, G.; Pinton, R.; Cesco, S.; Varanini, Z.

    1995-12-01

    Plasma membrane H+-ATPase was studied in maize (Zea mays L.) roots induced for NO3- uptake. Membrane vesicles were isolated by means of Suc density gradient from roots exposed for 24 h either to 1.5 mM NO3- or 1.5 mM SO4-. The two populations of vesicles had similar composition as shown by diagnostic inhibitors of membrane-associated ATPases. However, both ATP-dependent intravesicular H+ accumulation and ATP hydrolysis were considerably enhanced (60-100%) in vesicles isolated from NO3--induced roots. Km for Mg:ATP and pH dependency were not influenced by NO3- treatment of the roots. ATP hydrolysis in plasma membrane vesicles for both control and NO3--induced roots was not affected by 10 to 150 mM NO3- or Cl-. On the other hand, kinetics of NO3-- or Cl--stimulated ATP-dependent intravesicular H+ accumulation were modified in plasma membrane vesicles isolated from NO3-- induced roots. Immunoassays carried out with polyclonal antibodies against plasma membrane H+-ATPase revealed an increased steady-state level of the enzyme in plasma membrane vesicles isolated from NO3--induced roots. Results are consistent with the idea of an involvement of plasma membrane H+-ATPase in the overall response of roots to NO3-.

  10. Clinical evaluation of a flat-plate membrane plasma exchange system.

    PubMed

    Grossman, L; Benny, W B; Buchanan, J; Erickson, R R; Buffaloe, G W

    1983-01-01

    A new flat-plate membrane plasma separation system specifically designed for therapeutic plasma exchange (TPE) was clinically evaluated in both research and routine clinical settings. The study included a comparison to a currently available centrifugal cell separation system employed for TPE. A total of 267 membrane procedures were performed on 39 patients over a 14-month period. Both qualitative and quantitative studies showed that membrane plasma exchange procedures were equivalent to centrifugal procedures in the removal of plasma constituents from patients. A notable difference between the two types of procedure was the effect on the peripheral blood platelet count: the plasma filtrate from the membrane system was essentially cell-free and platelet counts fell only 11% during the procedure, compared to a 53% decrease during the centrifugation runs. Patient responses to both types of procedure were similar and the frequency of side-effects was low. A sampling of patient opinion revealed a preference for the membrane system for a variety of reasons. Procedure times were shorter with the membrane system because of higher achievable blood flow rates, and thus higher plasma exchange rates, while the overall nursing time requirement was lower. The results show that this flat-plate membrane TPE system enables rapid and effective plasma exchange therapy, and offered a number of monitoring and control functions that provided a safer, more efficient therapeutic procedure in the majority of patient treatments performed in this study.

  11. Modeling of Fluid-Membrane Interaction in Cellular Microinjection Process

    NASA Astrophysics Data System (ADS)

    Karzar-Jeddi, Mehdi; Diaz, Jhon; Olgac, Nejat; Fan, Tai-Hsi

    2009-11-01

    Cellular microinjection is a well-accepted method to deliver matters such as sperm, nucleus, or macromolecules into biological cells. To improve the success rate of in vitro fertilization and to establish the ideal operating conditions for a novel computer controlled rotationally oscillating intracytoplasmic sperm injection (ICSI) technology, we investigate the fluid-membrane interactions in the ICSI procedure. The procedure consists of anchoring the oocyte (a developing egg) using a holding pipette, penetrating oocyte's zona pellucida (the outer membrane) and the oolemma (the plasma or inner membrane) using an injection micropipette, and finally to deliver sperm into the oocyte for fertilization. To predict the large deformation of the oocyte membranes up to the piercing of the oolemma and the motion of fluids across both membranes, the dynamic fluid-pipette-membrane interactions are formulated by the coupled Stokes' equations and the continuum membrane model based on Helfrich's energy theory. A boundary integral model is developed to simulate the transient membrane deformation and the local membrane stress induced by the longitudinal motion of the injection pipette. The model captures the essential features of the membranes shown on optical images of ICSI experiments, and is capable of suggesting the optimal deformation level of the oolemma to start the rotational oscillations for piercing into the oolemma.

  12. Previtellogenic and vitellogenic oocytes in ovarian follicles of cultured siberian sturgeon Acipenser baerii (Chondrostei, Acipenseriformes).

    PubMed

    Żelazowska, Monika; Fopp-Bayat, Dorota

    2017-01-01

    Previtellogenic and vitellogenic oocytes in ovarian follicles from cultured Siberian sturgeon Acipenser baerii were examined. In previtellogenic oocytes, granular and homogeneous zones in the cytoplasm (the ooplasm) are distinguished. Material of nuclear origin, rough endoplasmic reticulum, Golgi complexes, complexes of mitochondria with cement and round bodies are numerous in the granular ooplasm. In vitellogenic oocytes, the ooplasm comprises three zones: perinuclear area, endoplasm and periplasm. The endoplasm contains yolk platelets, lipid droplets, and aggregations of mitochondria and granules immersed in amorphous material. In the nucleoplasm, lampbrush chromosomes, nucleoli, and two types of nuclear bodies are present. The first type of nuclear bodies is initially composed of fibrillar threads only. Their ultrastructure subsequently changes and they contain threads and medium electron dense material. The second type of nuclear bodies is only composed of electron dense particles. All nuclear bodies impregnate with silver, stain with propidium iodide, and are DAPI-negative. Their possible role is discussed. All oocytes are surrounded by follicular cells and a basal lamina which is covered by thecal cells. Egg envelopes are not present in previtellogenic oocytes. In vitellogenic oocytes, the plasma membrane (the oolemma) is covered by three envelopes: vitelline envelope, chorion, and extrachorion. Vitelline envelope comprises four sublayers: filamentous layer, trabecular layer 2 (t2), homogeneous layer, and trabecular layer 1 (t1). In the chorion, porous layer 1 and porous layer 2 are distinguished in most voluminous examined oocytes. Three micropylar cells that are necessary for the formation of micropyles are present between follicular cells at the animal hemisphere. J. Morphol. 278:50-61, 2017. ©© 2016 Wiley Periodicals,Inc.

  13. 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

    2002-11-05

    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.

  14. 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.

  15. Plasma membrane poration by opioid neuropeptides: a possible mechanism of pathological signal transduction.

    PubMed

    Maximyuk, O; Khmyz, V; Lindskog, C-J; Vukojević, V; Ivanova, T; Bazov, I; Hauser, K F; Bakalkin, G; Krishtal, O

    2015-03-12

    Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.

  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. 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

    2015-11-16

    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.

  18. 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.

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

    PubMed Central

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

    2016-01-01

    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

  20. Endocytic adaptors – social networking at the plasma membrane

    PubMed Central

    Reider, Amanda; Wendland, Beverly

    2011-01-01

    Receptor-mediated endocytosis is a dynamic process that is crucial for maintaining plasma membrane composition and controlling cell-signaling pathways. A variety of entry routes have evolved to ensure that the vast array of molecules on the cell surface can be differentially internalized by endocytosis. This diversity has extended to include a growing list of endocytic adaptor proteins, which are thought to initiate the internalization process. The key function of adaptors is to select the proteins that should be removed from the cell surface. Thus, they have a central role in defining the physiology of a cell. This has made the study of adaptor proteins a very active area of research that is ripe for exciting future discoveries. Here, we review recent work on how adaptors mediate endocytosis and address the following questions: what characteristics define an endocytic adaptor protein? What roles do these proteins fulfill in addition to selecting cargo and how might adaptors function in clathrin-independent endocytic pathways? Through the findings discussed in this Commentary, we hope to stimulate further characterization of known adaptors and expansion of the known repertoire by identification of new adaptors. PMID:21536832

  1. Calcium occlusion in plasma membrane Ca2+-ATPase.

    PubMed

    Ferreira-Gomes, Mariela S; González-Lebrero, Rodolfo M; de la Fuente, María C; Strehler, Emanuel E; Rossi, Rolando C; Rossi, Juan Pablo F C

    2011-09-16

    In this work, we set out to identify and characterize the calcium occluded intermediate(s) of the plasma membrane Ca(2+)-ATPase (PMCA) to study the mechanism of calcium transport. To this end, we developed a procedure for measuring the occlusion of Ca(2+) in microsomes containing PMCA. This involves a system for overexpression of the PMCA and the use of a rapid mixing device combined with a filtration chamber, allowing the isolation of the enzyme and quantification of retained calcium. Measurements of retained calcium as a function of the Ca(2+) concentration in steady state showed a hyperbolic dependence with an apparent dissociation constant of 12 ± 2.2 μM, which agrees with the value found through measurements of PMCA activity in the absence of calmodulin. When enzyme phosphorylation and the retained calcium were studied as a function of time in the presence of La(III) (inducing accumulation of phosphoenzyme in the E(1)P state), we obtained apparent rate constants not significantly different from each other. Quantification of EP and retained calcium in steady state yield a stoichiometry of one mole of occluded calcium per mole of phosphoenzyme. These results demonstrate for the first time that one calcium ion becomes occluded in the E(1)P-phosphorylated intermediate of the PMCA.

  2. Control of plasma membrane lipid homeostasis by the extended synaptotagmins

    PubMed Central

    Saheki, Yasunori; Bian, Xin; Schauder, Curtis M.; Sawaki, Yujin; Surma, Michal A.; Klose, Christian; Pincet, Frederic; Reinisch, Karin M.; De Camilli, Pietro

    2016-01-01

    Acute metabolic changes of plasma membrane (PM) lipids, such as those mediating signaling reactions, are rapidly compensated by homeostatic responses whose molecular basis is poorly understood. Here we show that the Extended-Synaptotagmins (E-Syts), ER proteins which function as PI(4,5)P2 and Ca2+-regulated tethers to the PM, participate in these responses. E-Syts transfer glycerolipids between bilayers in vitro and such transfer requires Ca2+ and their SMP domain, a lipid-harboring module. Genome edited cells lacking E-Syts do not exhibit abnormalities in the major glycerolipids at rest, but display enhanced and sustained accumulation of PM diacylglycerol (DAG) upon PI(4,5)P2 hydrolysis by PLC activation, which can be rescued by expression of E-Syt1, but not by mutant E-Syt1 lacking the SMP domain. The formation of E-Syts-dependent ER-PM tethers in response to stimuli that cleave PI(4,5)P2 and elevate Ca2+ may help reverse accumulation of DAG in the PM by transferring it to the ER for metabolic recycling. PMID:27065097

  3. The plasma membrane transport systems and adaptation to salinity.

    PubMed

    Mansour, Mohamed Magdy F

    2014-11-15

    Salt stress represents one of the environmental challenges that drastically affect plant growth and yield. Evidence suggests that glycophytes and halophytes have a salt tolerance mechanisms working at the cellular level, and the plasma membrane (PM) is believed to be one facet of the cellular mechanisms. The responses of the PM transport proteins to salinity in contrasting species/cultivars were discussed. The review provides a comprehensive overview of the recent advances describing the crucial roles that the PM transport systems have in plant adaptation to salt. Several lines of evidence were presented to demonstrate the correlation between the PM transport proteins and adaptation of plants to high salinity. How alterations in these transport systems of the PM allow plants to cope with the salt stress was also addressed. Although inconsistencies exist in some of the information related to the responses of the PM transport proteins to salinity in different species/cultivars, their key roles in adaptation of plants to high salinity is obvious and evident, and cannot be precluded. Despite the promising results, detailed investigations at the cellular/molecular level are needed in some issues of the PM transport systems in response to salinity to further evaluate their implication in salt tolerance.

  4. Golgi and vacuolar membrane proteins reach the vacuole in vps1 mutant yeast cells via the plasma membrane.

    PubMed

    Nothwehr, S F; Conibear, E; Stevens, T H

    1995-04-01

    The Vps1 protein of Saccharomyces cerevisiae is an 80-kD GTPase associated with the Golgi apparatus. Vps1p appears to play a direct role in the retention of late Golgi membrane proteins, which are mislocalized to the vacuolar membrane in its absence. The pathway by which late Golgi and vacuolar membrane proteins reach the vacuole in vps1 delta mutants was investigated by analyzing transport of these proteins in vps1 delta cells that also contained temperature sensitive mutations in either the SEC4 or END4 genes, which are required for a late step in secretion and the internalization step of endocytosis, respectively. Not only was vacuolar transport of a Golgi membrane protein blocked in the vps1 delta sec4-ts and vps1 delta end4-ts double mutant cells at the non-permissive temperature but vacuolar delivery of the vacuolar membrane protein, alkaline phosphatase was also blocked in these cells. Moreover, both proteins expressed in the vps1 delta end4-ts cells at the elevated temperature could be detected on the plasma membrane by a protease digestion assay indicating that these proteins are transported to the vacuole via the plasma membrane in vps1 mutant cells. These data strongly suggest that a loss of Vps1p function causes all membrane traffic departing from the late Golgi normally destined for the prevacuolar compartment to instead be diverted to the plasma membrane. We propose a model in which Vps1p is required for formation of vesicles from the late Golgi apparatus that carry vacuolar and Golgi membrane proteins bound for the prevacuolar compartment.

  5. Transmembrane Signal Transduction in Oocyte Maturation and Fertilization: Focusing on Xenopus laevis as a Model Animal

    PubMed Central

    Sato, Ken-ichi

    2014-01-01

    Fertilization is a cell biological phenomenon of crucial importance for the birth of new life in a variety of multicellular and sexual reproduction species such as algae, animal and plants. Fertilization involves a sequence of events, in which the female gamete “egg” and the male gamete “spermatozoon (sperm)” develop, acquire their functions, meet and fuse with each other, to initiate embryonic and zygotic development. Here, it will be briefly reviewed how oocyte cytoplasmic components are orchestrated to undergo hormone-induced oocyte maturation and sperm-induced activation of development. I then review how sperm-egg membrane interaction/fusion and activation of development in the fertilized egg are accomplished and regulated through egg coat- or egg plasma membrane-associated components, highlighting recent findings and future directions in the studies using Xenopus laevis as a model experimental animal. PMID:25546390

  6. Calcium influx factor (CIF) as a diffusible messenger for the activation of capacitative calcium entry in Xenopus oocytes.

    PubMed

    Kim, H Y; Hanley, M R

    1999-06-30

    Acid extracts of thapsigargin-treated Xenopus oocytes revealed Ca2(+)-dependent Cl- currents by microinjection into Xenopus oocytes. These currents were detected in highly purified fractions by carrying out a sequence of purification steps including gel filtration chromatography and high performance thin layer chromatography. The nature of the membrane currents evoked by the highly purified fractions were carried by chloride ions as blockade by the selective chloride channel blocker 1 mM niflumic acid. Injection of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) eradicated the current activities, indicating that the current responses are completely Ca2(+)-dependent. Moreover, the currents were sensitive to the removal of extracellular calcium, indicating the dependence on calcium entry through plasma membrane calcium entry channels. These results elucidate that the highly purified fractions aquired by thapsigargin-stimulated oocytes is an authentic calcium influx factor (CIF). Thus, the detection of increased CIF production from thapsigargin treatment in Xenopus oocytes would give strong support for the existence of CIF as a diffusible messenger for the activation of capacitative calcium entry pathways in Xenopus oocytes.

  7. 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.

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

    PubMed

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

    2014-03-01

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

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

    PubMed

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

    2014-03-01

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

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

    PubMed Central

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

    2014-01-01

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

  11. Role of G-protein-coupled estrogen receptor (GPER/GPR30) in maintenance of meiotic arrest in fish oocytes.

    PubMed

    Thomas, Peter

    2017-03-01

    An essential role for GPER (formerly known as GPR30) in regulating mammalian reproduction has not been identified to date, although it has shown to be involved in the regulation a broad range of other estrogen-dependent functions. In contrast, an important reproductive role for GPER in the maintenance of oocyte meiotic arrest has been identified in teleost fishes, which is briefly reviewed here. Recent studies have clearly shown that ovarian follicle production of estradiol-17β (E2) maintains meiotic arrest in several teleost species through activation of GPER coupled to a stimulatory G protein (Gs) on oocyte plasma membranes resulting in stimulation of cAMP production and maintenance of elevated cAMP levels. Studies with denuded zebrafish oocytes and with microinjection of GPER antisense oligonucleotides into oocytes have demonstrated the requirement for both ovarian follicle production of estrogens and expression of GPER on the oocyte surface for maintenance of meiotic arrest. This inhibitory action of E2 on the resumption of meiosis is mimicked by the GPER-selective agonist G-1, by the GPER agonists and nuclear ER antagonists, ICI 182,780 and tamoxifen, and also by the xenoestrogen bisphenol-A (BPA) and related alkylphenols. GPER also maintains meiotic arrest of zebrafish oocytes through estrogen- and BPA-dependent GPER activation of epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase (MAPK) signaling. Interestingly, progesterone receptor component 1 (PGRMC1) is also involved in estrogen maintenance of meiotic arrest through regulation of EGFR expression on the oocyte plasma membrane. The preovulatory surge in LH secretion induces the ovarian synthesis of progestin hormones that activate a membrane progestin receptor alpha (mPRα)/inhibitory G protein (Gi) pathway. It also increases ovarian synthesis of the catecholestrogen, 2-hydroxy-estradiol-17β (2-OHE2) which inhibits the GPER/Gs/adenylyl cyclase pathway. Both of these LH actions

  12. 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.

  13. Protein Kinases in Zucchini (Characterization of Calcium-Requiring Plasma Membrane Kinases).

    PubMed Central

    Verhey, S. D.; Gaiser, J. C.; Lomax, T. L.

    1993-01-01

    Using an in situ phosphorylation assay with zucchini (Cucurbita pepo L. cv Dark Green) seedling tissue, we have identified numerous polypeptides that are capable of acting as protein kinases. Total protein preparations from different organs contain different kinase profiles, but all are within the range of 55 to 70 kD. At least four kinases are associated with highly purified plasma membranes from etiolated zucchini hypocotyls. The major phosphorylated polypeptides from plasma membranes range in apparent molecular mass from 58 to 68 kD. The plasma membrane kinases are activated by micromolar concentrations of calcium and phosphorylate serine, and, to a lesser extent, threonine residues. These characteristics are similar to those of a soluble calcium-dependent protein kinase that has been purified to homogeneity from soybean suspension cultures. Three of the zucchini plasma membrane kinases share antigenic epitopes with the soluble soybean kinase. The presence of kinase activity at different apparent molecular masses may be indicative of separate kinases with similar characteristics. The zucchini hypocotyl protein kinases are not removed from plasma membrane vesicles by 0.5 M NaCl/5 mM ethylenediaminetetraacetate or by detergent concentrations below the critical micelle concentration of two types of detergent. This indicates that the plasma membrane protein kinases are tightly associated with the membrane in zucchini seedlings. PMID:12231949

  14. A theoretical formalism for aggregation of peroxidized lipids and plasma membrane stability during photolysis.

    PubMed Central

    Busch, N A; Yarmush, M L; Toner, M

    1998-01-01

    The objective of this investigation was to examine, from a theoretical perspective, the mechanism underlying the lysis of plasma membranes by photoinduced, chemically mediated damage such as is found in photolysis. Toward this end, a model is presented which relates the membrane lifetime to the thermodynamic parameters of the membrane components based upon the kinetic theory of aggregate formation. The formalism includes a standard birth/death process for the formation of damaged membrane components (i.e., peroxidized lipids) as well as a terminating condensation process for the formation of aggregates of peroxidized plasma membrane lipids. Our theory predicts that 1) the membrane lifetime is inversely correlated with predicted rate of membrane damage; 2) an upper limit on the duration of membrane damage exists, above which the mean and variance of the membrane lifetime is independent of further membrane damage; and 3) both the mean and variance of the time of membrane lifetime distribution are correlated with the number of sites that may be damaged to form a single membrane defect. The model provides a framework to optimize the lysis of cell membranes by photodynamic therapy. PMID:9826616

  15. Ultrastructural Alteration of Plant Plasma Membranes Induced by Auxin and Calcium Ions 1

    PubMed Central

    Morré, D. James; Bracker, Charles E.

    1976-01-01

    Ultrastructural changes in isolated and in situ plasma membranes of etiolated soybean hypocotyls (Glycine max L. cv. Wayne) were induced by indole-3-acetic acid (IAA), other auxins, and calcium chloride. Fixed and embedded preparations were stained by a phosphotungstate-chromate procedure to identify and accentuate plasma membrane. Measurements were on micrographs obtained with an electron optical system calibrated and corrected for reproducible and accurate size measurements. Plasma membranes treated for 20 minutes with 1 μm IAA were 10 to 15% thinner than controls. The response to IAA was rapid, reproducible, auxin-specific, temperature-dependent, and reversible. Comparable responses were obtained with isolated and in situ membranes. Membranes treated with 0.5 m calcium chloride for 20 minutes were 15 to 20% thicker than controls. Multiple cycles of alternating calcium and IAA treatments yielded membranes with dimensions that reflected the last treatment of the series. The findings show a direct response of plasma membranes to growth regulating agents and provide evidence for a cell-free response of isolated plasma membranes to a hormone. Images PMID:16659714

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

    PubMed

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

    2008-08-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2017-01-01

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

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

    PubMed

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

    2015-02-01

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

  19. 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

  20. The C-terminal Cytosolic Region of Rim21 Senses Alterations in Plasma Membrane Lipid Composition: INSIGHTS INTO SENSING MECHANISMS FOR PLASMA MEMBRANE LIPID ASYMMETRY.

    PubMed

    Nishino, Kanako; Obara, Keisuke; Kihara, Akio

    2015-12-25

    Yeast responds to alterations in plasma membrane lipid asymmetry and external alkalization via the sensor protein Rim21 in the Rim101 pathway. However, the sensing mechanism used by Rim21 remains unclear. Here, we found that the C-terminal cytosolic domain of Rim21 (Rim21C) fused with GFP was associated with the plasma membrane under normal conditions but dissociated upon alterations in lipid asymmetry or external alkalization. This indicates that Rim21C contains a sensor motif. Rim21C contains multiple clusters of charged residues. Among them, three consecutive Glu residues (EEE motif) were essential for Rim21 function and dissociation of Rim21C from the plasma membrane in response to changes in lipid asymmetry. In contrast, positively charged residues adjacent to the EEE motif were required for Rim21C to associate with the membrane. We therefore propose an "antenna hypothesis," in which Rim21C moves to or from the plasma membrane and functions as the sensing mechanism of Rim21.

  1. 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.

  2. 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

  3. 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

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

    DOE PAGES

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

    2013-04-22

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

  5. Laser induced wounding of the plasma membrane and methods to study the repair process.

    PubMed

    Jimenez, Ana J; Maiuri, Paolo; Lafaurie-Janvore, Julie; Perez, Franck; Piel, Matthieu

    2015-01-01

    Cells are constantly exposed to agents that can trigger the perforation of their plasma membrane. This damage occurs naturally, and the frequency and intensity depends on how much cells are exposed to damaging threats. The following protocol is a simple and powerful method to damage the plasma membrane using laser ablation. It allows the induction of a single and localized wound at the plasma membrane of cultured cells, which can be followed with fast time-lapse imaging. The first part of the protocol describes simple cell culture techniques and the material ideal to make the experiments. A second part of the protocol gives advice about the procedures to make effective wounds in cells while ensuring a good survival rate. We also propose different ways to follow the opening and closure of the plasma membrane. Finally, we describe the procedure to efficiently analyze the data acquired after single cell photodamage to characterize the wounding process.

  6. 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.

  7. Dopamine transporter: expression in Xenopus oocytes.

    PubMed

    Uhl, G R; O'Hara, B; Shimada, S; Zaczek, R; DiGiorgianni, J; Nishimori, T

    1991-01-01

    Xenopus oocytes can express biologically relevant transport activity after injection of mRNAs encoding several carrier molecules. mRNA from PC12 cells, as well as transcripts from a rat ventral midbrain library, can be expressed in these oocytes and allow them to display pharmacologically specific dopamine uptake. mRNA-injected oocytes incubated with tritiated dopamine contain tritiated dopamine and metabolites; lower amounts of radiolabeled dopamine and more radiolabeled metabolites are found in oocytes co-incubated with cocaine or in water-injected oocytes. Tritiated dopamine uptake into mRNA-injected oocytes is time, sodium, and temperature dependent. It is blocked by cocaine and mazindol, but not by haloperidol. It is not found after injection of mRNA from other brain regions. A size-selected rat midbrain library constructed in the plasma vector pCDM8 yields mRNA transcripts whose injection into oocytes causes cocaine-blockable [3H]dopamine uptake. These findings provide an assay for purification of the dopamine transporter cDNA by sib selection techniques.

  8. Evidence for annexin A6-dependent plasma membrane remodelling of lipid domains

    PubMed Central

    Alvarez-Guaita, Anna; Vilà de Muga, Sandra; Owen, Dylan M; Williamson, David; Magenau, Astrid; García-Melero, Ana; Reverter, Meritxell; Hoque, Monira; Cairns, Rose; Cornely, Rhea; Tebar, Francesc; Grewal, Thomas; Gaus, Katharina; Ayala-Sanmartín, Jesús; Enrich, Carlos; Rentero, Carles

    2015-01-01

    Background and Purpose Annexin A6 (AnxA6) is a calcium-dependent phospholipid-binding protein that can be recruited to the plasma membrane to function as a scaffolding protein to regulate signal complex formation, endo- and exocytic pathways as well as distribution of cellular cholesterol. Here, we have investigated how AnxA6 influences the membrane order. Experimental Approach We used Laurdan and di-4-ANEPPDHQ staining in (i) artificial membranes; (ii) live cells to investigate membrane packing and ordered lipid phases; and (iii) a super-resolution imaging (photoactivated localization microscopy, PALM) and Ripley's K second-order point pattern analysis approach to assess how AnxA6 regulates plasma membrane order domains and protein clustering. Key Results In artificial membranes, purified AnxA6 induced a global increase in membrane order. However, confocal microscopy using di-4-ANEPPDHQ in live cells showed that cells expressing AnxA6, which reduces plasma membrane cholesterol levels and modifies the actin cytoskeleton meshwork, displayed a decrease in membrane order (∼15 and 30% in A431 and MEF cells respectively). PALM data from Lck10 and Src15 membrane raft/non-raft markers revealed that AnxA6 expression induced clustering of both raft and non-raft markers. Altered clustering of Lck10 and Src15 in cells expressing AnxA6 was also observed after cholesterol extraction with methyl-β-cyclodextrin or actin cytoskeleton disruption with latrunculin B. Conclusions and Implications AnxA6-induced plasma membrane remodelling indicated that elevated AnxA6 expression decreased membrane order through the regulation of cellular cholesterol homeostasis and the actin cytoskeleton. This study provides the first evidence from live cells that support current models of annexins as membrane organizers. PMID:25409976

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

    PubMed

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

    2017-02-17

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

  10. Determinants of plasma membrane wounding by deforming stress.

    PubMed

    Oeckler, Richard A; Lee, Won-Yeon; Park, Mun-Gi; Kofler, Othmar; Rasmussen, Deborah L; Lee, Heung-Bum; Belete, Hewan; Walters, Bruce J; Stroetz, Randolph W; Hubmayr, Rolf D

    2010-12-01

    Once excess liquid gains access to air spaces of an injured lung, the act of breathing creates and destroys foam and thereby contributes to the wounding of epithelial cells by interfacial stress. Since cells are not elastic continua, but rather complex network structures composed of solid as well as liquid elements, we hypothesize that plasma membrane (PM) wounding is preceded by a phase separation, which results in blebbing. We postulate that interventions such as a hypertonic treatment increase adhesive PM-cytoskeletal (CSK) interactions, thereby preventing blebbing as well as PM wounds. We formed PM tethers in alveolar epithelial cells and fibroblasts and measured their retractive force as readout of PM-CSK adhesive interactions using optical tweezers. A 50-mOsm increase in media osmolarity consistently increased the tether retractive force in epithelial cells but lowered it in fibroblasts. The osmo-response was abolished by pretreatment with latrunculin, cytochalasin D, and calcium chelation. Epithelial cells and fibroblasts were exposed to interfacial stress in a microchannel, and the fraction of wounded cells were measured. Interventions that increased PM-CSK adhesive interactions prevented blebbing and were cytoprotective regardless of cell type. Finally, we exposed ex vivo perfused rat lungs to injurious mechanical ventilation and showed that hypertonic conditioning reduced the number of wounded subpleural alveolus resident cells to baseline levels. Our observations support the hypothesis that PM-CSK adhesive interactions are important determinants of the cellular response to deforming stress and pave the way for preclinical efficacy trials of hypertonic treatment in experimental models of acute lung injury.

  11. Pulmonary lipid phosphate phosphohydrolase in plasma membrane signalling platforms.

    PubMed Central

    Nanjundan, M; Possmayer, F

    2001-01-01

    Lipid phosphate phosphohydrolase (LPP) has recently been proposed to have roles in signal transduction, acting sequentially to phospholipase D (PLD) and in attenuating the effects of phospholipid growth factors on cellular proliferation. In this study, LPP activity is reported to be enriched in lipid-rich signalling platforms isolated from rat lung tissue, isolated rat type II cells and type II cell-mouse lung epithelial cell lines (MLE12 and MLE15). Lung and cell line caveolin-enriched domains (CEDs), prepared on the basis of their detergent-insolubility in Triton X-100, contain caveolin-1 and protein kinase C isoforms. The LPP3 isoform was predominantly localized to rat lung CEDs. These lipid-rich domains, including those from isolated rat type II cells, were enriched both in phosphatidylcholine plus sphingomyelin (PC+SM) and cholesterol. Saponin treatment of MLE15 cells shifted the LPP activity, cholesterol, PC+SM and caveolin-1 from lipid microdomains to detergent-soluble fractions. Elevated LPP activity and LPP1/1a protein are present in caveolae from MLE15 cells prepared using the cationic-colloidal-silica method. In contrast, total plasma membranes had a higher abundance of LPP1/1a protein with low LPP activity. Phorbol ester treatment caused a 3.8-fold increase in LPP specific activity in MLE12 CEDs. Thus the activated form of LPP1/1a may be recruited into caveolae/rafts. Transdifferentiation of type II cells into a type I-like cell demonstrated enrichment in caveolin-1 levels and LPP activity. These results indicate that LPP is localized in caveolae and/or rafts in lung tissue, isolated type II cells and type II cell lines and is consistent with a role for LPP in both caveolae/raft signalling and caveolar dynamics. PMID:11535125

  12. The lateral organization of influenza virus proteins in the budozone region of the plasma membrane.

    PubMed

    Leser, George P; Lamb, Robert A

    2017-02-15

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

  13. 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.

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

    PubMed

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

    2015-12-04

    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.

  15. LPS-Induced Macrophage Activation and Plasma Membrane Fluidity Changes are Inhibited Under Oxidative Stress.

    PubMed

    de la Haba, Carlos; Morros, Antoni; Martínez, Paz; Palacio, José R

    2016-12-01

    Macrophage activation is essential for a correct and efficient response of innate immunity. During oxidative stress membrane receptors and/or membrane lipid dynamics can be altered, leading to dysfunctional cell responses. Our aim is to analyze membrane fluidity modifications and cell function under oxidative stress in LPS-activated macrophages. Membrane fluidity of individual living THP-1 macrophages was evaluated by the technique two-photon microscopy. LPS-activated macrophage function was determined by TNFα secretion. It was shown that LPS activation causes fluidification of macrophage plasma membrane and production of TNFα. However, oxidative stress induces rigidification of macrophage plasma membrane and inhibition of cell activation, which is evidenced by a decrease of TNFα secretion. Thus, under oxidative conditions macrophage proinflammatory response might develop in an inefficient manner.

  16. T-Type Ca2+ Current Activity during Oocyte Growth and Maturation in the Ascidian Styela plicata

    PubMed Central

    Gallo, Alessandra; Russo, Gian Luigi; Tosti, Elisabetta

    2013-01-01

    Voltage-dependent calcium currents play a fundamental role during oocyte maturation, mostly L-type calcium currents, whereas T-type calcium currents are involved in sperm physiology and cell growth. In this paper, using an electrophysiological and pharmacological approach, we demonstrated, for the first time in oocytes, that T-type calcium currents are present with functional consequences on the plasma membrane of growing immature oocytes of the ascidian Styela plicata. We classified three subtypes of immature oocytes at the germinal vesicle stage on the basis of their size, morphology and accessory cellular structures. These stages were clearly associated with an increased activity of T-type calcium currents and hyperpolarization of the plasma membrane. We also observed that T-type calcium currents oscillate in the post-fertilization embryonic stages, with minimal amplitude of the currents in the zygote and maximal at 8-cell stage. In addition, chemical inhibition of T-type calcium currents, obtained by applying specific antagonists, induced a significant reduction in the rate of cleavage and absence of larval formation. We suggest that calcium entry via T-type calcium channels may act as a potential pacemaker in regulating cytosolic calcium involved in fertilization and early developmental events. PMID:23349937

  17. 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.

  18. [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.

  19. 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

  20. Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase1

    PubMed Central

    Olivari, Claudio; Meanti, Cristina; De Michelis, Maria Ida; Rasi-Caldogno, Franca

    1998-01-01

    Different approaches were utilized to investigate the mechanism by which fusicoccin (FC) induces the activation of the H+-ATPase in plasma membrane (PM) isolated from radish (Raphanus sativus L.) seedlings treated in vivo with (FC-PM) or without (C-PM) FC. Treatment of FC-PM with different detergents indicated that PM H+-ATPase and the FC-FC-binding-protein (FCBP) complex were solubilized to a similar extent. Fractionation of solubilized FC-PM proteins by a linear sucrose-density gradient showed that the two proteins comigrated and that PM H+-ATPase retained the activated state induced by FC. Solubilized PM proteins were also fractionated by a fast-protein liquid chromatography anion-exchange column. Comparison between C-PM and FC-PM indicated that in vivo treatment of the seedlings with FC caused different elution profiles; PM H+-ATPase from FC-PM was only partially separated from the FC-FCBP complex and eluted at a higher NaCl concentration than did PM H+-ATPase from C-PM. Western analysis of fast-protein liquid chromatography fractions probed with an anti-N terminus PM H+-ATPase antiserum and with an anti-14–3-3 antiserum indicated an FC-induced association of FCBP with the PM H+-ATPase. Analysis of the activation state of PM H+-ATPase in fractions in which the enzyme was partially separated from FCBP suggested that the establishment of an association between the two proteins was necessary to maintain the FC-induced activation of the enzyme. PMID:9489010

  1. The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains Segregated Bioenergetic Domains[C][W

    PubMed Central

    Rexroth, Sascha; Mullineaux, Conrad W.; Ellinger, Dorothea; Sendtko, Esther; Rögner, Matthias; Koenig, Friederike

    2011-01-01

    The light reactions of oxygenic photosynthesis almost invariably take place in the thylakoid membranes, a highly specialized internal membrane system located in the stroma of chloroplasts and the cytoplasm of cyanobacteria. The only known exception is the primordial cyanobacterium Gloeobacter violaceus, which evolved before the appearance of thylakoids and harbors the photosynthetic complexes in the plasma membrane. Thus, studies on G. violaceus not only shed light on the evolutionary origin and the functional advantages of thylakoid membranes but also might include insights regarding thylakoid formation during chloroplast differentiation. Based on biochemical isolation and direct in vivo characterization, we report here structural and functional domains in the cytoplasmic membrane of a cyanobacterium. Although G. violaceus has no internal membranes, it does have localized domains with apparently specialized functions in its plasma membrane, in which both the photosynthetic and the respiratory complexes are concentrated. These bioenergetic domains can be visualized by confocal microscopy, and they can be isolated by a simple procedure. Proteomic analysis of these domains indicates their physiological function and suggests a protein sorting mechanism via interaction with membrane-intrinsic terpenoids. Based on these results, we propose specialized domains in the plasma membrane as evolutionary precursors of thylakoids. PMID:21642550

  2. Activation of the progesterone signaling pathway by methyl-β-cyclodextrin or steroid in Xenopus laevis oocytes involves release of 45-kDa Gαs

    PubMed Central

    Sadler, Susan E.; Archer, Mallory R.; Spellman, Kirsten M.

    2008-01-01

    Treatment of Xenopus laevis oocytes with cholesterol-depleting methyl-β-cyclodextrin (MeβCD) stimulates phosphorylation of mitogen-activated protein kinase (MAPK) and oocyte maturation, as reported previously (Sadler and Jacobs, 2004). Here we report that treatment of oocytes with MeβCD increased levels of immunodetectable 39-kDa mos protein. The protein synthesis inhibitor, cycloheximide, blocked the appearance of Mos, blocked MeβCD-stimulated phosphorylation of MAPK, and inhibited MeβCD-induced oocyte maturation. These observations suggest that MeβCD activates the progesterone signaling pathway. Chemical inhibition of steroid synthesis and mechanical removal of follicle cells were used to verify that MeβCD acts at the level of the oocyte and does not require production of steroid by surrounding follicle cells. Cortical Gαs is contained in low-density membrane; and treatment of oocytes with progesterone or MeβCD reduced immunodetectable levels of Gαs protein in cortices and increased internal levels of 45-kDa Gαs in cortical-free extracts. Dose-dependent increases in internal Gαs after treatment of oocytes with progesterone correlated with the steroid-induced maturation response, and the increase in internal Gαs after hormone treatment was comparable to the decrease in cortical Gαs. These results are consistent with a model in which release of Gαs from the plasma membrane is involved in activation of the progesterone signaling pathway that leads to amphibian oocyte maturation. PMID:18706402

  3. 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.

  4. Latrunculin A depolarizes starfish oocytes.

    PubMed

    Moccia, F

    2007-12-01

    Depolymerization of the actin cytoskeleton may liberate Ca2+ from InsP3-sensitive stores in some cell types, including starfish oocytes, while inhibiting Ca2+ influx in others. However, no information is available on the modulation of membrane potential (V(m)) by actin. The present study was aimed to ascertain whether the widely employed actin depolymerizing drug, latrunculin A (Lat A), affects V(m) in mature oocytes of the starfish Astropecten aranciacus. Lat A induced a membrane depolarization which was mimicked by cytochalasin D, another popular actin disruptor, and prevented by jasplakinolide, a stabilizer of the actin network. Lat A-elicited depolarization consisted in a positive shift in V(m) which reached the threshold of activation of voltage-gated Ca2+ channels (VGCC), thus triggering an action potential. Lat A-promoted depolarization lacked the action potential in Ca2+-free sea water, while it was abolished upon removal of external Na+. Moreover, membrane depolarization was prevented by pre-injection of BAPTA and heparin, but not ryanodine. These data indicate that Lat A induces a membrane depolarization by releasing Ca2+ from InsP3Rs. The Ca2+ signal in turn activates a Ca2+-dependent Na+ entry, which causes the positive shift in V(m) and stimulates the VGCC.

  5. Plasma membrane redox system in the control of stress-induced apoptosis.

    PubMed

    Villalba, J M; Navas, P

    2000-01-01

    The plasma membrane of animal cells contains an electron transport system based on coenzyme Q (CoQ) reductases. Cytochrome b5 reductase is NADH-specific and reduces CoQ through a one-electron reaction mechanism. DT-diaphorase also reduces CoQ, although through a two-electron reaction mechanism using both NADH and NADPH, which may be particularly important under oxidative stress conditions. Because reduced CoQ protects membranes against peroxidations, and also maintains the reduced forms of exogenous antioxidants such as alpha-tocopherol and ascorbate, this molecule can be considered a central component of the plasma membrane antioxidant system. Stress-induced apoptosis is mediated by the activation of plasma membrane-bound neutral sphingomyelinase, which releases ceramide to the cytosol. Ceramide-dependent caspase activation is part of the apoptosis pathway. The reduced components of the plasma membrane antioxidant system, mainly CoQ, prevent both lipid peroxidation and sphingomyelinase activation. This results in the prevention of ceramide accumulation and caspase 3 activation and, as consequence, apoptosis is inhibited. We propose the hypothesis that antioxidant protective function of the plasma membrane redox system can be enough to protect cells against the externally induced mild oxidative stress. If this system is overwhelmed, intracellular mechanisms of protection are required to avoid activation of the apoptosis pathway.

  6. Molecular characterization of a cold-induced plasma membrane protein gene from wheat.

    PubMed

    Koike, Michiya; Sutoh, Keita; Kawakami, Akira; Torada, Atsushi; Oono, Kiyoharu; Imai, Ryozo

    2005-12-01

    As a means to study the function of plasma membrane proteins during cold acclimation, we have isolated a cDNA clone for wpi6 which encodes a putative plasma membrane protein from cold-acclimated winter wheat. The wpi6 gene encodes a putative 5.9 kDa polypeptide with two predicted membrane-spanning domains, the sequence of which shows high sequence similarity with BLT101-family proteins from plants and yeast. Strong induction of wpi6 mRNA was observed during an early stage of cold acclimation in root and shoot tissues of both winter and spring wheat cultivars. In contrast to blt101 in barley, wpi6 mRNA was also induced by drought and salinity stresses, and exogenous application of ABA. Expression of wpi6 in a Deltapmp3 mutant of Saccharomyces cerevisiae, which is disturbed in plasma membrane potential due to the lack of a BLT101-family protein, partially complemented NaCl sensitivity of the mutant. Transient expression analysis of a WPI6::GFP fusion protein in onion epidermal cells revealed that WPI6 is localized in the plasma membrane. Taken together, these data suggested that WPI6 may have a protective role in maintaining plasma membrane function during cold acclimation in wheat.

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

    PubMed

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

    2017-03-01

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

  8. Changes in lipid composition of hepatocyte plasma membrane induced by overfeeding in duck.

    PubMed

    Molee, W; Bouillier-Oudot, M; Auvergne, A; Babilé, R

    2005-08-01

    This experiment was carried out to examine the influence of overfeeding ducks with corn on the lipid composition of hepatocyte plasma membrane. Seventy-day-old male Mule ducks (Cairina moschata x Anas platyrhynchos) were overfed with corn for 12.5 days in order to induce fatty livers. The cholesterol and phospholipid contents were approximately 50% higher in hepatocyte plasma membranes from fatty livers compared to those of lean livers obtained from non-overfed ducks. However, the cholesterol/phospholipids molar ratio did not differ between both groups. Overfeeding induced a significant change in phospholipid composition of hepatocyte plasma membrane with a decrease in phosphatidylcholine proportion and conversely an increase in phosphatidylethanolamine. The fatty acid profile of phospholipids was also altered. In fatty hepatocyte plasma membrane, the overall proportion of polyunsaturated fatty acids (PUFA) was decreased and this was due to the decrease of some of, but not all, the PUFA. In addition, the proportions of oleic acid and n-9 series unsaturated fatty acids were higher in fatty than in lean liver membranes. This study provides evidence that overfeeding with a carbohydrate-rich corn-based diet induces a de novo hepatic lipogenesis in Mule duck which predominates over dietary lipid intake to change the lipid composition of the hepatocyte plasma membrane.

  9. Isolation and characterization of plasma membranes from wild type Neurospora crassa.

    PubMed

    Bowman, E J; Bowman, B J; Slayman, C W

    1981-12-10

    A method has been developed to isolate plasma membranes with high ATPase activity from wild type Neurospora. Cells are treated with snail enzyme to weaken their cell walls, disrupted by gentle homogenization in a medium designed to keep mitochondria and other organelles intact, and fractionated by differential centrifugation. After removal of mitochondria, several higher speed particulate fractions (particularly one sedimenting at 40,000 X g) contain an ATPase that can be identified as the plasma membrane enzyme on the basis of sensitivity to vanadate and kinetic properties. Its [S]0.5 for Mg.ATP, specificity for nucleotides and divalent cations, and pH optimum are virtually identical with those reported previously for plasma membrane ATPase from the slime mutant of Neurospora (Bowman, B. J., and Slayman, C. W. (1977) J. Biol. Chem. 252, 3357-3363). By contrast, ATPase specific activities in the wild type plasma membranes are much higher than in slime, ranging up to 7.3 mumol/min/mg of protein (the highest value yet reported for Neurospora). The best preparations appear homogeneous upon sucrose density gradient centrifugation, and band at an equilibrium density of 1.15 g/cm3. Two other markers, chitin synthetase and [acetyl-3H] concanavalin A binding, show approximate co-purification with the plasma membrane ATPase through membrane fractionation and sucrose gradient centrifugation.

  10. Removal of seminal plasma enhances membrane stability on fresh and cooled stallion spermatozoa.

    PubMed

    Barrier-Battut, I; Bonnet, C; Giraudo, A; Dubois, C; Caillaud, M; Vidament, M

    2013-02-01

    Fertility is reduced after semen cooling for a considerable number of stallions. The main hypotheses include alterations in plasma membrane following cooling and deleterious influence of seminal plasma. However, interindividual variability is controversial. We hypothesized that the removal of seminal plasma could enhance motility in some 'poor cooler' stallions, but could also affect, negatively or positively, membrane quality in some stallions. This study examined the effect of centrifugation, followed or not by removal of seminal plasma, on parameters indicating semen quality after 48 h at 4 °C: motility, plasma membrane integrity as evaluated by hypo-osmotic swelling test, acrosome integrity and response to a pharmacological induction of acrosome reaction using ionophore A23187. Sixty-six ejaculates from 14 stallions were used, including stallions showing high or low sperm motility after cooled storage. Centrifugation without removal of seminal plasma did not affect sperm parameters. Removal of seminal plasma did not affect motility, but significantly stabilized sperm membranes, as demonstrated by a higher response to the osmotic challenge, and a reduced reactivity of the acrosome. Moreover, for the same semen sample, the response to an induction of acrosome reaction was significantly higher when the induction was performed in the presence of seminal plasma, compared with the induction in the absence of seminal plasma. This was observed both for fresh and cooled semen. When the induction of acrosome reaction with ionophore A23187 is used to evaluate sperm quality, care must therefore be taken to standardize the proportion of seminal plasma between samples. For the 10 stallions serving at least 25 mares, the only variable significantly correlated with fertility was motility. The influence of membrane stabilization regarding fertility requires further investigations.

  11. Embryonal cell surface recognition. Extraction of an active plasma membrane component.

    PubMed

    Merrell, R; Gottlieb, D I; Glaser, L

    1975-07-25

    Plasma membranes obtained from different neural regions of the chicken embryo have previously been shown to specifically bind to homotypic cells and prevent cell aggregation (Merrell, R., and Glaser, L. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 2794-2798). Proteins responsible for the specific inhibition of cell aggregation have been solubilized from the plasma membrane of neural retina and optic tectum by delipidation with acetone followed by extraction with lithium diiodosalicylate. The extracts show the same regional and temporal specificity as previously shown for plasma membrane recognition by the same cells (Gottlieb, D. I., Merrell, R., and Glaser, L. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 1800-1802). Two micrograms of the most purified protein fraction inhibits the aggregation of 2.5 times 10(-4) cells under standard assay conditions. This represents a 20-fold increase in specific activity compared to whole membranes.

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

    PubMed Central

    Fujimoto, Toyoshi; Parmryd, Ingela

    2017-01-01

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

  13. A novel biotinylated lipid raft reporter for electron microscopic imaging of plasma membrane microdomains[S

    PubMed Central

    Krager, Kimberly J.; Sarkar, Mitul; Twait, Erik C.; Lill, Nancy L.; Koland, John G.

    2012-01-01

    The submicroscopic spatial organization of cell surface receptors and plasma membrane signaling molecules is readily characterized by electron microscopy (EM) via immunogold labeling of plasma membrane sheets. Although various signaling molecules have been seen to segregate within plasma membrane microdomains, the biochemical identity of these microdomains and the factors affecting their formation are largely unknown. Lipid rafts are envisioned as submicron membrane subdomains of liquid ordered structure with differing lipid and protein constituents that define their specific varieties. To facilitate EM investigation of inner leaflet lipid rafts and the localization of membrane proteins therein, a unique genetically encoded reporter with the dually acylated raft-targeting motif of the Lck kinase was developed. This reporter, designated Lck-BAP-GFP, incorporates green fluorescent protein (GFP) and biotin acceptor peptide (BAP) modules, with the latter allowing its single-step labeling with streptavidin-gold. Lck-BAP-GFP was metabolically biotinylated in mammalian cells, distributed into low-density detergent-resistant membrane fractions, and was readily detected with avidin-based reagents. In EM images of plasma membrane sheets, the streptavidin-gold-labeled reporter was clustered in 20–50 nm microdomains, presumably representative of inner leaflet lipid rafts. The utility of the reporter was demonstrated in an investigation of the potential lipid raft localization of the epidermal growth factor receptor. PMID:22822037

  14. A novel biotinylated lipid raft reporter for electron microscopic imaging of plasma membrane microdomains.

    PubMed

    Krager, Kimberly J; Sarkar, Mitul; Twait, Erik C; Lill, Nancy L; Koland, John G

    2012-10-01

    The submicroscopic spatial organization of cell surface receptors and plasma membrane signaling molecules is readily characterized by electron microscopy (EM) via immunogold labeling of plasma membrane sheets. Although various signaling molecules have been seen to segregate within plasma membrane microdomains, the biochemical identity of these microdomains and the factors affecting their formation are largely unknown. Lipid rafts are envisioned as submicron membrane subdomains of liquid ordered structure with differing lipid and protein constituents that define their specific varieties. To facilitate EM investigation of inner leaflet lipid rafts and the localization of membrane proteins therein, a unique genetically encoded reporter with the dually acylated raft-targeting motif of the Lck kinase was developed. This reporter, designated Lck-BAP-GFP, incorporates green fluorescent protein (GFP) and biotin acceptor peptide (BAP) modules, with the latter allowing its single-step labeling with streptavidin-gold. Lck-BAP-GFP was metabolically biotinylated in mammalian cells, distributed into low-density detergent-resistant membrane fractions, and was readily detected with avidin-based reagents. In EM images of plasma membrane sheets, the streptavidin-gold-labeled reporter was clustered in 20-50 nm microdomains, presumably representative of inner leaflet lipid rafts. The utility of the reporter was demonstrated in an investigation of the potential lipid raft localization of the epidermal growth factor receptor.

  15. Mass spectrometric approach for identifying putative plasma membrane proteins of Arabidopsis leaves associated with cold acclimation.

    PubMed

    Kawamura, Yukio; Uemura, Matsuo

    2003-10-01

    Although enhancement of freezing tolerance in plants during cold acclimation is closely associated with an increase in the cryostability of plasma membrane, the molecular mechanism for the increased cryostability of plasma membrane is still to be elucidated. In Arabidopsis, enhanced freezing tolerance was detectable after cold acclimation at 2 degrees C for as short as 1 day, and maximum freezing tolerance was attained after 1 week. To identify the plasma membrane proteins that change in quantity in response to cold acclimation, a highly purified plasma membrane fraction was isolated from leaves before and during cold acclimation, and the proteins in the fraction were separated with gel electrophoresis. We found that there were substantial changes in the protein profiles after as short as 1 day of cold acclimation. Subsequently, using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS), we identified 38 proteins that changed in quantity during cold acclimation. The proteins that changed in quantity during the first day of cold acclimation include those that are associated with membrane repair by membrane fusion, protection of the membrane against osmotic stress, enhancement of CO2 fixation, and proteolysis.

  16. Characterization of phospholipid composition and its control in the plasma membrane of developing soybean root

    SciTech Connect

    Whitman, C.E.

    1985-01-01

    The phospholipid composition of plasma membrane enriched fractions from developing soybean root and several mechanisms which may regulate it have been examined. Plasma membrane vesicles were isolated from meristematic and mature sections of four-day-old dark grown soybean roots (Glycine max (L.) Merr. Cult. Wells II). Analysis of lipid extracts revealed two major phospholipid classes: phosphatidylcholine and phosphatidylethanolamine. Minor phospholipid classes were phosphatidylinositol, phosphatidylserine, phosphatidylgylcerol and diphosphatidylgylcerol. Phospholipid composition was similar at each developmental stage. Fatty acids of phosphatidylcholine and phosphatidylethanolamine were 16:0, 18:0, 18:2, and 18:3. Fatty acid composition varied with both phospholipid class and the developmental stage of the root. The degradation of phosphatidylcholine by endogenous phospholipase D during membrane isolation indicated that this enzyme might be involved in phospholipid turnover within the membrane. Phospholipase D activity was heat labile and increasing the pH of the enzyme assay from 5.3 to 7.8 resulted in 90% inhibition of activity. The turnover of fatty acids within the phospholipids of the plasma membrane was studied. Mature root sections were incubated with (1-/sup 14/C) acetate, 1 mM Na acetate and 50 ..mu..g/ml chloramphenicol. Membrane lipid extracts analyzed for phospholipid class and acyl chain composition revealed that the long incubation times did not alter the phospholipid composition of the plasma membrane enriched fraction.

  17. 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.

  18. 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.

  19. High-throughput electrophysiology with Xenopus oocytes

    PubMed Central

    Papke, Roger L.; Smith-Maxwell, Cathy

    2010-01-01

    Voltage-clamp techniques are typically used to study the plasma membrane proteins, such as ion channels and transporters that control bioelectrical signals. Many of these proteins have been cloned and can now be studied as potential targets for drug development. The two approaches most commonly used for heterologous expression of cloned ion channels and transporters involve either transfection of the genes into small cells grown in tissue culture or the injection of the genetic material into larger cells. The standard large cells used for the expression of cloned cDNA or synthetic RNA are the egg progenitor cells (oocytes) of the African frog, Xenopus laevis. Until recently, cellular electrophysiology was performed manually, one cell at a time by a single operator. However, methods of high-throughput electrophysiology have been developed which are automated and permit data acquisition and analysis from multiple cells in parallel. These methods are breaking a bottleneck in drug discovery, useful in some cases for primary screening as well as for thorough characterization of new drugs. Increasing throughput of high-quality functional data greatly augments the efficiency of academic research and pharmaceutical drug development. Some examples of studies that benefit most from high-throughput electrophysiology include pharmaceutical screening of targeted compound libraries, secondary screening of identified compounds for subtype selectivity, screening mutants of ligand-gated channels for changes in receptor function, scanning mutagenesis of protein segments, and mutant-cycle analysis. We describe here the main features and potential applications of OpusXpress, an efficient commercially available system for automated recording from Xenopus oocytes. We show some types of data that have been gathered by this system and review realized and potential applications. PMID:19149490

  20. 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.

  1. Organization of dimethyl sulfoxide reductase in the plasma membrane of Escherichia coli.

    PubMed Central

    Sambasivarao, D; Scraba, D G; Trieber, C; Weiner, J H

    1990-01-01

    Dimethyl sulfoxide reductase is a trimeric, membrane-bound, iron-sulfur molybdoenzyme induced in Escherichia coli under anaerobic growth conditions. The enzyme catalyzes the reduction of dimethyl sulfoxide, trimethylamine N-oxide, and a variety of S- and N-oxide compounds. The topology of dimethyl sulfoxide reductase subunits was probed by a combination of techniques. Immunoblot analysis of the periplasmic proteins from the osmotic shock and chloroform wash fluids indicated that the subunits were not free in the periplasm. The reductase was susceptible to proteases in everted membrane vesicles, but the enzyme in outer membrane-permeabilized cells became protease sensitive only after detergent solubilization of the E. coli plasma membrane. Lactoperoxidase catalyzed the iodination of each of the three subunits in an everted membrane vesicle preparation. Antibodies to dimethyl sulfoxide reductase and fumarate reductase specifically agglutinated the everted membrane vesicles. No TnphoA fusions could be found in the dmsA or -B genes, indicating that these subunits were not translocated to the periplasm. Immunogold electron microscopy of everted membrane vesicles and thin sections by using antibodies to the DmsABC, DmsA, DmsB subunits resulted in specific labeling of the cytoplasmic surface of the inner membrane. These results show that the DmsA (catalytic subunit) and DmsB (electron transfer subunit) are membrane-extrinsic subunits facing the cytoplasmic side of the plasma membrane. Images PMID:2170332

  2. Oocyte cryopreservation and ovarian tissue banking.

    PubMed

    Ledda, S; Leoni, G; Bogliolo, L; Naitana, S

    2001-04-01

    Oocyte cryopreservation, despite its impact on conservation of genetic resources, is not yet an established technology. Several problems need to be solved before this technology can be applied regularly. Chilling membrane susceptibility and formation of ice due to the large volume of the cell are the major problems observed. However, during the last years, several studies were done to obtain viable oocytes after cryopreservation. The addition of molecules known to stabilize membranes and the creation of freezing systems with rapid cooling throughout the transition phase have yielded a good percentage of viable immature and mature oocytes More recently, storage of female gametes was achieved by cryopreservation of cortical ovarian tissue. The possibility of restoring fertility by transplantation of frozen ovarian tissue or its long-term culture in vitro represents an important future means of preserving the fertility of patients and of storing the gametes of rare animals.

  3. 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.

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

    PubMed Central

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

    2017-01-01

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

  5. Ultrastructural observation of oocytes in six types of stony corals.

    PubMed

    Tsai, Sujune; Chang, Wei-Chieh; Chavanich, Suchana; Viyakarn, Voranop; Lin, Chiahsin

    2016-08-01

    In this study, the ultrastructure of the oocytes of 6 types of scleractinian corals was observed by transmission electron microscopy (TEM). Moreover, histological and ultrastructural analyses were performed to improve our understanding of the organelles involved in coral oocyte formation. In all 6 stony coral species, the microvilli were tubular and directly grew from the surface of the oocyte membrane; yolk bodies, lipid granules, and cortical alveoli accounted for most of the volume inside the oocytes, suggesting that they are associated with energy storage and buoyancy. Clear differences were observed in the size of yolk bodies and lipid granules in the oocytes of the 6 stony coral species, which occupied approximately 55%-80% of the inner space of the oocytes. Galaxea fascicularis exhibited the largest lipid granule volume, but the oocytes contained only an average number of 12.45 lipid granules per unit area. Only Montipora incrassata oocytes contained symbiotic algae. The smallest size and proportion of lipid granules in M. incrassata oocytes may be attributed to the presence of symbiotic algae and large yolk bodies, which may help oocytes produce energy and function as a nutritional source. This study is crucial for improving the understanding of the basic biology of coral reproduction, and the ensuing datasets is critical for conservation-oriented studies seeking to cryopreserve corals during these times of dramatic global climate change.

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

    PubMed

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

    2008-04-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

    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.

  8. Num1 anchors mitochondria to the plasma membrane via two domains with different lipid binding specificities.

    PubMed

    Ping, Holly A; Kraft, Lauren M; Chen, WeiTing; Nilles, Amy E; Lackner, Laura L

    2016-06-06

    The mitochondria-ER cortex anchor (MECA) is required for proper mitochondrial distribution and functions by tethering mitochondria to the plasma membrane. The core component of MECA is the multidomain protein Num1, which assembles into clusters at the cell cortex. We show Num1 adopts an extended, polarized conformation. Its N-terminal coiled-coil domain (Num1CC) is proximal to mitochondria, and the C-terminal pleckstrin homology domain is associated with the plasma membrane. We find that Num1CC interacts directly with phospholipid membranes and displays a strong preference for the mitochondria-specific phospholipid cardiolipin. This direct membrane interaction is critical for MECA function. Thus, mitochondrial anchoring is mediated by a protein that interacts directly with two different membranes through lipid-specific binding domains, suggesting a general mechanism for interorganelle tethering.

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

    NASA Astrophysics Data System (ADS)

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

    2017-01-01

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

  10. Visualization of Src activity at different compartments of the plasma membrane by FRET imaging

    PubMed Central

    Seong, Jihye; Lu, Shaoying; Ouyang, Mingxing; Huang, He; Zhang, Jin; Frame, Margaret C.; Wang, Yingxiao

    2009-01-01

    Summary Membrane compartments function as segregated signaling platforms for different cellular functions. It is not clear how Src is regulated at different membrane compartments. To visualize local Src activity in live cells, a FRET-based Src biosensor was targeted in or outside of lipid rafts at the plasma membrane, via acylation or prenylation modifications on targeting tags either directly fused to the biosensor or coupled to the biosensor through an inducible heterodimerization system. In response to growth factors and pervanadate, the induction of Src activity in rafts was slower and weaker, dependent on actin and possibly its mediated transportation of Src from perinuclear regions to the plasma membrane. In contrast, the induction of Src activity in non-rafts was faster and stronger, dependent on microtubules. Hence, the Src activity is differentially regulated via cytoskeleton at different membrane compartments. PMID:19171305

  11. Num1 anchors mitochondria to the plasma membrane via two domains with different lipid binding specificities

    PubMed Central

    Ping, Holly A.; Kraft, Lauren M.; Chen, WeiTing; Nilles, Amy E.

    2016-01-01

    The mitochondria–ER cortex anchor (MECA) is required for proper mitochondrial distribution and functions by tethering mitochondria to the plasma membrane. The core component of MECA is the multidomain protein Num1, which assembles into clusters at the cell cortex. We show Num1 adopts an extended, polarized conformation. Its N-terminal coiled-coil domain (Num1CC) is proximal to mitochondria, and the C-terminal pleckstrin homology domain is associated with the plasma membrane. We find that Num1CC interacts directly with phospholipid membranes and displays a strong preference for the mitochondria-specific phospholipid cardiolipin. This direct membrane interaction is critical for MECA function. Thus, mitochondrial anchoring is mediated by a protein that interacts directly with two different membranes through lipid-specific binding domains, suggesting a general mechanism for interorganelle tethering. PMID:27241910

  12. Capillary high-performance liquid chromatography/mass spectrometric analysis of proteins from affinity-purified plasma membrane.

    PubMed

    Zhao, Yingxin; Zhang, Wei; White, Michael A; Zhao, Yingming

    2003-08-01

    Proteomics analysis of plasma membranes is a potentially powerful strategy for the discovery of proteins involved in membrane remodeling under diverse cellular environments and identification of disease-specific membrane markers. A key factor for successful analysis is the preparation of plasma membrane fractions with low contamination from subcellular organelles. Here we report the characterization of plasma membrane prepared by an affinity-purification method, which involves biotinylation of cell-surface proteins and subsequent affinity enrichment with strepavidin beads. Western blotting analysis showed this method was able to achieve a 1600-fold relative enrichment of plasma membrane versus mitochondria and a 400-fold relative enrichment versus endoplasmic reticulum, two major contaminants in plasma membrane fractions prepared by conventional ultracentrifugation methods. Capillary-HPLC/MS analysis of 30 microg of affinity-purified plasma membrane proteins led to the identification of 918 unique proteins, which include 16.4% integral plasma membrane proteins and 45.5% cytosol proteins (including 8.6% membrane-associated proteins). Notable among the identified membrane proteins include 30 members of ras superfamily, receptors (e.g., EGF receptor, integrins), and signaling molecules. The low number of endoplasmic reticulum and mitochondria proteins (approximately 3.3% of the total) suggests the plasma membrane preparation has minimum contamination from these organelles. Given the importance of integral membrane proteins for drug design and membrane-associated proteins in the regulation cellular behaviors, the described approach will help expedite the characterization of plasma membrane subproteomes, identify signaling molecules, and discover therapeutic membrane-protein targets in diseases.

  13. [Mitochondrial and oocyte development].

    PubMed

    Deng, Wei-Ping; Ren, Zhao-Rui

    2007-12-01

    Oocyte development and maturation is a complicated process. The nuclear maturation and cytoplasmic maturation must synchronize which can ensure normal oocyte fertilization and following development. Mitochondrial is the most important cellular organell in cytoplasm, and the variation of its distribution during oocyte maturation, the capacity of OXPHOS generating ATP as well as the content or copy number or transcription level of mitochondrial DNA play an important role in oocyte development and maturation. Therefore, the studies on the variation of mitochondrial distribution, function and mitochondrial DNA could enhance our understanding of the physiology of reproduction and provide new insight to solve the difficulties of assisted reproduction as well as cloning embryo technology.

  14. Endogenous glycosphingolipid acceptor specificity of sialosyltransferase systems in intact golgi membranes, synaptosomes, and synaptic plasma membranes from rat brain

    SciTech Connect

    Durrie, R.; Saito, M.; Rosenberg, A.

    1988-05-17

    Preparations highly enriched in Golgi complex membranes, synaptosomes, and synaptic plasma membranes (SPM) by marker enzyme analysis and electron microscopic morphology were made from the brains of 28-day-old rats. These were incubated with cytidine 5'-monophosphate-N-acetyl(/sup 14/C)neuraminic acid (CMP-NeuAc) in a physiologic buffer, without detergents. Glycolipid sialosyltransferase activities (SATs) were measured by analyzing incorporation of radiolabeled NeuAc into endogenous membrane gangliosides. Golgi SAT was diversified in producing all the various molecular species of labeled gangliosides. Synaptosomal SAT exhibited a lower activity, but it was highly specific in its labeling pattern, with a marked preference for labeling NeuAc..cap alpha..2 ..-->.. 8NeuAc..cap alpha..2 ..-->.. 3Gal..beta..1 ..-->.. 4Glc..beta..1 ..-->.. 1Cer (GD3 ganglioside). SPM prepared from the synaptosomes retained the GD3-related SAT (or SAT-2), and the total specific activity increased, which suggests that the location of the synaptosomal activity is in the SPM. These results indicate that SAT activity in Golgi membranes differs from that in synaptosomes with regard to endogenous acceptor substrate specificity and SAT activity of synaptosomes should be located in the synaptosomal plasma membrane. This SAT could function as an ectoenzyme in concert with ecto-sialidase to modulate the GD3 and other ganglioside population in situ at the SPM of the central nervous system.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-10-28

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

  17. 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

  18. Preparation of immuno-affinity membranes for cholesterol removal from human plasma.

    PubMed

    Denizli, Adil

    2002-06-05

    Anti-low density lipoprotein antibody (anti-LDL) immobilized polyhydroxyethylmethacrylate (pHEMA) based membrane was prepared for selective removal of cholesterol from hypercholesterolemic human plasma. In order to further increase blood-compatibility, a newly synthesized comonomer, methacryloylamidophenylalanine (MAPA) was included in the membrane formulation. p(HEMA-MAPA) membranes were produced by a photopolymerization and then characterized by swelling tests, SEM and contact angle studies. Blood-compatibility tests were also investigated. The water swelling ratio of the p(HEMA-MAPA) membrane increases significantly (133.2.9%) compared with pHEMA (58%). p(HEMA-MAPA) membranes have large pores around in the range of 5-10 microm. All the clotting times increased when compared with pHEMA membranes. Loss of platelets and leukocytes was very low. The maximum anti-LDL antibody immobilization was achieved around pH 7.0. Immobilization of anti-LDL antibody was 12.6 mg/ml. There was a very low non-specific cholesterol adsorption onto the plain p(HEMA-MAPA) membranes, about 0.36 mg/ml. Anti-LDL antibody immobilized membranes adsorbed in the range of 4.5-7.2 mg cholesterol/ml from hypercholesterolemic human plasma. Up to 95% of the adsorbed LDL antibody was desorbed. The adsorption-desorption cycle was repeated 10 times using the same membrane. There was no significant loss in the adsorption capacity.

  19. MPP1 as a Factor Regulating Phase Separation in Giant Plasma Membrane-Derived Vesicles

    PubMed Central

    Podkalicka, Joanna; Biernatowska, Agnieszka; Majkowski, Michał; Grzybek, Michał; Sikorski, Aleksander F.

    2015-01-01

    The existence of membrane-rafts helps to conceptually understand the spatiotemporal organization of membrane-associated events (signaling, fusion, fission, etc.). However, as rafts themselves are nanoscopic, dynamic, and transient assemblies, they cannot be directly observed in a metabolizing cell by traditional microscopy. The observation of phase separation in giant plasma membrane-derived vesicles from live cells is a powerful tool for studying lateral heterogeneity in eukaryotic cell membranes, specifically in the context of membrane rafts. Microscopic phase separation is detectable by fluorescent labeling, followed by cooling of the membranes below their miscibility phase transition temperature. It remains unclear, however, if this lipid-driven process is tuneable in any way by interactions with proteins. Here, we demonstrate that MPP1, a member of the MAGUK family, can modulate membrane properties such as the fluidity and phase separation capability of giant plasma membrane-derived vesicles. Our data suggest that physicochemical domain properties of the membrane can be modulated, without major changes in lipid composition, through proteins such as MPP1. PMID:25954878

  20. The plasma membrane proteome of Saccharomyces cerevisiae and its response to the antifungal calcofluor.

    PubMed

    Delom, Frédéric; Szponarski, Wojciech; Sommerer, Nicolas; Boyer, Jean-Christophe; Bruneau, Jean-Michel; Rossignol, Michel; Gibrat, Rémy

    2006-05-01

    Calcofluor is an antifungal compound known to induce structural perturbations of the cell wall by interfering with the synthesis of chitin microfibril. Proteins from a stripped plasma membrane fraction were solubilized with the neutral and non-denaturing detergent, the n-dodecyl beta-D-maltoside. Proteins were then resolved using a recently described ion-exchange chromatography (IEC)/lithium dodecyl sulfate (LDS)-PAGE procedure. Nearly 90 proteins were identified and clustered, based on their pI, molecular weight, abundance and/or hydrophobicity. This method was then applied to profile the plasma membrane response to calcofluor. The LDS-PAGE patterns obtained from whole plasma membrane proteins were similar for the non-treated and calcofluor-treated samples. However, IEC/LDS-PAGE analysis revealed subtle changes in the expression of several proteins of low abundance, in response to calcofluor. These proteins include Pil1p and Lsp1p, two sphingolipid long-chain base-responsive inhibitors of protein kinases involved in signaling pathways for cell wall integrity and Rho1p, a small GTPase. It was recently hypothesized that Pil1p and Lsp1p could associate with, and regulate, the plasma membrane beta-1-3-glucan synthase, responsible for the synthesis of another major microfibril for yeast cell wall. Results are discussed with respect to both calcofluor effects on the plasma membrane proteins and the power of the IEC/LDS-PAGE procedure in the search for new potential therapeutics targets.

  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. 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

    2016-07-01

    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.

  3. A phytochrome–phototropin light signaling complex at the plasma membrane

    PubMed Central

    Jaedicke, Katharina; Lichtenthäler, Anna Lena; Meyberg, Rabea; Zeidler, Mathias; Hughes, Jon

    2012-01-01

    Phytochromes are red/far-red photochromic photoreceptors central to regulating plant development. Although they are known to enter the nucleus upon light activation and, once there, regulate transcription, this is not the complete picture. Various phytochrome effects are manifested much too rapidly to derive from changes in gene expression, whereas others seem to occur without phytochrome entering the nucleus. Phytochromes also guide directional responses to light, excluding a genetic signaling route and implying instead plasma membrane association and a direct cytoplasmic signal. However, to date, no such association has been demonstrated. Here we report that a phytochrome subpopulation indeed associates physically with another photoreceptor, phototropin, at the plasma membrane. Yeast two-hybrid methods using functional photoreceptor molecules showed that the phytochrome steering growth direction in Physcomitrella protonemata binds several phototropins specifically in the photoactivated Pfr state. Split-YFP studies in planta showed that the interaction occurs exclusively at the plasma membrane. Coimmunoprecipitation experiments provided independent confirmation of in vivo phy-phot binding. Consistent with this interaction being associated with a cellular signal, we found that phytochrome-mediated tropic responses are impaired in Physcomitrella phot− mutants. Split-YFP revealed a similar interaction between Arabidopsis phytochrome A and phototropin 1 at the plasma membrane. These associations additionally provide a functional explanation for the evolution of neochrome photoreceptors. Our results imply that the elusive phytochrome cytoplasmic signal arises through binding and coaction with phototropin at the plasma membrane. PMID:22773817

  4. 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

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

    PubMed Central

    Ray, Supriyo; Kassan, Adam; Busija, Anna R.; Rangamani, Padmini

    2016-01-01

    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

  6. 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

  7. 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

  8. Effects of hormonal supplementation on nuclear maturation and cortical granules distribution of canine oocytes during various reproductive stages.

    PubMed

    Apparicio, M; Alves, A E; Pires-Butler, E A; Ribeiro, A P C; Covizzi, G J; Vicente, W R R

    2011-10-01

    The aim of this study was to evaluate the effects of hCG, progesterone and oestradiol supplementation on nuclear and cytoplasmic maturation of canine oocytes cultured for 24, 48, 72 and 96 h. Oocytes obtained from 18 healthy bitches were divided into three groups according to their reproductive status (follicular, luteal and anoestrus stages) and cultured in TCM 199 + 25 UI/ml of hCG + 1 μg/ml of progesterone + 1 μg/ml of 17-β oestradiol or without hormonal supplementation (control) for different periods. Then, they were stained with FITC-LCA-Hoescht for chromatin configuration and cortical granules distribution and evaluated under an epifluorescence microscope. Culture time and the influence of different stages of the oestrous cycle were also evaluated. The present study demonstrated that there was no significant difference among the reproductive stages. With regards to culture medium, only oocytes from the supplemented medium were able to complete meiosis; however, significant difference was only noticed in the percentage of MI stage oocytes (p < 0.05) in the follicular and luteal group at 72 h of culture. Most oocytes in germinal vesicle, germinal vesicle breakdown and metaphase I stage had cortical granules distributed throughout the cytoplasm (immature pattern), irrespective of the culture period (p < 0.05). Cortical granules distributed immediately beneath the plasma membrane (mature) was only observed in metaphase II stage oocytes, but not all of them presented matured cytoplasm. Our results reveal that cortical granules distribution in canine oocytes matured in vitro did not progressed in correspondence with nuclear stage changes and are in accordance with those from other species.

  9. Water homeostasis in the fish oocyte: new insights into the role and molecular regulation of a teleost-specific aquaporin.

    PubMed

    Cerdà, J; Zapater, C; Chauvigné, F; Finn, R N

    2013-02-01

    The discovery of the role of a teleost-specific aquaporin (Aqp1ab) during the process of oocyte hydration in marine fish producing pelagic (floating) eggs, recently confirmed by molecular approaches, has revealed that this mechanism is more sophisticated than initially thought. Recent phylogenetic and genomic studies suggest that Aqp1ab likely evolved by tandem duplication from a common ancestor and further neofunctionalized in oocytes for water transport. Investigations into the regulation of Aqp1ab during oogenesis indicate that the mRNA and protein product are highly accumulated during early oocyte growth, possibly through the transcriptional activation of the aqp1ab promoter by the classical nuclear progesterone receptor and perhaps by Sry-related high mobility group [HMG]-box (Sox) transcription factors. During oocyte growth and maturation, Aqp1ab intracellular trafficking may be regulated by phosphorylation and/or dephosphorylation of specific C-terminal residues in Aqp1ab, as well as by signal-mediated sorting processes. These mechanisms possibly regulate the temporal insertion of Aqp1ab into the oocyte plasma membrane during oocyte hydration, although the intracellular signaling pathways involved are yet unknown. Interestingly, in some freshwater species that spawn partially hydrated eggs, high accumulation of transcripts encoding functional Aqp1ab channels have also been found in the ovary. These findings suggest that the Aqp1ab-mediated mechanism for oocyte hydration is likely conserved in teleosts. The tight regulation of Aqp1ab during oogenesis, at both the transcriptional and posttranslational levels, highlights the essential physiological role of this water channel and opens new research avenues for understanding the molecular basis of egg formation in fish.

  10. Partial purification and characterization of oestrogen receptors in subfractions of hepatocyte plasma membranes

    PubMed Central

    Pietras, Richard J.; Szego, Clara M.

    1980-01-01

    To assess the subcellular distribution of oestrogen-binding components in their native state, plasma membrane and other cell fractions were prepared from hepatocytes in the absence of [3H]oestradiol-17β. Cells from livers of ovariectomized rats were disrupted, with submaximal homogenization in buffered isotonic sucrose with CaCl2 and proteinase inhibitor, and fractionated by using isotonic media. Fractions were characterized by determinations of enzyme activities, biochemical constituents and ligand binding. Specific binding of 2nm-[3H]oestradiol-17β to intact cells and their fractions was detemined after equilibration for 1.5h at 4°C. More than 92% of the radioactivity from representative preparations was verified as authentic oestradiol by thin-layer chromatography. Activities of plasma-membrane marker enzymes as well as binding sites for oestrogen and for wheat germ agglutinin were present principally in particulate fractions, rather than in 105000g-supernatant fractions. However, by using alternative homogenization procedures (i.e. hypotonic media), known to fragment and strip structural components, oestradiol-binding sites and activities of plasma-membrane marker enzymes were distributed predominantly into cytosol. By using the more conservative procedures, plasma membranes of low (ρ=1.13–1.16) and high (ρ=1.16–1.18) density were purified from crude nuclear fractions. A second low-density subfraction of plasma membrane was prepared from microsome-rich fractions. Activities of plasma-membrane marker enzymes were enriched to about 28 and four times that of the homogenate in plasma membranes of low and high density respectively. Binding sites for wheat germ agglutinin and oestradiol were concentrated in low-density plasma membranes to 46–63 times that of the homogenate. Specific binding of oestrogen in low-density plasma membranes purified from crude nuclei was saturable, with an apparent association constant of 3.5nm. At saturation, such oestradiol

  11. ATP-dependent calcium transport across basal plasma membranes of human placental trophoblast

    SciTech Connect

    Fisher, G.J.; Kelley, L.K.; Smith, C.H.

    1987-01-01

    As a first step in understanding the cellular basis of maternal-fetal calcium transfer, the authors examined the characteristics of calcium uptake by a highly purified preparation of the syncytiotrophoblast basal (fetal facing) plasma membrane. In the presence of nanomolar concentrations of free calcium, basal membranes demonstrated substantial ATP-dependent calcium uptake. This uptake required magnesium, was not significantly affected by Na/sup +/ or K/sup +/ (50 mM), or sodium azide (10 mM). Intravesicular calcium was rapidly and completely released by the calcium ionophore rapidly and completely released by the calcium ionophore A23187. Calcium transport was significantly stimulated by the calcium-dependent regulatory protein calmodulin. Placental membrane fractions enriched in endoplasmic reticulum (ER) and mitochondria also demonstrated ATP-dependent calcium uptake. In contrast to basal membrane, mitochondrial calcium uptake was completely inhibited by azide. The rate of calcium uptake was completely inhibited by azide. The rate of calcium uptake by the ER was only 20% of that of basal membranes. They conclude that the placental basal plasma membrane possesses a high-affinity calcium transport system similar to that found in plasma membranes of a variety of cell types. This transporter is situated to permit it to function in vivo in maternal-fetal calcium transfer.

  12. 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.

  13. 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.

  14. 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.

  15. Heterogeneous distribution of enzymes among plasma-membrane fragments sedimenting with the microsomal fraction of rat liver

    PubMed Central

    Norris, Kenneth A.; Dobrota, Miloslav; Issa, Faiz S.; Hinton, Richard H.; Reid, Eric

    1974-01-01

    Plasma-membrane fragments recovered in the microsomal fraction of rat liver homogenates were shown to be heterogeneous in density. It was demonstrated that 5′-nucleotidase, the most commonly used plasma-membrane marker, is concentrated in the lightest subfraction. Two of the published procedures for the isolation of plasma-membrane fragments from the microsomal fraction (Touster et al., 1970; Hinton et al., 1971) are shown to give products which are not representative of all the plasma-membrane fragments of microsomal size, and it is argued that a third procedure (House & Weidemann, 1970) is likely to give a similar product. PMID:4377214

  16. Fc gamma-receptor activity of isolated human placental syncytiotrophoblast plasma membrane.

    PubMed Central

    Brown, P J; Johnson, P M

    1981-01-01

    Fc gamma-receptor activity of isolated human placental syncytiotrophoblast microvillous plasma membrane (StMPM) vesicle preparations has been determined in an immunoradiometric assay using Sepharose-immobilized protein A to separate free 125I-labelled human IgG from membrane-bound 125I-IgG. This receptor assay has been optimalized in terms of buffer pH and molarity, and used to demonstrate that prior 60 min washing of isolated membranes in 3 M KCl to remove extrinsic membrane-bound protein substantially increases the membrane-binding capacity for IgG. Inhibition studies have determined the syncytiotrophoblast Fc gamma-receptor equilibrium constant for association (Ka) as 4.0 x 10(7) M-1 at 37 degrees and the number of available Fc gamma-receptor sites as 1.5 x 10(14) per mg membrane protein. PMID:7461733

  17. Two pathways regulate cortical granule translocation to prevent polyspermy in mouse oocytes.

    PubMed

    Cheeseman, Liam P; Boulanger, Jérôme; Bond, Lisa M; Schuh, Melina

    2016-12-19

    An egg must be fertilized by a single sperm only. To prevent polyspermy, the zona pellucida, a structure that surrounds mammalian eggs, becomes impermeable upon fertilization, preventing the entry of further sperm. The structural changes in the zona upon fertilization are driven by the exocytosis of cortical granules. These translocate from the oocyte's centre to the plasma membrane during meiosis. However, very little is known about the mechanism of cortical granule translocation. Here we investigate cortical granule transport and dynamics in live mammalian oocytes by using Rab27a as a marker. We show that two separate mechanisms drive their transport: myosin Va-dependent movement along actin filaments, and an unexpected vesicle hitchhiking mechanism by which cortical granules bind to Rab11a vesicles powered by myosin Vb. Inhibiting cortical granule translocation severely impaired the block to sperm entry, suggesting that translocation defects could contribute to miscarriages that are caused by polyspermy.

  18. Two pathways regulate cortical granule translocation to prevent polyspermy in mouse oocytes

    PubMed Central

    Cheeseman, Liam P.; Boulanger, Jérôme; Bond, Lisa M.; Schuh, Melina

    2016-01-01

    An egg must be fertilized by a single sperm only. To prevent polyspermy, the zona pellucida, a structure that surrounds mammalian eggs, becomes impermeable upon fertilization, preventing the entry of further sperm. The structural changes in the zona upon fertilization are driven by the exocytosis of cortical granules. These translocate from the oocyte's centre to the plasma membrane during meiosis. However, very little is known about the mechanism of cortical granule translocation. Here we investigate cortical granule transport and dynamics in live mammalian oocytes by using Rab27a as a marker. We show that two separate mechanisms drive their transport: myosin Va-dependent movement along actin filaments, and an unexpected vesicle hitchhiking mechanism by which cortical granules bind to Rab11a vesicles powered by myosin Vb. Inhibiting cortical granule translocation severely impaired the block to sperm entry, suggesting that translocation defects could contribute to miscarriages that are caused by polyspermy. PMID:27991490

  19. 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.

  20. 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

    2014-09-04

    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

  1. 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

  2. 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.

  3. Denitrification by plant roots? New aspects of plant plasma membrane-bound nitrate reductase.

    PubMed

    Eick, Manuela; Stöhr, Christine

    2012-10-01

    A specific form of plasma membrane-bound nitrate reductase in plants is restricted to roots. Two peptides originated from plasma membrane integral proteins isolated from Hordeum vulgare have been assigned as homologues to the subunit NarH of respiratory nitrate reductase of Escherichia coli. Corresponding sequences have been detected for predicted proteins of Populus trichocarpa with high degree of identities for the subunits NarH (75%) and NarG (65%), however, with less accordance for the subunit NarI. These findings coincide with biochemical properties, particularly in regard to the electron donors menadione and succinate. Together with the root-specific and plasma membrane-bound nitrite/NO reductase, nitric oxide is produced under hypoxic conditions in the presence of nitrate. In this context, a possible function in nitrate respiration of plant roots and an involvement of plants in denitrification processes are discussed.

  4. 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.

  5. Treatment of Immobilized Collagen on Poly(tetrafluoroethylene) Nanoporous Membrane with Plasma

    NASA Astrophysics Data System (ADS)

    Bratescu, Maria Antoaneta; Saito, Nagahiro; Takai, Osamu

    2006-10-01

    In this work we treated type I collagen immobilized on different nanoporous membranes with microwave (MW) argon plasma. Hydrophobic and hydrophilic nanoporous substrates of poly(tetrafluoroethylene), with thickness varying from 35 to 70 μm and a 100 nm pore size, were employed as support for collagen immobilization. On the hydrophilic nanoporous membrane, after the MW plasma treatment, the immobilized collagen changed its morphology and showed a tendency to self-assemble in quasi-regular forms as microellipsoids. The presence of collagen immobilized on the nanoporous membrane after the MW plasma treatment was analyzed by detecting in the Raman spectrum an α-helix form, NH deformation vibration, amide II band at 1550 cm-1, a characteristic group frequency of the collagen macromolecule.

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

    PubMed

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

    2013-04-01

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

  7. 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.

  8. Using fluorometry and ion-sensitive microelectrodes to study the functional expression of heterologously-expressed ion channels and transporters in Xenopus oocytes

    PubMed Central

    Musa-Aziz, Raif; Boron, Walter F.; Parker, Mark D.

    2010-01-01

    The Xenopus laevis oocyte is a model system for the electrophysiological study of exogenous ion transporters. Three main reasons make the oocyte suitable for this purpose: (a) it has a large cell size (~1 mm diameter), (b) it has an established capacity to produce—from microinjected mRNAs or cRNAs—exogenous ion transporters with close-to-physiological post-translational modifications and actions, and (c) its membranes contain endogenous ion-transport activities which are usually smaller in magnitude than the activities of exogenously-expressed ion transporters. The expression of ion-transporters as green-fluorescent-protein fusions allows the fluorometric assay of transporter yield in living oocytes. Monitoring of transporter-mediated movement of ions such as Cl−, H+ (and hence base equivalents like OH−1 and HCO3−), K+, and Na+ is achieved by positioning the tips of ion-sensitive microelectrodes inside the oocyte and/or at the surface of the oocyte plasma membrane. The use of ion-sensitive electrodes is critical for studying net ion-movements mediated by electroneutral transporters. The combined use of fluorometry and electrophysiology expedites transporter study by allowing measurement of transporter yield prior to electrophysiological study and correlation of relative transporter yield with transport rates. PMID:20051266

  9. Optical tweezers study of viscoelastic properties in the outer hair cell plasma membrane

    NASA Astrophysics Data System (ADS)

    Murdock, David R.; Ermilov, Sergey A.; Qian, Feng; Brownell, William E.; Anvari, Bahman

    2004-06-01

    An optical tweezers system was used to study the mechanical characteristics of the outer hair cell (OHC) lateral wall by forming plasma membrane tethers. A 2nd order generalized Kelvin model was applied to describe the viscoelastic behavior of OHC membrane tethers. The measured parameters included equilibrium tethering force, (Feq), force relaxation times (τ), stiffness values (κ), and coefficients of friction (μ). An analysis of force relaxation in membrane tethers indicated that the force decay is a biphasic process containing both an elastic and a viscous phase. In general, we observed an overall negative trend in the measured parameters upon application of the cationic amphipath chlorpromazine (CPZ). CPZ was found to cause up to a 40 pN reduction in Feq in OHCs. A statistically significant reduction in relaxation times and coefficients of friction was also observed, suggesting an increase in rate of force decay and a decrease in plasma membrane viscosity.

  10. 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

  11. A Pea Plasma Membrane Protein Exhibiting Blue Light-Induced Phosphorylation Retains Photosensitivity following Triton Solubilization.

    PubMed Central

    Short, T. W.; Reymond, P.; Briggs, W. R.

    1993-01-01

    Phosphorylation of a polypeptide of approximately 120 kD in pea (Pisum sativum L.) plasma membranes in response to blue light has been shown to be involved in phototropic curvature, but the relationship of this protein to the kinase and photoreceptor acting upon it is uncertain. Using two-phase aqueous partitioning to isolate right-side-out plasma membrane vesicles, we have obtained evidence suggesting that the photoreceptor, kinase, and substrate are localized to the plasma membrane fraction. Latent phosphorylation accessible through Triton X-100 or freeze/thaw treatments of purified plasma membrane vesicles indicates that at least the kinase moiety is present on the internal face of the plasma membrane. Effects of solubilization of vesicles on fluence-response characteristics and on phosphorylation levels provide evidence that the receptor, kinase, and protein substrate are present together in individual mixed detergent micelles, either as a stable complex or as domains of a single polypeptide. In vivo blue-light irradiation results in a small but significant decrease in mobility of the 120-kD phosphorylated protein on sodium dodecylsulfate gel electrophoresis. This mobility shift is evident on Coomassie-stained gels and on western blots probed with polyclonal antibodies raised against the 120-kD protein. Among the plasma membrane proteins bound to the reactive nucleotide analog fluorosulfonylbenzoyladenine (FSBA), a distinct protein band at 120 kD can be detected on blots probed with anti-FSBA antibodies. This band exhibits an in vivo light-dependent mobility shift identical to that observed for the protein band and antibodies specific for the 120-kD protein, implying that the 120-kD protein has an integral nucleotide binding site and consistent with the possibility that the substrate protein is also a kinase. PMID:12231721

  12. 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

  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. Monoclonal antibodies to antigens in the peribacteroid membrane from Rhizobium-induced root nodules of pea cross-react with plasma membranes and Golgi bodies

    PubMed Central

    Brewin, N. J.; Robertson, J. G.; Wood, E. A.; Wells, B.; Larkins, A. P.; Galfre, G.; Butcher, G. W.

    1985-01-01

    Three rat hybridoma lines that produced monoclonal antibodies reacting with the peribacteroid membrane from Pisum sativum were isolated, and these all appeared to recognise the same antigenic structure. Using one of these monoclonal antibodies, AFRC MAC 64, electron microscopy of immunogold-stained thin sections of nodule tissue revealed that the antigen, present in the peribacteroid membrane, was also found in the plant plasma membranes and in the Golgi bodies, but not in the endoplasmic reticulum. When peribacteroid membrane proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose by electro-blotting, it was found that MAC 64 bound to a series of protease-sensitive bands that migrated in the mol. wt. range 50-85 K. The epitope was sensitive to periodate oxidation and its structure may therefore involve the carbohydrate component of a membrane glycoprotein. We suggest that this structure originates in the Golgi apparatus and is subsequently transferred to the peribacteriod membranes and plasma membranes. The monoclonal antibody also reacted with peribacteroid membranes from nodules of Vicia and lupin, and with plasma membranes and Golgi membranes from uninfected plant cells, including root tip cells from onion (Allium cepa), indicating that the antigen is highly conserved in the plasma membranes of plant cells. ImagesFig. 1.Fig. 2.Fig. 3.Fig. 4.Fig. 5.Fig. 6. PMID:15926221

  15. 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

  16. Membrane proteins associated with sperm-oocyte interaction: A proteomic comparison between Kedah Kelantan (Bos indicus) and Mafriwal (Bos taurus × Bos indicus) sperm

    NASA Astrophysics Data System (ADS)

    Ashrafzadeh, Ali; Nathan, Sheila; Othman, Iekhsan; Yee, Tee Ting; Karsani, Saiful Anuar

    2013-11-01

    Production performance of European cattle breeds has significantly improved through various breeding programs. However, European breeds are more susceptible to heat stress compared to zebu cattle (Bos indicus) as their conception rate can range between 20 to 30% in hot seasons compared to winter. To identify cattle sperm proteins associated with zebu cattle higher fertility and heat tolerance in tropical environments, we utilised a proteomics-based approach to compare sperm from the highly fertile Malaysian indigenous breed, Kedah Kelantan (Bos indicus), with sperm from the sub-fertile crossbreed, Mafriwal (Bos taurus × Bos indicus). Frozen semen of three high performance bulls from each breed was processed to obtain live and pure sperm. Proteins were separated and gel bands were processed by in-gel tryptic digestion. For each breed, mass spectrometry data was acquired over 11 replicates. The analyzed data identified peptides with different expression levels (99% confidence level) and protein identification was determined by targeted MS/MS. Among the identified proteins associated with sperm-oocyte interaction, two proteins were up-regulated in Kedah Kelantan sperm and 7 proteins were up-regulated in or specific to Mafriwal. Our results suggest that the higher fertility of zebu cattle in tropical areas may not be related to more efficient sperm-oocyte interaction. Further analysis of the other regulated proteins in these two breeds may contribute further knowledge on the physiological reason/s for higher fertility and heat tolerance of Zebu cattle in tropical areas.

  17. 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.

  18. 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.

  19. Comparison of solubilized and purified plasma membrane and nuclear insulin receptors

    SciTech Connect

    Wong, K.Y.; Hawley, D.; Vigneri, R.; Goldfine, I.D.

    1988-01-12

    Prior studies have detected biochemical and immunological differences between insulin receptors in plasma membranes and isolated nuclei. To further investigate these receptors, they were solubilized in Triton X-100 partially purified by wheat germ agglutinin-agarose chromatography. In these preparations, the nuclear and plasma membrane receptors had very similar pH optima (pH 8.0) and reactivities to a group of polyclonal antireceptor antibodies. Further, both membrane preparations had identical binding activities when labeled insulin was competed for by unlabeled insulin (50% inhibition at 800 pM). Next, nuclear and plasma membranes were solubilized and purified to homogeneity by wheat germ agglutinin-agarose and insulin-agarose chromatography. In both receptors, labeled insulin was covalently cross-linked to a protein of 130 kilodaltons representing the insulin receptor ..cap alpha.. subunit. When preparations of both receptors were incubated with insulin and then adenosine 5'-(..gamma..-/sup 32/P)triphosphate, a protein of 95 kilodaltons representing the insulin receptor ..beta.. subunit was phosphorylated in a dose-dependent manner. These studies indicate, therefore, that solubilized plasma membrane and nuclear insulin receptors have similar structures and biochemical properties, and they suggest that they are the same (or very similar) proteins.

  20. Nonidet P-40 extraction of lymphocyte plasma membrane. Characterization of the insoluble residue.

    PubMed

    Davies, A A; Wigglesworth, N M; Allan, D; Owens, R J; Crumpton, M J

    1984-04-01

    Purified preparations of lymphocyte plasma membrane were extracted exhaustively with Nonidet P-40 in Dulbecco's phosphate-buffered saline medium. The insoluble fraction, as defined by sedimentation at 10(6) g-min, contained about 10% of the membrane protein as well as cholesterol and phospholipid. The lipid/protein ratio, cholesterol/phospholipid ratio and sphingomyelin content were increased in the residue. Density-gradient centrifugation suggested that the lipid and protein form a common entity. As judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the Nonidet P-40-insoluble fractions of the plasma membranes of human B lymphoblastoid cells and pig mesenteric lymph-node lymphocytes possessed similar qualitative polypeptide compositions but differed quantitatively. Both residues comprised major polypeptides of Mr 28 000, 33 000, 45 000 and 68 000, together with a prominent band of Mr 120 000 in the human and of Mr 200 000 in the pig. The polypeptides of Mr 28 000, 33 000, 68 000 and 120 000 were probably located exclusively in the Nonidet P-40-insoluble residue, which also possessed a 4-fold increase in 5'-nucleotidase specific activity. The results indicate that a reproducible fraction of lymphocyte plasma membrane is insoluble in non-ionic detergents and that this fraction possesses a unique polypeptide composition. By analogy with similar studies with erythrocyte ghosts, it appears likely that the polypeptides are located on the plasma membrane's cytoplasmic face.

  1. 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.

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

    PubMed

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

    2017-02-16

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

  3. Characterization of antibody binding to cell surface antigens using a plasma membrane-bound plate assay.

    PubMed

    Vater, C A; Reid, K; Bartle, L M; Goldmacher, V S

    1995-01-01

    A procedure has been developed for measuring antibody binding to cell surface antigens using an immobilized plasma membrane fraction. In this method, isolated plasma membranes are dried onto wells of a 96-well microtiter plate and incubated with antibodies that recognize a cell surface protein. Bound antibody is detected indirectly using an enzyme-linked or fluorescently tagged second antibody. Alternatively, the primary antibody itself can be labeled and its binding can be detected directly. The assay is simple and fast and provides several advantages over whole cell binding assays currently in widespread use.

  4. Distribution of adenylate cyclase and GTP-binding proteins in hepatic plasma membranes.

    PubMed

    Dixon, B S; Sutherland, E; Alexander, A; Nibel, D; Simon, F R

    1993-10-01

    Hepatic membrane subfractions prepared from control rats demonstrated forskolin (FSK)-stimulated adenylate cyclase activity in the basolateral (sinusoidal) but not apical (canalicular) plasma membrane. After bile duct ligation (BDL) for 12 or 24 h, there was an increase in FSK-stimulated adenylate cyclase activity in the apical membrane (54.2 +/- 3.9 pmol.mg-1 x min-1). The mechanism for this increase was explored further. ATP hydrolysis was found to be much higher in the apical than the basolateral membrane. Increasing the ATP levels in the assay enhanced apical membrane adenylate cyclase activity (10.5 +/- 0.2 pmol.mg-l.min-1); however, total adenosinetriphosphatase (ATPase) activity was not altered after BDL. Extraction of the apical membrane with bile acids or other detergents resulted in a two- to threefold increase in adenylate cyclase activity (30.6 +/- 3.6 pmol.mg-1 x min-1; detergent C12E8) This suggested that bile duct ligation was acting via the detergent-like action of bile acids to uncover latent adenylate cyclase activity on apical membranes. Further studies demonstrated that both BDL and detergent extraction also enhanced toxin-directed ADP-ribosylation of Gs alpha (cholera toxin) and Gi alpha (pertussis toxin) in the apical but not the basolateral membrane. After BDL, Gi alpha was found to be twofold greater in the apical membrane than the basolateral membrane. Immunoblotting using specific G protein antibodies further confirmed that apical membranes from control rats had a higher concentration of Gi1, 2 alpha and beta and slightly elevated levels of Gi3 alpha and Gs alpha compared with the basolateral membrane. The results demonstrate that adenylate cyclase and heterotrimeric GTP-binding proteins are present on the apical membrane, but measurement of their functional activity requires detergent permeabilization of apical membrane vesicles and is limited by the presence of high ATPase activity.

  5. 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.

  6. Prostasomes of canine seminal plasma - zinc-binding ability and effects on motility characteristics and plasma membrane integrity of spermatozoa.

    PubMed

    Mogielnicka-Brzozowska, M; Strzeżek, R; Wasilewska, K; Kordan, W

    2015-06-01

    Prostasomes are small lipid membrane-confined vesicles that are involved in various fertilization-related processes. The aim of this study was to demonstrate canine seminal plasma prostasomes' ability to bind zinc ions, as well as examining their effects on sperm motility characteristics and plasma membrane integrity during cold storage. Ejaculates, collected from five cross-bred dogs (n = 50), were subjected to ultracentrifugation followed by gel filtration (GF) on a Superose 6 column. Prostasomes appeared as a single fraction in the elution profile. Transmission electron microscopy (TEM) analysis of canine prostasomes revealed the presence of membrane vesicles with diameters ranging from 20.3 to 301 nm. The zinc-affinity chromatography on a Chelating Sepharose Fast Flow - Zn(2 +) showed that from 93 to 100% of the prostasome proteins bind zinc ions (P(+) Zn). SDS-PAGE revealed that canine P(+) Zn comprised four protein bands, with low molecular weights (10.2-12 kDa). We have also shown a positive effect of prostasomes (p < 0.05), especially variant B (2% of total seminal plasma protein) on canine sperm motility parameters after 2 h storage at 5°C (TMOT%, 44.75 ± 5.18) and PMOT%, 12.42 ± 1.59) and VAP, VSL, VCL, when compared with Control (TMOT%, 7.30 ± 1.41 and PMOT%, 1.70 ± 0.42). Higher percentage of spermatozoa with intact plasma membrane (SYBR/PI dual staining) and intact acrosome (Giemsa stained), after 2 h storage at 5°C, was showed, in variant A (1.5% of total seminal plasma protein) and B, when compared with Control and variant C (2.5% of total seminal plasma protein). The prostasomes' effect on motility and plasma membrane integrity of canine cold-stored spermatozoa may be related to their ability to bind zinc ions and regulate their availability to the sperm.

  7. CFTR channel in oocytes from Xenopus laevis and its regulation by xShroom1 protein.

    PubMed

    Palma, Alejandra G; Galizia, Luciano; Kotsias, Basilio A; Marino, Gabriela I

    2016-05-01

    Shroom is a family of related proteins linked to the actin cytoskeleton. xShroom1 is constitutively expressed in Xenopus laevis oocytes, and it is required for the expression of the epithelial sodium channel (ENaC). As there is a close relationship between ENaC and the cystic fibrosis transmembrane regulator (CFTR), we examined the action of xShroom1 on CFTR expression and activity. Biotinylation was used to measure CFTR surface expression, and currents were registered with voltage clamp when stimulated with forskolin and 3-isobutyl-1-methylxanthine. Oocytes were coinjected with CFTR complementary RNAs (cRNAs) and xShroom1 sense or antisense oligonucleotides. We observed an increment in CFTR currents and CFTR surface expression in oocytes coinjected with CFTR and xShroom1 antisense oligonucleotides. MG-132, a proteasome inhibitor, did not prevent the increment in currents when xShroom1 was suppressed by antisense oligonucleotides. In addition, we inhibited the delivery of newly synthesized proteins to the plasma membrane with BFA and we found that the half-life of plasma membrane CFTR was prolonged when coinjected with the xShroom1 antisense oligonucleotides. Chloroquine, an inhibitor of the late endosome/lysosome, did not significantly increase CFTR currents when xShroom1 expression was inhibited. The higher expression of CFTR when xShroom1 is suppressed is in concordance with the functional studies suggesting that the suppression of the xShroom1 protein resulted in an increment in CFTR currents by promoting the increase of the half-life of CFTR in the plasma membrane. The role of xShroom1 in regulating CFTR expression could be relevant in the understanding of the channel malfunction in several diseases.

  8. Evidence that the platelet plasma membrane does not contain a (Ca2+ + Mg2+)-dependent ATPase.

    PubMed

    Steiner, B; Lüscher, E F

    1985-09-10

    The present study was designed to determine the subcellular distribution of the platelet (Ca2+ + Mg2+)-ATPase. Human platelets were surface labeled by the periodate-boro[3H]hydride method. Plasma membrane vesicles were then isolated to a purity of approx. 90% by a procedure utilizing wheat germ agglutinin affinity chromatography. These membranes were found to be 2.6-fold enriched in surface glycoproteins compared to an unfractionated vesicle fraction and almost 7-fold enriched compared to intact platelets. In contrast, the isolated plasma membranes showed a decreased specific activity of the (Ca2+ + Mg2+)-ATPase compared to the unfractionated vesicle fraction. This decrease in specific activity was found to be similar to that of an endoplasmic reticulum marker, glucose-6-phosphatase, and to that of a platelet inner membrane marker, phospholipase A2. We conclude, therefore, that the (Ca2+ + Mg2+)-ATPase is not located in the platelet plasma membrane but is restricted to membranes of intracellular origin.

  9. 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.

  10. Effect of oxidative stress on plasma membrane fluidity of THP-1 induced macrophages.

    PubMed

    de la Haba, Carlos; Palacio, José R; Martínez, Paz; Morros, Antoni

    2013-02-01

    Plasma membrane is one of the preferential targets of reactive oxygen species which cause lipid peroxidation. This process modifies membrane properties such as membrane fluidity, a very important physical feature known to modulate membrane protein localization and function. The aim of this study is to evaluate the effect of oxidative stress on plasma membrane fluidity regionalization of single living THP-1 macrophages. These cells were oxidized with H(2)O(2) at different concentrations, and plasma membrane fluidity was analyzed by two-photon microscopy in combination with the environment-sensitive probe Laurdan. Results show a significant H(2)O(2) concentration dependent increase in the frequency of rigid lipid regions, mainly attributable to lipid rafts, at the expense of the intermediate fluidity regions. A novel statistical analysis evaluated changes in size and number of lipid raft domains under oxidative stress conditions, as lipid rafts are platforms aiding cell signaling and are thought to have relevant roles in macrophage functions. It is shown that H(2)O(2) causes an increase in the number, but not the size, of raft domains. As macrophages are highly resistant to H(2)O(2), these new raft domains might be involved in cell survival pathways.

  11. 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

    2013-11-05

    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.

  12. Meiotic maturation and developmental capability of ovine oocytes at germinal vesicle stage following vitrification using different cryodevices.

    PubMed

    Quan, Guo Bo; Wu, Guo Quan; Wang, Ya Jing; Ma, Yuan; Lv, Chun Rong; Hong, Qiong Hua

    2016-02-01

    oocytes. The cleavage rate of oocytes vitrified with Cryoloop was similar to that of oocytes vitrified with open-pulled straws. However, the cleavage rate of vitrified oocytes in the CS group was significantly less than that in the OPS or Cryoloop group (P < 0.05). None of oocytes vitrified using conventional plastic straw developed to the blastocyst stage following IVF. There was no significant difference existing between OPS and Cryoloop with respect to the blastocyst rate. After staining with cFDA and PI, cumulus cells surrounding oocytes were partly damaged by vitrification and thawing while the membrane of vitrified oocyte still remained intact. In conclusion, vitrification can seriously damage ovine immature oocytes and cumulus cells surrounding oocytes, which may subsequently affect their developmental capability. Finally, this study further proves that increasing the freezing and thawing velocity benefits survival of vitrified immature oocytes.

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

    PubMed Central

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

    2016-01-01

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

  14. Binding of plasma membrane lipids recruits the yeast integral membrane protein Ist2 to the cortical ER.

    PubMed

    Fischer, Marcel André; Temmerman, Koen; Ercan, Ebru; Nickel, Walter; Seedorf, Matthias

    2009-08-01

    Recruitment of cytosolic proteins to individual membranes is governed by a combination of protein-protein and protein-membrane interactions. Many proteins recognize phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] at the cytosolic surface of the plasma membrane (PM). Here, we show that a protein-lipid interaction can also serve as a dominant signal for the sorting of integral membrane proteins. Interaction with phosphatidly-inositolphosphates (PIPs) at the PM is involved in the targeting of the polytopic yeast protein Ist2 to PM-associated domains of the cortical endoplasmic reticulum (ER). Moreover, binding of PI(4,5)P(2) at the PM functions as a dominant mechanism that targets other integral membrane proteins to PM-associated domains of the cortical ER. This sorting to a subdomain of the ER abolishes proteasomal degradation and trafficking along the classical secretory (sec) pathway. In combination with the localization of IST2 mRNA to the bud tip and other redundant signals in Ist2, binding of PIPs leads to efficient accumulation of Ist2 at domains of the cortical ER from where the protein may reach the PM independently of the function of the sec-pathway.

  15. A mutant cytochrome b5 with a lengthened membrane anchor escapes from the endoplasmic reticulum and reaches the plasma membrane.

    PubMed Central

    Pedrazzini, E; Villa, A; Borgese, N

    1996-01-01

    Many resident membrane proteins of the endoplasmic reticulum (ER) do not have known retrieval sequences. Among these are the so-called tail-anchored proteins, which are bound to membranes by a hydrophobic tail close to the C terminus and have most of their sequence as a cytosolically exposed N-terminal domain. Because ER tail-anchored proteins generally have short (< or = 17 residues) hydrophobic domains, we tested whether this feature is important for localization, using cytochrome b5 as a model. The hydrophobic domain of cytochrome b5 was lengthened by insertion of five amino acids (ILAAV), and the localization of the mutant was analyzed by immunofluorescence in transiently transfected mammalian cells. While the wild-type cytochrome was localized to the ER, the mutant was relocated to the surface. This relocation was not due to the specific sequence introduced, as demonstrated by the ER localization of a second mutant, in which the original length of the membrane anchor was restored, while maintaining the inserted ILAAV sequence. Experiments with brefeldin A and with cycloheximide demonstrated that the extended anchor mutant reached the plasma membrane by transport along the secretory pathway. We conclude that the short membrane anchor of cytochrome b5 is important for its ER residency, and we discuss the relevance of this finding for other ER tail-anchored proteins. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8633042

  16. Is the mammalian cell plasma membrane a barrier to oxygen transport?

    PubMed Central

    1992-01-01

    Oxygen transport in the Chinese hamster ovary (CHO) plasma membrane has been studied by observing the collision of molecular oxygen with nitroxide radical spin labels placed in the lipid bilayer portion of the membrane at various distances from the membrane surface using the long-pulse saturation-recovery electron spin resonance (ESR) technique. The collision rate was estimated for 5-, 12-, and 16-doxylstearic acids from spin-lattice relaxation times (T1) measured in the presence and absence of molecular oxygen. Profiles of the local oxygen transport parameters across the membrane were obtained showing that the oxygen diffusion-concentration product is lower than in water for all locations at 37 degrees C. From oxygen transport parameter profiles, the membrane oxygen permeability coefficients were estimated according to the procedure developed earlier by Subczynski et al. (Subczynski, W. K., J. S. Hyde, and A. Kusumi. 1989. Proceedings of the National Academy of Sciences, USA. 86:4474-4478). At 37 degrees C, the oxygen permeability coefficient for the plasma membrane was found to be 42 cm/s, about two times lower than for a water layer of the same thickness as the membrane. The oxygen concentration difference across the CHO plasma membrane at physiological conditions is in the nanomolar range. It is concluded that oxygen permeation across the cell plasma membrane cannot be a rate-limiting step for cellular respiration. Correlations of the form PM = cKs between membrane permeabilities PM of small nonelectrolyte solutes of mol wt less than 50, including oxygen, and their partition coefficients K into hexadecane and olive oil are reported. Hexadecane: c = 26 cm/s, s = 0.95; olive oil: c = 23 cm/s, s = 1.56. These values of c and s differ from those reported in the literature for solutes of 50 less than mol wt less than 300 (Walter, A., and J. Gutknecht. 1986. Journal of Membrane Biology. 90:207-217). It is concluded that oxygen permeability through membranes can be

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

    PubMed

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

    2015-06-01

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

  18. Plasma membrane characterization, by scanning electron microscopy, of multipotent myoblasts-derived populations sorted using dielectrophoresis

    SciTech Connect

    Muratore, Massimo; Mitchell, Steve; Waterfall, Martin

    2013-09-06

    Highlights: •Dielectrophoretic separation/sorting of multipotent cells. •Plasma membrane microvilli structure of C2C12 and fibroblasts by SEM microscopy. •Cell cycle determination by Ki-67 in DEP-sorted cells. •Plasma membrane differences responsible for changes in membrane capacitance. -- Abstract: Multipotent progenitor cells have shown promise for use in biomedical applications and regenerative medicine. The implementation of such cells for clinical application requires a synchronized, phenotypically and/or genotypically, homogenous cell population. Here we have demonstrated the implementation of a biological tag-free dielectrophoretic device used for discrimination of multipotent myoblastic C2C12 model. The multipotent capabilities in differentiation, for these cells, diminishes with higher passage number, so for cultures above 70 passages only a small percentage of cells is able to differentiate into terminal myotubes. In this work we demonstrated that we could recover, above 96% purity, specific cell types from a mixed population of cells at high passage number without any biological tag using dielectrophoresis. The purity of the samples was confirmed by cytometric analysis using the cell specific marker embryonic myosin. To further investigate the dielectric properties of the cell plasma membrane we co-culture C2C12 with similar size, when in suspension, GFP-positive fibroblast as feeder layer. The level of separation between the cell types was above 98% purity which was confirmed by flow cytometry. These levels of separation are assumed to account for cell size and for the plasma membrane morphological differences between C2C12 and fibroblast unrelated to the stages of the cell cycle which was assessed by immunofluorescence staining. Plasma membrane conformational differences were further confirmed by scanning electron microscopy.

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

    PubMed

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

    2016-11-01

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

  20. 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.

  1. The plasma membrane flattens out to fuel cell surface growth during Drosophila cellularization

    PubMed Central

    Figard, Lauren; Xu, Heng; Garcia, Hernan G.; Golding, Ido; Sokac, Anna Marie

    2014-01-01

    Summary Cell shape change demands cell surface growth, but how growth is fueled and choreographed is still debated. Here, we use cellularization, the first complete cytokinetic event in Drosophila embryos, to show that cleavage furrow ingression is kinetically coupled to the loss of surface microvilli. We modulate furrow kinetics with RNAi against the Rho1-GTPase regulator slam, and show that furrow ingression controls the rate of microvillar depletion. Finally, we directly track microvillar membrane and see it move along the cell surface and into ingressing furrows, independent of endocytosis. Together, our results demonstrate that the kinetics of the ingressing furrow regulate the utilization of a microvillar membrane reservoir. Since the membrane of the furrow and microvilli are contiguous, we suggest that ingression drives unfolding of the microvilli and incorporation of microvillar membrane into the furrow. We conclude that plasma membrane folding/unfolding can contribute to the cell shape changes that promote embryonic morphogenesis. PMID:24316147

  2. Effects of liquid preservation of sperm on their ability to activate oocytes and initiate preimplantational development after intracytoplasmic sperm injection in the pig.

    PubMed

    Binh, N T; Van Thuan, N; Miyake, M

    2009-06-01

    The objective of this study was to clarify the effects of liquid preservation conditions on the ability of pig sperm to activate oocytes, form a male pronucleus, and initiate preimplantational development of embryos after intracytoplasmic sperm injection (ICSI). Porcine ejaculates were preserved at 4, 14, and 24 degrees C for up to 48h, and then damage to the plasma membrane, morphologic changes of the acrosome, and the amount of phospholipase Czeta (PLCzeta) in the sperm were assessed by SYBR-14/propidium iodide staining, fluorescein isothiocyanate-conjugated peanut agglutinin staining, indirect immunofluorescence, and Western blots, respectively. The proportion of sperm with a disintegrated plasma membrane or damaged acrosome increased in all samples as the duration of preservation increased, although the time courses of the increases varied among preservation temperatures. The immunolocalization and immunoreactivity of PLCzeta in the sperm showed its reduction concurrent with disintegration of the plasma membrane and acrosome. Rates of oocyte activation, male-pronuclear formation, and blastocyst formation after ICSI using sperm preserved for 18h at 24 degrees C (78%, 62%, and 35%, respectively) and for 48h at 14 degrees C (63%, 53%, and 28%, respectively) were significantly higher than those of any other sperm sample. We concluded that the damage to the plasma membrane and acrosome, and a sufficient amount of PLCzeta in the sperm head, enhanced successful oocyte activation, fertilization, and early development of the oocytes after ICSI. Moreover, we inferred that appropriate liquid preservation of sperm improved the efficiency of blastocyst production in vitro after ICSI in pigs.

  3. Proteomic analysis of plasma membrane and secretory vesicles from human neutrophils

    PubMed Central

    Jethwaney, Deepa; Islam, Md Rafiqul; Leidal, Kevin G; de Bernabe, Daniel Beltran-Valero; Campbell, Kevin P; Nauseef, William M; Gibson, Bradford W

    2007-01-01

    Background Polymorphonuclear neutrophils (PMN) constitute an essential cellular component of innate host defense against microbial invasion and exhibit a wide array of responses both to particulate and soluble stimuli. As the cells recruited earliest during acute inflammation, PMN respond rapidly and release a variety of potent cytotoxic agents within minutes of exposure to microbes or their products. PMN rely on the redistribution of functionally important proteins, from intracellular compartments to the plasma membrane and phagosome, as the means by which to respond quickly. To determine the range of membrane proteins available for rapid recruitment during PMN activation, we analyzed the proteins in subcellular fractions enriched for plasma membrane and secretory vesicles recovered from the light membrane fraction of resting PMN after Percoll gradient centrifugation and free-flow electrophoresis purification using mass spectrometry-based proteomics methods. Results To identify the proteins light membrane fractions enriched for plasma membrane vesicles and secretory vesicles, we employed a proteomic approach, first using MALDI-TOF (peptide mass fingerprinting) and then by HPLC-MS/MS using a 3D ion trap mass spectrometer to analyze the two vesicle populations from resting PMN. We identified several proteins that are functionally important but had not previously been recovered in PMN secretory vesicles. Two such proteins, 5-lipoxygenase-activating protein (FLAP) and dysferlin were further validated by immunoblot analysis. Conclusion Our data demonstrate the broad array of proteins present in secretory vesicles that provides the PMN with the capacity for remarkable and rapid reorganization of its plasma membrane after exposure to proinflammatory agents or stimuli. PMID:17692124

  4. Environmental impacts on the gonadotropic system in female Atlantic salmon (Salmo salar) during vitellogenesis: Photothermal effects on pituitary gonadotropins, ovarian gonadotropin receptor expression, plasma sex steroids and oocyte growth.

    PubMed

    Taranger, Geir Lasse; Muncaster, Simon; Norberg, Birgitta; Thorsen, Anders; Andersson, Eva

    2015-09-15

    The gonadotropic system and ovarian growth and development were studied during vitellogenesis in female Atlantic salmon subjected to either simulated natural photoperiod and ambient water temperature (NL-amb), or an accelerating photoperiod (short day of LD8:16 from May 10) combined with either warmed (ca 2°C above ambient; 8L-warm) or cooled water (ca 2°C below ambient; 8L-cold) from May to September. Monthly samples were collected from 10 females/group for determination of transcript levels of pituitary gonadotropin subunits (fshb and lhb) and ovarian gonadotropin receptors (fshr and lhr), plasma sex steroids (testosterone: T and estradiol-17β: E2), gonadosomatic index (GSI) and oocyte size. Short day in combination with either warmed or cooled water induced an earlier increase in pituitary fshb and lhb levels compared with NL-amb controls, and advanced ovarian growth and the seasonal profiles of T, E2. By contrast only minor effects were seen of the photothermal treatments on ovarian fshr and lhr. The 8L-cold had earlier increase in fshb, lhb and E2, but similar oocyte and gonadal growth as 8L-warm, suggesting that the 8L-cold group tried to compensate for the lower water temperature during the period of rapid gonadal growth by increasing fshb and E2 production. Both the 8L-warm and 8L-cold groups showed incomplete ovulation in a proportion of the females, possibly due to the photoperiod advancement resulting in earlier readiness of spawning occurring at a higher ambient temperature, or due to some reproductive dysfunction caused by photothermal interference with normal neuroendocrine regulation of oocyte development and maturation.

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

    NASA Technical Reports Server (NTRS)

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

    1989-01-01

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

  6. Partition of Membrane Particles in Aqueous Two-Polymer Phase System and Its Practical Use for Purification of Plasma Membranes from Plants 1

    PubMed Central

    Yoshida, Shizuo; Uemura, Matsuo; Niki, Teruo; Sakai, Akira; Gusta, Lawrence V.

    1983-01-01

    A simplified method for the isolation of a plasma membrane-enriched fraction from plants utilizing an aqueous two-polymer phase system is outlined. Mainly, the plant used was Orchard grass (Dactylis glomerata L.). The two-phase system consisted of 5.6% (w/w) of dextran T500 and 5.6% (w/w) of polyethyleneglycol 4000 in 0.5 molar sorbitol-15 millimolar Tris-maleate (pH 7.3), and 30 millimolar NaCl. In this system, the plasma membranes and the other membranes were preferentially partitioned into the top phase and into the lower phase, respectively. The purity of the isolated plasma membrane was sufficiently high even after a single partition (i.e. about 85% purity) and more than 90% purity was obtained after repeating the partition in a newly prepared lower phase. The plasma membrane was identified with the aid of phosphotungstic acid-chromic acid stain and the association of vanadate-sensitive Mg2+-ATPase. The plasma membrane-associated ATPase had a pH optimum at 6.5 and showed a high specificity for Mg2+ and ATP. KCl stimulation was low (6% stimulation) at the pH optimum, but a relatively high stimulation (23%) occurred at pH 5.5. This method for plasma membrane isolation may be applicable to a wide variety of plants and plant tissue including green leaves. Images Fig. 8 PMID:16662942

  7. Expression of a Translationally Fused TAP-Tagged Plasma Membrane Proton Pump in Arabidopsis thaliana

    PubMed Central

    2015-01-01

    The Arabidopsis thaliana plasma membrane proton ATPase genes, AHA1 and AHA2, are the two most highly expressed isoforms of an 11 gene family and are collectively essential for embryo development. We report the translational fusion of a tandem affinity-purification tag to the 5′ end of the AHA1 open reading frame in a genomic clone. Stable expression of TAP-tagged AHA1 in Arabidopsis rescues the embryonic lethal phenotype of endogenous double aha1/aha2 knockdowns. Western blots of SDS-PAGE and Blue Native gels show enrichment of AHA1 in plasma membrane fractions and indicate a hexameric quaternary structure. TAP-tagged AHA1 rescue lines exhibited reduced vertical root growth. Analysis of the plasma membrane and soluble proteomes identified several plasma membrane-localized proteins with alterred abundance in TAP-tagged AHA1 rescue lines compared to wild type. Using affinity-purification mass spectrometry, we uniquely identified two additional AHA isoforms, AHA9 and AHA11, which copurified with TAP-tagged AHA1. In conclusion, we have generated transgenic Arabidopsis lines in which a TAP-tagged AHA1 transgene has complemented all essential endogenous AHA1 and AHA2 functions and have shown that these plants can be used to purify AHA1 protein and to identify in planta interacting proteins by mass spectrometry. PMID:24397334

  8. Molecular Characterization of the Plasma Membrane H+-ATPase, an Antifungal Target in Cryptococcus neoformans

    PubMed Central

    Soteropoulos, Patricia; Vaz, Tanya; Santangelo, Rosaria; Paderu, Padmaja; Huang, David Y.; Tamás, Markus J.; Perlin, David S.

    2000-01-01

    The Cryptococcus neoformans PMA1 gene, encoding a plasma membrane H+-ATPase, was isolated from a genomic DNA library of serotype A strain ATCC 6352. An open reading frame of 3,380 nucleotides contains six introns and encodes a predicted protein consisting of 998 amino acids with a molecular mass of approximately 108 kDa. Plasma membranes were isolated, and the H+-ATPase was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be slightly larger than the S. cerevisiae H+-ATPase, consistent with its predicted molecular mass. The plasma membrane-bound enzyme exhibited a pH 6.5 optimum for ATP hydrolysis, Km and Vmax values of 0.5 mM and 3.1 μmol mg−1 min−1, respectively, and an apparent Ki for vanadate inhibition of 1.6 μM. ATP hydrolysis in plasma membranes and medium acidification by whole cells were inhibited by ebselen, a nonspecific H+-ATPase antagonist which was also fungicidal. The predicted C. neoformans protein is 35% identical to proton pumps of both pathogenic and nonpathogenic fungi but exhibits more than 50% identity to PMA1 genes from plants. Collectively, this study provides the basis for establishing the Cryptococcus H+-ATPase as a viable target for antifungal drug discovery. PMID:10952578

  9. Method of preparing water purification membranes. [polymerization of allyl amine as thin films in plasma discharge

    NASA Technical Reports Server (NTRS)

    Hollahan, J. R.; Wydeven, T. J., Jr. (Inventor)

    1974-01-01

    Allyl amine and chemically related compounds are polymerized as thin films in the presence of a plasma discharge. The monomer compound can be polymerized by itself or in the presence of an additive gas to promote polymerization and act as a carrier. The polymerized films thus produced show outstanding advantages when used as reverse osmosis membranes.

  10. Purification of the synaptosomal plasma membrane (Ca(2+) + Mg(2+))-ATPase from pig brain.

    PubMed Central

    Salvador, J M; Mata, A M

    1996-01-01

    The Ca(2+)-ATPase from the synaptosomal plasma membrane has been purified nearly to homogeneity from pig brain by a new procedure involving the calmodulin-affinity-chromatography technique. This is a convenient alternative to the standard methods for the purification of the plasma membrane Ca(2+)-ATPase from different sources that were unsuitable to purify the enzyme from pig brain. The main feature of this procedure is the use of 15% (v/v) glycerol as stabilizing agent, instead of acidic phospholipid. By using this protocol the enzyme was purified 36-fold with respect to the plasma membrane vesicle fraction, showing a specific activity of 2.3 i.u. in the presence of acidic phospholipid. In SDS/PAGE, it appears as a single protein band around Mr140 000 that can be phosphorylated by [gamma-(32)P]ATP in the presence of La(3+) and recognized by specific antibodies against the plasma membrane Ca(2+)-ATPase from pig antral smooth muscle. Calmodulin activates the enzyme 1.5-1.8-fold in the presence of phosphatidylcholine but not in the presence of phosphatidylserine. PMID:8670105

  11. The apoptotic microtubule network preserves plasma membrane integrity during the execution phase of apoptosis.

    PubMed

    Sánchez-Alcázar, José A; Rodríguez-Hernández, Angeles; Cordero, Mario D; Fernández-Ayala, Daniel J M; Brea-Calvo, Gloria; Garcia, Katherina; Navas, Plácido

    2007-07-01

    It has recently been shown that the microtubule cytoskeleton is reformed during the execution phase of apoptosis. We demonstrate that this microtubule reformation occurs in many cell types and under different apoptotic stimuli. We confirm that the apoptotic microtubule network possesses a novel organization, whose nucleation appears independent of conventional gamma-tubulin ring complex containing structures. Our analysis suggests that microtubules are closely associated with the plasma membrane, forming a cortical ring or cellular "cocoon". Concomitantly other components of the cytoskeleton, such as actin and cytokeratins disassemble. We found that colchicine-mediated disruption of apoptotic microtubule network results in enhanced plasma membrane permeability and secondary necrosis, suggesting that the reformation of a microtubule cytoskeleton plays an important role in preserving plasma membrane integrity during apoptosis. Significantly, cells induced to enter apoptosis in the presence of the pan-caspase inhibitor z-VAD, nevertheless form microtubule-like structures suggesting that microtubule formation is not dependent on caspase activation. In contrast we found that treatment with EGTA-AM, an intracellular calcium chelator, prevents apoptotic microtubule network formation, suggesting that intracellular calcium may play an essential role in the microtubule reformation. We propose that apoptotic microtubule network is required to maintain plasma membrane integrity during the execution phase of apoptosis.

  12. Early glycation products of endothelial plasma membrane proteins in experimental diabetes.

    PubMed

    Nguyen, Sarah; Pascariu, Mirela; Ghitescu, Lucian

    2006-01-01

    The participation of glucose and two intermediates of glucose metabolism: glucose-6-phosphate (G6P) and glyceraldehyde-3-phosphate (Gald3P) to the formation of early glycation products was comparatively evaluated in the endothelial plasma membrane of streptozotocin-induced diabetic rats. Antibodies risen to a carrier protein reductively glycated by each of the sugars mentioned above were used to probe by immunoblotting the proteins of the lung microvascular endothelium plasmalemma purified from normal and diabetic rats. The amount of glycated endothelial plasma membrane proteins was below the limit of detection in normoglycemic animals but increased dramatically in diabetic animals for glucose and G6P. In contrast, no signal was found in diabetic rats for Gald3P, indicating that either the contribution of this phosphotriose to the glycation of intracellular proteins is negligible in vivo, or the Schiff base generated by this sugar transforms very rapidly into products of advanced glycation. Globally, the endothelial plasma membrane proteins bound on average 300 times more glucose than G6P proving that, in spite of its low in vitro potency as glycating agent, glucose represents the main contributor to the intracellular formation of early glycation products. The most abundant glycated proteins of the lung endothelial plasma membrane were separated by two dimensional electrophoresis and identified by mass spectrometry.

  13. G protein-coupled receptor 30 is an estrogen receptor in the plasma membrane

    SciTech Connect

    Funakoshi, Takeshi; Yanai, Akie; Shinoda, Koh; Kawano, Michio M.; Mizukami, Yoichi . E-mail: mizukami@yamaguchi-u.ac.jp

    2006-08-04

    Recently, GPR30 was reported to be a novel estrogen receptor; however, its intracellular localization has remained controversial. To investigate the intracellular localization of GPR30 in vivo, we produced four kinds of polyclonal antibodies for distinct epitopes on GPR30. Immunocytochemical observations using anti-GPR30 antibody and anti-FLAG antibody show that FLAG-GPR30 localizes to the plasma membrane 24 h after transfection. Treatment with estrogen (17{beta}-estradiol or E2) causes an elevation in the intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}) within 10 s in HeLa cells expressing FLAG-GPR30. In addition, E2 induces the translocation of GPR30 from the plasma membrane to the cytoplasm by 1 h after stimulation. Immunohistochemical analysis shows that GPR30 exists on the cell surface of CA2 pyramidal neuronal cells. The images on transmission electron microscopy show that GPR30 is localized to a particular region associated with the plasma membranes of the pyramidal cells. These data indicate that GPR30, a transmembrane receptor for estrogen, is localized to the plasma membrane of CA2 pyramidal neuronal cells of the hippocampus in rat brain.

  14. Modified by air plasma polymer tack membranes as drainage material for antiglaucomatous operations

    NASA Astrophysics Data System (ADS)

    Ryazantseva, T. V.; Kravets, L. I.; Elinson, V. M.

    2014-06-01

    The morphological and clinical studies of poly(ethylene terephthalate) track membranes modified by air plasma as drainage materials for antiglaucomatous operations were performed. It was demonstrated their compatibility with eye tissues. Moreover, it was shown that a new drainage has a good lasting hypotensive effect and can be used as operation for refractory glaucoma surgery.

  15. Plasma chemical modification of track-etched membrane surface layer for improvement of their biomedical properties

    NASA Astrophysics Data System (ADS)

    Kravets, Liubov I.; Ryazantseva, Tatyana V.

    2013-12-01

    The morphological and clinical studies of poly(ethylene terephthalate) track-etched membrane modified by plasma of non-polymerizing gases as drainage materials for antiglaucomatous operations were performed. It was demonstrated their compatibility with eye tissues. Moreover, it was shown that a new drainage has a good lasting hypotensive effect and can be used as operation for refractory glaucoma surgery.

  16. Video Views and Reviews: Golgi Export, Targeting, and Plasma Membrane Caveolae

    ERIC Educational Resources Information Center

    Watters, Christopher

    2004-01-01

    In this article, the author reviews videos from "Molecular Biology of the Cell (MBC)" depicting various aspects of plasma membrane (PM) dynamics, including the targeting of newly synthesized components and the organization of those PM invaginations called caveolae. The papers accompanying these videos describe, respectively, the constitutive…

  17. Comparison of nanowire pellicles for plasma membrane enrichment: coating nanowires on cell

    PubMed Central

    Kim, Sung-Kyoung; Rose, Rebecca; Choksawangkarn, Waeowalee; Graham, Lauren; Hu, Junkai; Fenselau, Catherine; Lee, Sang Bok

    2014-01-01

    A study is reported on the effect of nanowire density on the ease of pellicle formation and the enrichment of plasma membrane proteins for analysis by mass spectrometry. An optimized synthesis is reported for iron silicate nanowires with a narrow size range of 900 ±400 nm in length and 200 nm diameter. The nanowires were coated with Al2O3 and used to form pellicles around suspended multiple myeloma cells, which acted as a model for cells recovered from tissue samples. Lighter alumina-coated silica nanowires were also synthesized (Kim et al. 2013), which allowed a comparison of the construction of the two pellicles and of the effect of nanowire density on plasma membrane enrichment. Evidence is offered that the dense nanowire pellicle does not crush or distort these mammalian cells. Finally, the pellicles were incorporated into a mass-spectrometry-based proteomic workflow to analyze transmembrane proteins in the plasma membrane. In contrast to a prior comparison of the effect of density with nanoparticles pellicles (Choksawangkarn et al. 2013), nanowire density was not found to significantly affect the enrichment of the plasma membrane. However, nanowires with a favorable aspect for pellicle formation are more easily and reliably produced with iron silicate than with silica. Additionally, the method for pellicle formation was optimized through the use of iron silicate nanowires (ISNW), which is crucial to the improvement of PM protein enrichment and analysis. PMID:24465155

  18. Independent Localization of Plasma Membrane and Chloroplast Components during Eyespot Assembly

    PubMed Central

    Mittelmeier, Telsa M.; Thompson, Mark D.; Öztürk, Esra

    2013-01-01

    Like many algae, Chlamydomonas reinhardtii is phototactic, using two anterior flagella to swim toward light optimal for photosynthesis. The flagella are responsive to signals initiated at the photosensory eyespot, which comprises photoreceptors in the plasma membrane and layers of pigment granules in the chloroplast. Phototaxis depends on placement of the eyespot at a specific asymmetric location relative to the flagella, basal bodies, and bundles of two or four highly acetylated microtubules, termed rootlets, which extend from the basal bodies toward the posterior of the cell. Previous work has shown that the eyespot is disassembled prior to cell division, and new eyespots are assembled in daughter cells adjacent to the nascent four-membered rootlet associated with the daughter basal body (D4), but the chronology of these assembly events has not been determined. Here we use immunofluorescence microscopy to follow assembly and acetylation of the D4 rootlet, localization of individual eyespot components in the plasma membrane or chloroplast envelope, and flagellar emergence during and immediately following cell division. We find that the D4 rootlet is assembled before the initiation of eyespot assembly, which occurs within the same time frame as rootlet acetylation and flagellar outgrowth. Photoreceptors in the plasma membrane are correctly localized in eyespot mutant cells lacking pigment granule layers, and chloroplast components of the eyespot assemble in mutant cells in which photoreceptor localization is retarded. The data suggest that plasma membrane and chloroplast components of the eyespot are independently responsive to a cytoskeletal positioning cue. PMID:23873865

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

    PubMed

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

    1993-05-01

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

  20. Moesin, ezrin, and p205 are actin-binding proteins associated with neutrophil plasma membranes.

    PubMed Central

    Pestonjamasp, K; Amieva, M R; Strassel, C P; Nauseef, W M; Furthmayr, H; Luna, E J

    1995-01-01

    Actin-binding proteins in bovine neutrophil plasma membranes were identified using blot overlays with 125I-labeled F-actin. Along with surface-biotinylated proteins, membranes were enriched in major actin-binding polypeptides of 78, 81, and 205 kDa. Binding was specific for F-actin because G-actin did not bind. Further, unlabeled F-actin blocked the binding of 125I-labeled F-actin whereas other acidic biopolymers were relatively ineffective. Binding also was specifically inhibited by myosin subfragment 1, but not by CapZ or plasma gelsolin, suggesting that the membrane proteins, like myosin, bind along the sides of the actin filaments. The 78- and 81-kDa polypeptides were identified as moesin and ezrin, respectively, by co-migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoprecipitation with antibodies specific for moesin and ezrin. Although not present in detectable amounts in bovine neutrophils, radixin (a third and closely related member of this gene family) also bound 125I-labeled F-actin on blot overlays. Experiments with full-length and truncated bacterial fusion proteins localized the actin-binding site in moesin to the extreme carboxy terminus, a highly conserved sequence. Immunofluorescence micrographs of permeabilized cells and cell "footprints" showed moesin co-localization with actin at the cytoplasmic surface of the plasma membrane, consistent with a role as a membrane-actin-linking protein. Images PMID:7612961

  1. Water/O2-plasma-assisted treatment of PCL membranes for biosignal immobilization.

    PubMed

    Saşmazel, Hilal Türkoğlu; Manolache, Sorin; Gümüşderelioğlu, Menemşe

    2009-01-01

    The main purpose of this study was to obtain COOH functionalities on the surface of poly-epsilon-caprolactone (PCL) membranes using low-pressure water/O(2)-plasma-assisted treatment. PCL membranes were prepared using the solvent-casting technique. Then, low-pressure water/O(2) plasma treatments were performed in a cylindrical, capacitively coupled RF-plasma-reactor in three steps: H(2)O/O(2)-plasma treatment; in situ (oxalyl chloride vapors) gas/solid reaction to convert -OH functionalities into -COCl groups; and hydrolysis for final -COOH functionalities. Optimization of plasma modification processes was done using the DoE software program. COOH and OH functionalities on modified surfaces were detected quantitatively using the fluorescent labeling technique and an UVX 300G sensor. Chemical structural information of untreated, plasma treated and oxalyl chloride functionalized PCL membranes were acquired using pyrolysis GC/MS and ESCA analysis. High-resolution AFM images revealed that nanopatterns were more affected than micropatterns by plasma treatments. AFM images recorded with amino-functionalized tips presented increased size of the features on the surface that suggests higher density of the carboxyls on the nanotopographical elements. Low-pressure water/O(2)-plasma-treated and oxalyl chloride functionalized samples were biologically activated with insulin and/or heparin biosignal molecules using a PEO (polyoxyethylene bis amine) spacer. The success of the immobilization process was checked qualitatively by ESCA analysis. In addition, fluorescent labeling techniques were used for the quantitative determination of immobilized biomolecules. Cell-culture experiments indicated that biomolecule immobilization onto PCL scaffolds was effective on L929 cell adhesion and proliferation, especially in the presence of heparin.

  2. Coating cells with cationic silica-magnetite nanocomposites for rapid purification of integral plasma membrane proteins.

    PubMed

    Zhang, Wei; Zhao, Chao; Wang, Sheng; Fang, Caiyun; Xu, Yawei; Lu, Haojie; Yang, Pengyuan

    2011-09-01

    This study developed a simple and rapid purification method for plasma membrane with high yields from adherent cells. The plasma membrane (PM) sheets could be absorbed specifically by the cationic silica-magnetite nanocomposites (CSMN) under acidic conditions, and recovered directly in cell-lysis-buffer with no need for precipitation. The binding between CSMN and PM sheets was confirmed by electron microscopy. Western blot analysis demonstrated a >10-fold relative enrichment factor. Up to 422 integral membrane proteins were identified from 10(7) Huh7 cells. Notably, we found 29 Ras family proteins by classification according to their biological functions. The whole enrichment procedure took <30 min. The CSMN-based procedure demonstrates a simple, economical and efficient enrichment of integral PM proteins in proteomic study.

  3. Differential effects of plasma membrane electric excitation on H+ fluxes and photosynthesis in characean cells.

    PubMed

    Bulychev, Alexander A; Kamzolkina, Natalia A

    2006-10-01

    Cells of characean algae exposed to illumination arrange plasma-membrane H(+) fluxes and photosynthesis in coordinated spatial patterns (bands). This study reveals that H(+) transport and photosynthesis patterns in these excitable cells are affected not only by light conditions but also by electric excitation of the plasma membrane. It is shown that generation of action potential (AP) temporally eliminates alkaline bands, suppresses O(2) evolution, and differentially affects primary reactions of photosystem II (PSII) in different cell regions. The quantum yield of PSII electron transport decreased after AP in the alkaline but not in acidic cell regions. The effects of electric excitation on fluorescence and the PSII electron flow were most pronounced at light-limiting conditions. Evidence was obtained that the shift in chlorophyll fluorescence after AP is due to the increase in DeltapH at thylakoid membranes. It is concluded that the AP-triggered pathways affecting ion transport and photosynthetic energy conversion are linked but not identical.

  4. The Structure of the Yeast Plasma Membrane SNARE Complex Reveals Destabilizing Water Filled Cavities

    SciTech Connect

    Strop, P.; Kaiser, S.E.; Vrljic, M.; Brunger, A.T.

    2009-05-26

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

  5. Inhibition of cell adhesion by xARVCF indicates a regulatory function at the plasma membrane.

    PubMed

    Reintsch, Wolfgang E; Mandato, Craig A; McCrea, Pierre D; Fagotto, François

    2008-09-01

    The cytoplasmic tail of cadherins is thought to regulate the strength and dynamics of cell-cell adhesion. Part of its regulatory activity has been attributed to a membrane-proximal region, the juxtamembrane domain (JMD), and its interaction with members of the p120 catenin subfamily. We show that titration of xARVCF, a member of this family, to the plasma membrane disrupts adhesion in the early embryo. Adhesion can be restored by coexpression of constitutively active Rac, suggesting that intracellular signaling is the primary cause in the loss of adhesion phenotype. Our observations suggest that the recruitment of p120 type catenins to the plasma membrane by the cadherin cytoplasmic tail may create protein complexes, which actively modulate the adhesion "status" of embryonic cells.

  6. Alternative mechanism of Eimeria bovis sporozoites to invade cells in vitro by breaching the plasma membrane.

    PubMed

    Behrendt, J H; Clauss, W; Zahner, H; Hermosilla, C

    2004-10-01

    In vitro Eimeria bovis sporozoites invade a wide range of cell types, and in the case of bovine cells, they may develop to first-generation schizonts. Often, however, they subsequently leave their host cell to invade a new one, which seems contrary to the classical way of infecting a cell by forming a parasitophorous vacuole. Using a standard, "cell wound assay," we show that E. bovis can invade bovine endothelial cells by breaching the plasma membrane and may again leave the surviving cell. Eimeria bovis sporozoites also infected VERO and HT29 cells but obviously without damaging the plasma membrane. The same held true when bovine endothelial cells were exposed to tachyzoites of Toxoplasma gondii and Neospora caninum. According to a literature report dealing with Plasmodium yoelii sporozoites, breaching the membrane of certain host cells may be a common phenomenon for coccidian sporozoites but may not be for merozoites.

  7. Evidence for a plasma-membrane-bound nitrate reductase involved in nitrate uptake of Chlorella sorokiniana

    NASA Technical Reports Server (NTRS)

    Tischner, R.; Ward, M. R.; Huffaker, R. C.

    1989-01-01

    Anti-nitrate-reductase (NR) immunoglobulin-G (IgG) fragments inhibited nitrate uptake into Chlorella cells but had no affect on nitrate uptake. Intact anti-NR serum and preimmune IgG fragments had no affect on nitrate uptake. Membrane-associated NR was detected in plasma-membrane (PM) fractions isolated by aqueous two-phase partitioning. The PM-associated NR was not removed by sonicating PM vesicles in 500 mM NaCl and 1 mM ethylenediaminetetraacetic acid and represented up to 0.8% of the total Chlorella NR activity. The PM NR was solubilized by Triton X-100 and inactivated by Chlorella NR antiserum. Plasma-membrane NR was present in ammonium-grown Chlorella cells that completely lacked soluble NR activity. The subunit sizes of the PM and soluble NRs were 60 and 95 kDa, respectively, as determined by sodium-dodecyl-sulfate electrophoresis and western blotting.

  8. Phospholipid Flippase ATP10A Translocates Phosphatidylcholine and Is Involved in Plasma Membrane Dynamics*

    PubMed Central

    Naito, Tomoki; Takatsu, Hiroyuki; Miyano, Rie; Takada, Naoto; Nakayama, Kazuhisa; Shin, Hye-Won

    2015-01-01

    We showed previously that ATP11A and ATP11C have flippase activity toward aminophospholipids (phosphatidylserine (PS) and phosphatidylethanolamine (PE)) and ATP8B1 and that ATP8B2 have flippase activity toward phosphatidylcholine (PC) (Takatsu, H., Tanaka, G., Segawa, K., Suzuki, J., Nagata, S., Nakayama, K., and Shin, H. W. (2014) J. Biol. Chem. 289, 33543–33556). Here, we show that the localization of class 5 P4-ATPases to the plasma membrane (ATP10A and ATP10D) and late endosomes (ATP10B) requires an interaction with CDC50A. Moreover, exogenous expression of ATP10A, but not its ATPase-deficient mutant ATP10A(E203Q), dramatically increased PC flipping but not flipping of PS or PE. Depletion of CDC50A caused ATP10A to be retained at the endoplasmic reticulum instead of being delivered to the plasma membrane and abrogated the increased PC flipping activity observed by expression of ATP10A. These results demonstrate that ATP10A is delivered to the plasma membrane via its interaction with CDC50A and, specifically, flips PC at the plasma membrane. Importantly, expression of ATP10A, but not ATP10A(E203Q), dramatically altered the cell shape and decreased cell size. In addition, expression of ATP10A, but not ATP10A(E203Q), delayed cell adhesion and cell spreading onto the extracellular matrix. These results suggest that enhanced PC flipping activity due to exogenous ATP10A expression alters the lipid composition at the plasma membrane, which may in turn cause a delay in cell spreading and a change in cell morphology. PMID:25947375

  9. Voltage-dependent calcium-permeable channels in the plasma membrane of a higher plant cell.

    PubMed Central

    Thuleau, P; Ward, J M; Ranjeva, R; Schroeder, J I

    1994-01-01

    Numerous biological assays and pharmacological studies on various higher plant tissues have led to the suggestion that voltage-dependent plasma membrane Ca2+ channels play prominent roles in initiating signal transduction processes during plant growth and development. However, to date no direct evidence has been obtained for the existence of such depolarization-activated Ca2+ channels in the plasma membrane of higher plant cells. Carrot suspension cells (Daucus carota L.) provide a well-suited system to determine whether voltage-dependent Ca2+ channels are present in the plasma membrane of higher plants and to characterize the properties of putative Ca2+ channels. It is known that both depolarization, caused by raising extracellular K+, and exposure to fungal toxins or oligogalacturonides induce Ca2+ influx into carrot cells. By direct application of patch-clamp techniques to isolated carrot protoplasts, we show here that depolarization of the plasma membrane positive to -135 mV activates Ca(2+)-permeable channels. These voltage-dependent ion channels were more permeable to Ca2+ than K+, while displaying large permeabilities to Ba2+ and Mg2+ ions. Ca(2+)-permeable channels showed slow and reversible inactivation. The single-channel conductance was 13 pS in 40 mM CaCl2. These data provide direct evidence for the existence of voltage-dependent Ca2+ channels in the plasma membrane of a higher plant cell and point to physiological mechanisms for plant Ca2+ channel regulation. The depolarization-activated Ca(2+)-permeable channels identified here could constitute a regulated pathway for Ca2+ influx in response to physiologically occurring stimulus-induced depolarizations in higher plant cells. PMID:8039493

  10. Graphene Oxide Induced Perturbation to Plasma Membrane and Cytoskeletal Meshwork Sensitize Cancer Cells to Chemotherapeutic Agents.

    PubMed

    Zhu, Jianqiang; Xu, Ming; Gao, Ming; Zhang, Zhihong; Xu, Yong; Xia, Tian; Liu, Sijin

    2017-03-28

    The outstanding physicochemical properties endow graphene materials (e.g., graphene oxide, GO) with beneficial potentials in diverse biomedical fields such as bioimaging, drug delivery, and biomolecular detection. GO recently emerged as a chemosensitizer; however, the detailed molecular basis underlying GO-conducted sensitization and corresponding biological effects are still elusive. Based on our recent findings that GO treatment at sublethal concentrations could impair the general cellular priming state, including disorders of plasma membrane and cytoskeleton construction, we aimed here to explore the mechanism of GO as a sensitizer to make cancer cells more susceptible to chemotherapeutic agents. We discovered that GO could not only compromise plasma membrane and cytoskeleton in J774A.1 macrophages and A549 lung cancer cells at sublethal concentrations without incurring significant cell death but also dampen a number of biological processes. Using the toxicogenomics approaches, we laid out the gene expression signature affected by GO and further defined those genes involved in membrane and cytoskeletal impairments responding to GO. The mechanistic investigation uncovered that the interactions of GO-integrin occurred on the plasma membrane and consequently activated the integrin-FAK-Rho-ROCK pathway and suppressed the expression of integrin, resulting in compromised cell membrane and cytoskeleton and a subsequent cellular priming state. By making use of this mechanism, the efficacy of chemotherapeutic agents (e.g., doxorubicin and cisplatin) could be enhanced by GO pretreatment in killing cancer cells. This study unveiled a feature of GO in cancer therapeutics: sensitizing cancer cells to chemotherapeutic agents by undermining the resistance capability of tumor cells against chemotherapeutic agents, at least partially, by compromising plasma membrane and cytoskeleton meshwork.

  11. Solubilization and Partial Purification of the Adenosine Triphosphatase from a Corn Root Plasma Membrane Fraction

    PubMed Central

    Dupont, Frances M.; Leonard, Robert T.

    1980-01-01

    The K+-stimulated ATPase was partially purified from a plasma membrane fraction from corn roots (WF9 × Mo 17) by solubilization with 30 millimolar octyl-β-d-glucopyranoside followed by precipitation with dilute ammonium sulfate. The specific activity of the enzyme was increased about five times by this procedure. The molecular weight of the detergent-extracted ATPase complex was estimated to be at least 500,000 daltons by chromatography on a Bio-Gel A-5m column. Negative staining electron microscopy indicated that the detergent-extracted material consisted of amorphous particles, while the ammonium sulfate precipitate was composed of uniform vesicles with an average diameter of 100 nanometers. The protein composition of the ammonium sulfate precipitate was significantly different from that of the plasma membrane fraction when compared by sodium dodecyl sulfate gel electrophoresis. The characteristics of the partially purified ATPase resembled those of the plasma membrane associated enzyme. The ATPase required Mg2+, was further stimulated by K+, was almost completely inhibited by 0.1 millimolar diethylstilbestrol, and was not affected by 5.0 micrograms per milliliter oligomycin. Although the detergents sodium cholate, deoxycholate, Triton X-100 and Lubrol WX also solubilized some membrane protein, none solubilized the K+-stimulated ATPase activity. Low concentrations of each detergent, including octyl-β-d-glucopyranoside, activated the ATPase and higher concentrations inactivated the enzyme. These results suggest that the plasma membrane ATPase is a large, integral membrane protein or protein complex that requires lipids to maintain its activity. Images PMID:16661309

  12. Active Trans-Plasma Membrane Water Cycling in Yeast Is Revealed by NMR

    PubMed Central

    Zhang, Yajie; Poirier-Quinot, Marie; Springer, Charles S.; Balschi, James A.

    2011-01-01

    Plasma membrane water transport is a crucial cellular phenomenon. Net water movement in response to an osmotic gradient changes cell volume. Steady-state exchange of water molecules, with no net flux or volume change, occurs by passive diffusion through the phospholipid bilayer and passage through membrane proteins. The hypothesis is tested that plasma membrane water exchange also correlates with ATP-driven membrane transport activity in yeast (Saccharomyces cerevisiae). Longitudinal 1H2O NMR relaxation time constant (T1) values were measured in yeast suspensions containing extracellular relaxation reagent. Two-site-exchange analysis quantified the reversible exchange kinetics as the mean intracellular water lifetime (τi), where τi−1 is the pseudo-first-order rate constant for water efflux. To modulate cellular ATP, yeast suspensions were bubbled with 95%O2/5%CO2 (O2) or 95%N2/5%CO2 (N2). ATP was high during O2, and τi−1 was 3.1 s−1 at 25°C. After changing to N2, ATP decreased and τi−1 was 1.8 s−1. The principal active yeast ion transport protein is the plasma membrane H+-ATPase. Studies using the H+-ATPase inhibitor ebselen or a yeast genetic strain with reduced H+-ATPase found reduced τi−1, notwithstanding high ATP. Steady-state water exchange correlates with H+-ATPase activity. At volume steady state, water is cycling across the plasma membrane in response to metabolic transport activity. PMID:22261073

  13. Water permeability of polyethylene terephthalate track membranes modified in plasma of dimethylaniline

    NASA Astrophysics Data System (ADS)

    Kravets, Lyubov; Dmitriev, Serguei; Gilman, Alla; Drachev, Alexander

    2004-09-01

    The surface properties and hydrodynamic characteristics of composite membranes consisting of a porous substrate, on which a polymer layer from a direct current discharge in a mixture of air and vapours of dimethylaniline was deposited, have been investigated. As a substrate, we used poly(ethylene) terephthalate track membrane (PET TM) of the thickness of 10 μ m and the effective pore diameter of 0.215 μ m (pore density is 2\\cdot 10^8 cm-2). The performed researches show that when treating the membranes in plasma, two competing processes are observed: deposition of the polymer layer on a membrane surface, that testifies increase of the mass of sample, and etching of a polymeric matrix which causes growth of effective pore diameter. The last process is stipulated by presence of oxygen in the gas mixture. Decreasing the degree of overweight of the sample at increasing the treatment time leads us to a supposition that a dominating process in this case becomes the process of gas-discharge etching. In all cases, if treating PET TM, a drop of the water contact angle occurs, i.e. hydrophilization of the membrane surface takes place that is connected first of all with a grafting of polymer layer containing polar functional groups. The research in the hydrodynamic characteristics of the initial PET TM and the membranes modified in plasma at neutral and subacid pH value of filtrate leads to a linear dependence of their permeability upon the quantity of applied pressure. It is connected with a viscous character of the flow, that is, when the diameter of the pores of the membrane is much more than the size of the water molecules. This fact shows that the macromolecules of the deposited polymer layer in this case have a compact conformation, which does not hinder the water molecules infiltration. At a lower pH value of the filtrate, the picture cardinally changes. For modified in plasma membranes a diversion from the linear relation is observed. This means that in this case

  14. Plasma Membrane Sterol Distribution Resembles the Surface Topography of Living Cells

    PubMed Central

    2007-01-01

    Cholesterol is an important constituent of cellular membranes. It has been suggested that cholesterol segregates into sterol-rich and -poor domains in the plasma membrane, although clear evidence for this is lacking. By fluorescence imaging of the natural sterol dehydroergosterol (DHE), the lateral sterol distribution has been visualized in living cells. The spatial labeling pattern of DHE coincided with surface structures such as ruffles, microvilli, and filopodia with correlation lengths in the range of 0.8–2.5 μm. DHE staining of branched tubules and of nanotubes connecting two cells was detected. Dynamics of DHE in folded and plane membrane regions was comparable as determined by fluorescence recovery after photobleaching. DHE colocalized with fluid membrane-preferring phospholipids in surface structures and at sites of cell attachment as well as in the cleavage furrow of dividing cells, but it was not particularly enriched in those regions. Fluorescent sterol showed homogeneous staining in membrane blebs induced by F-actin disruption. Cross-linking the ganglioside GM1—a putative raft marker—did not affect the cell surface distribution of DHE. The results suggest that spatial heterogeneities of plasma membrane staining of DHE resolvable by light microscopy reflect the cell surface topography but not phase-separated sterol domains in the bilayer plane. PMID:17065557

  15. Polyethylene glycol acrylate-grafted polysulphone membrane for artificial lungs: plasma modification and haemocompatibility improvement.

    PubMed

    Wang, Weiping; Huang, Xin; Yin, Haiyan; Fan, Wenling; Zhang, Tao; Li, Lei; Mao, Chun

    2015-12-14

    In this study, polyethylene glycol acrylate (PEGA) was introduced onto the surface of polysulphone (PSF) membrane to prepare PSF-PEGA membranes through low-temperature plasma technology for haemocompatibility improvement of artificial lungs. The effects of plasma power, PEGA solution concentration and dipcoating temperature on surface modification were systematically investigated. Results of Fourier transform infrared spectroscopy, x-ray photoelectron spectroscopy and PEGA grafting degree confirmed that PEGA was successfully grafted onto the PSF membranes. Contact angle values showed that the hydrophilicity of the PSF-PEGA membrane surface increased by 21.5%. The results of the protein adsorption, platelet adhesion and coagulation tests further showed the excellent haemocompatibility of the modified membrane. Gas exchange tests also revealed that at a porcine blood flow rate of 5 l min(-1), O2 and CO2 exchange rates through the PSF-PEGA membrane were 198.6 and 170.9 ml min(-1), respectively; approximately this is the gas exchange capacity of commercial respiratory assistance devices.

  16. Short-Lived Cages Restrict Protein Diffusion in the Plasma Membrane

    PubMed Central

    Goiko, Maria; de Bruyn, John R.; Heit, Bryan

    2016-01-01

    The plasma membrane is a heterogeneous environment characterized by anomalous diffusion and the presence of microdomains that are molecularly distinct from the bulk membrane. Using single particle tracking of the C-type lectin CD93, we have identified for the first time the transient trapping of transmembrane proteins in cage-like microdomains which restrict protein diffusion. These cages are stabilized by actin-dependent confinement regions, but are separate structures with sizes and lifespans uncorrelated to those of the underlying actin corral. These membrane cages require cholesterol for their strength and stability, with cholesterol depletion decreasing both. Despite this, cages are much larger in size and are longer lived than lipid rafts, suggesting instead that cholesterol-dependent effects on membrane fluidity or molecular packing play a role in cage formation. This diffusional compartment in the plasma membrane has characteristics of both a diffusional barrier and a membrane microdomain, with a size and lifespan intermediate between short-lived microdomains such as lipid rafts and long-lasting diffusional barriers created by the actin cytoskeleton. PMID:27725698

  17. Forward transport of proteins in the plasma membrane of migrating cerebellar granule cells.

    PubMed

    Wang, Dong; She, Liang; Sui, Ya-nan; Yuan, Xiao-bing; Wen, Yunqing; Poo, Mu-ming

    2012-12-18

    Directional flow of membrane components has been detected at the leading front of fibroblasts and the growth cone of neuronal processes, but whether there exists global directional flow of plasma membrane components over the entire migrating neuron remains largely unknown. By analyzing the trajectories of antibody-coated single quantum dots (QDs) bound to two membrane proteins, overexpressed myc-tagged synaptic vesicle-associated membrane protein VAMP2 and endogenous neurotrophin receptor TrkB, we found that these two proteins exhibited net forward transport, which is superimposed upon Brownian motion, in both leading and trailing processes of migrating cerebellar granule cells in culture. Furthermore, no net directional transport of membrane proteins was observed in nonmigrating cells with either growing or stalling leading processes. Analysis of the correlation of motion direction between two QDs on the same process in migrating neurons also showed a higher frequency of correlated forward than rearward movements. Such correlated QD movements were markedly reduced in the presence of myosin II inhibitor blebbistatin,suggesting the involvement of myosin II-dependent active transport processes. Thus, a net forward transport of plasma membrane proteins exists in the leading and trailing processes of migrating neurons, in line with the translocation of the soma.

  18. Mixed matrix hollow fiber membranes for removal of protein-bound toxins from human plasma.

    PubMed

    Tijink, Marlon S L; Wester, Maarten; Glorieux, Griet; Gerritsen, Karin G F; Sun, Junfen; Swart, Pieter C; Borneman, Zandrie; Wessling, Matthias; Vanholder, Raymond; Joles, Jaap A; Stamatialis, Dimitrios

    2013-10-01

    In end stage renal disease (ESRD) waste solutes accumulate in body fluid. Removal of protein bound solutes using conventional renal replacement therapies is currently very poor while their accumulation is associated with adverse outcomes in ESRD. Here we investigate the application of a hollow fiber mixed matrix membrane (MMM) for removal of these toxins. The MMM hollow fiber consists of porous macro-void free polymeric inner membrane layer well attached to the activated carbon containing outer MMM layer. The new membranes have permeation properties in the ultrafiltration range. Under static conditions, they adsorb 57% p-cresylsulfate, 82% indoxyl sulfate and 94% of hippuric acid from spiked human plasma in 4 h. Under dynamic conditions, they adsorb on average 2.27 mg PCS/g membrane and 3.58 mg IS/g membrane in 4 h in diffusion experiments and 2.68 mg/g membrane PCS and 12.85 mg/g membrane IS in convection experiments. Based on the dynamic experiments we estimate that our membranes would suffice to remove the daily production of these protein bound solutes.

  19. A method to modify PVDF microfiltration membrane via ATRP with low-temperature plasma pretreatment

    NASA Astrophysics Data System (ADS)

    Han, Yu; Song, Shuijun; Lu, Yin; Zhu, Dongfa

    2016-08-01

    The hydrophilic modification of a polyvinylidene fluoride (PVDF) microfiltration membrane via pretreatment with argon plasma and direct surface-initiated atom transfer radical polymerization (ATRP) was studied. Both modified and unmodified PVDF membranes were characterized by Fourier transform infrared spectroscopy (FTIR), water contact angle, scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and pore size distribution measurements. FTIR and XPS spectra confirmed that sulfobetaine methacrylate (SBMA) had been grafted onto the membrane surface. The initial contact angle decreased from 87.0° to 29.8° and a water drop penetrated into the modified membrane completely in 8 s. The pore size distribution of the modified membrane exhibited a smaller mean value than that of the original membrane. The antifouling properties of the modified PVDF membrane were evaluated by a filtration test using bovine serum albumin (BSA) solution. The results showed that the initial flux of the modified membrane increased from 2140.1 L/m2 h to 2812.7 L/m2 h and the equilibrium flux of BSA solution increased from 31 L/m2 h to 53 L/m2 h.

  20. Membrane fusion-competent virus-like proteoliposomes and proteinaceous supported bilayers made directly from cell plasma membranes.

    PubMed

    Costello, Deirdre A; Hsia, Chih-Yun; Millet, Jean K; Porri, Teresa; Daniel, Susan

    2013-05-28

    Virus-like particles are useful materials for studying virus-host interactions in a safe manner. However, the standard production of pseudovirus based on the vesicular stomatitis virus (VSV) backbone is an intricate procedure that requires trained laboratory personnel. In this work, a new strategy for creating virus-like proteoliposomes (VLPLs) and virus-like supported bilayers (VLSBs) is presented. This strategy uses a cell blebbing technique to induce the formation of nanoscale vesicles from the plasma membrane of BHK cells expressing the hemagglutinin (HA) fusion protein of influenza X-31. These vesicles and supported bilayers contain HA and are used to carry out single particle membrane fusion events, monitored using total internal reflection fluorescence microscopy. The results of these studies show that the VLPLs and VLSBs contain HA proteins that are fully competent to carry out membrane fusion, including the formation of a fusion pore and the release of fluorophores loaded into vesicles. This new strategy for creating spherical and planar geometry virus-like membranes has many potential applications. VLPLs could be used to study fusion proteins of virulent viruses in a safe manner, or they could be used as therapeutic delivery particles to transport beneficial proteins coexpressed in the cells to a target cell. VLSBs could facilitate high throughput screening of antiviral drugs or pathogen-host cell interactions.

  1. Effects of vitamin E supplementation on plasma membrane permeabilization and fluidization induced by chlorpromazine in the rat brain.

    PubMed

    Maruoka, Nobuyuki; Murata, Tetsuhito; Omata, Naoto; Takashima, Yasuhiro; Fujibayashi, Yasuhisa; Wada, Yuji

    2008-03-01

    Neurotransmitter receptors play a key role in most research on antipsychotic drugs, but little is known about the effects of these drugs on the plasma membrane in the central nervous system. Therefore, we investigated whether chlorpromazine (CPZ), a typical phenothiazine antipsychotic drug, affects the plasma membrane integrity in the rat brain, and if so, whether these membrane alterations can be prevented by dietary supplementation with vitamin E, which has been shown to be an antioxidant and also a membrane-stabilizer. Leakage of [(18)F]2-fluoro-2-deoxy-D-glucose ([(18)F]FDG)-6-phosphate from rat striatal slices and decrease in 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy were used as indexes for plasma membrane permeabilization and fluidization, respectively. CPZ induced leakage of [(18)F]FDG-6-phosphate from striatal slices, and the leakage was delayed in the vitamin E-supplemented group compared to that in the normal diet group. The decrease in plasma membrane anisotropy induced by CPZ was significantly attenuated by vitamin E supplementation. Chronic treatment with alpha-phenyl-N-tert-butyl nitrone, a free radical scavenger, had no effect on CPZ-induced plasma membrane permeabilization, and the treatment with CPZ did not induce lipid peroxidation. CPZ can reduce plasma membrane integrity in the brain, and this reduction can be prevented by vitamin E via its membrane-stabilizing properties, not via its antioxidant activity.

  2. H+-Pumping Driven by the Plasma Membrane ATPase in Membrane Vesicles from Radish: Stimulation by Fusicoccin 1

    PubMed Central

    Rasi-Caldogno, Franca; De Michelis, Maria I.; Pugliarello, Maria C.; Marrè, Erasmo

    1986-01-01

    The effect of fusicoccin on Mg:ATP-dependent H+-pumping in microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings was investigated by measuring the initial rate of decrease in the absorbance of the ΔpH probe acridine orange. Fusicoccin stimulated Mg:ATP-dependent H+-pumping when the pH of the assay medium was in the range 7.0 to 7.6 while no effect of fusicoccin was detected between pH 6.6 and pH 6.0. Both basal and fusicoccin-stimulated H+-pumping were completely inhibited by vanadate and almost unaffected by nitrate. Fusicoccin did not change membrane permeability to protons and fusicoccin-induced stimulation of Mg:ATP-dependent H+-pumping was not affected by changes in the buffer capacity of the incubation medium. Deacetylfusicoccin stimulated H+-pumping as much as fusicoccin, while the physiologically inactive derivative 8-oxo-9-epideacetylfusicoccin did not. Stimulation of H+-pumping was saturated by 100 nanomolar fusicoccin. These data indicate that fusicoccin activates the plasma membrane H+-ATPase by acting at the membrane level independently of the involvement of other cell components. The percent stimulation by fusicoccin was the same at all ATP concentrations tested (0.5-5.0 millimolar), thus suggesting that with fusicoccin there is an increase in Vmax of the plasma membrane H+-ATPase rather than a decrease in its apparent Km for Mg:ATP. PMID:16664978

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

    PubMed

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

    2017-03-01

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

  4. Identification of detergent-resistant plasma membrane microdomains in dictyostelium: enrichment of signal transduction proteins.

    PubMed Central

    Xiao, Z; Devreotes, P N

    1997-01-01

    Unlike most other cellular proteins, the chemoattractant receptor, cAR1, of Dictyostelium is resistant to extraction by the zwitterionic detergent, CHAPS. We exploited this property to isolate a subcellular fraction highly enriched in cAR1 by flotation of CHAPS lysates of cells in sucrose density gradients. Immunogold electron microscopy studies revealed a homogeneous preparation of membrane bilayer sheets. This preparation, designated CHAPS-insoluble floating fraction (CHIEF), also contained a defined set of 20 other proteins and a single uncharged lipid. Cell surface biotinylation and preembedding immunoelectron microscopy both confirmed the plasma membrane origin of this preparation. The cell surface phosphodiesterase (PDE) and a downstream effector of cAR1, adenylate cyclase (ACA), were specifically localized in these structures, whereas the cell adhesion molecule gp80, most of the major cell surface membrane proteins, cytoskeletal components, the actin-binding integral membrane protein ponticulin, and G-protein alpha- and beta-subunits were absent. Overall, CHIFF represents about 3-5% of cell externally exposed membrane proteins. All of these results indicate that CHIFF is derived from specialized microdomains of the plasma membrane. The method of isolation is analogous to that of caveolae. However, we were unable to detect distinct caveolae-like structures on the cell surface associated with cAR1, which showed a diffuse staining profile. The discovery of CHIFF facilitates the purification of cAR1 and related signaling proteins and the biochemical characterization of receptor-mediated processes such as G-protein activation and desensitization. It also has important implications for the "fluid mosaic" model of the plasma membrane structures. Images PMID:9168471

  5. Single-channel response of hamster oocytes to fertilization with homologous spermatozoa.

    PubMed

    Ituarte, Leonor M E; Viera, Teresa B; Saldeña, Teobaldo A; de Rosas, Juan C; Fóscolo, Mabel; Ibáñez, Jorge E; Saraví, Fernando D

    2006-04-01

    Electrophysiological events occur early after fertilization, along with changes in intracellular Ca2+ concentration. Passive electrical parameters were determined in golden hamster oocytes by whole cell patch-clamp method. In separate experiments the effect of 4-aminopyridine on resting oocytes was tested. The single-channel patch clamp configuration was employed to assess the electrical response to fertilization with homologous sperm. Structure of oocytes submitted to patch clamp was evaluated with scanning electron microscopy and found to be preserved. Oocyte diameter was 70.2 +/- 2.2 microm; their resting parameters were: membrane potential 23.8 +/- 0.8 mV; total membrane specific resistance 519.1 +/- 94.6 ohms.cm2, and specific capacity 0.99 +/- 0.03 microF.cm(-2). Total membrane current was decreased by 42 % by 4-aminopyridine. Control oocytes and oocytes exposed to sperm differed in their membrane currents in response to a voltage ramp clamping membrane potential from - 100 mV to + 100 mV. In both cases, currents were largest at the most negative potentials, but sperm-exposed oocytes had larger currents. Additionally, while in control oocytes the current was inward at negative potentials but outward at positive potentials, in the presence of spermatozoa oocytes was inward within the whole voltage range tested. This latter current may represent Ca2+ entry.

  6. On-chip enucleation of an oocyte by untethered microrobots

    NASA Astrophysics Data System (ADS)

    Ichikawa, Akihiko; Sakuma, Shinya; Sugita, Masakuni; Shoda, Tatsuro; Tamakoshi, Takahiro; Akagi, Satoshi; Arai, Fumihito

    2014-09-01

    We propose a novel on-chip enucleation of an oocyte with zona pellucida by using a combination of untethered microrobots. To achieve enucleation within the closed space of a microfluidic chip, two microrobots, a microknife and a microgripper were integrated into the microfluidic chip. These microrobots were actuated by an external magnetic force produced by permanent magnets placed on the robotic stage. The tip of the microknife was designed by considering the biological geometric feature of an oocyte, i.e. the oocyte has a polar body in maturation stage II. Moreover, the microknife was fabricated by using grayscale lithography, which allows fabrication of three-dimensional microstructures. The microgripper has a gripping function that is independent of the driving mechanism. On-chip enucleation was demonstrated, and the enucleated oocytes are spherical, indicating that the cell membrane of the oocytes remained intact. To confirm successful enucleation using this method, we investigated the viability of oocytes after enucleation. The results show that the production rate, i.e. the ratio between the number of oocytes that reach the blastocyst stage and the number of bovine oocytes after nucleus transfer, is 100%. The technique will contribute to complex cell manipulation such as cell surgery in lab-on-a-chip devices.

  7. Co-overexpressing a plasma membrane and a vacuolar membrane sodium/proton antiporter significantly improves salt tolerance in transgenic Arabidopsis plants.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Arabidopsis gene AtNHX1 encodes a vacuolar membrane bound sodium/proton (Sodium/Hydrogen) antiporter that transports sodium into the vacuole and exports hydrogen into the cytoplasm. The Arabidopsis gene SOS1 encodes a plasma membrane bound sodium/hydrogen antiporter that exports sodium to the ex...

  8. Effect of bovine oviduct epithelial cell apical plasma membranes on sperm function assessed by a novel flow cytometric approach.

    PubMed

    Boilard, Mathieu; Bailey, Janice; Collin, Simon; Dufour, Maurice; Sirard, Marc-André

    2002-10-01

    In the bovine, as in many mammalian species, sperm are temporarily stored in the oviduct before fertilization by binding to the oviduct epithelial cell apical plasma membranes. As the oviduct is able to maintain motility and viability of sperm and modulate capacitation, we propose that proteins present on the apical plasma membrane of oviduct epithelial cells contribute to these effects. To verify this hypothesis, the motility of frozen-thawed sperm was determined after incubation for 6 h with purified apical plasma membranes from fresh or cultured oviduct epithelial cells or from bovine mammary gland cells as a control. Analysis of intracellular calcium levels was performed by flow cytometry on sperm incubated with fresh membranes using Indo-1 to assess the membrane effect on intracellular calcium concentration. The coculture of sperm with fresh and cultured apical membranes maintained initial motility for 6 h (65% and 84%, respectively). This effect was significantly different from control sperm incubated without oviduct epithelial cell apical membranes (23%), with mammary gland cell apical membranes (23%), or with boiled epithelial cell apical membranes (21%). Apical membranes from oviduct epithelial cells diminished the percentage of sperm that reached a lethal calcium concentration over a 4-h period (18.7%) compared with the control (53.8%) and maintained lower intracellular calcium levels in viable sperm. These results show that the apical plasma membrane of bovine oviduct epithelial cells contains anchored proteinic factors that contribute to maintaining motility and viability and possibly to modulating capacitation of bovine sperm.

  9. STUDIES ON THE HUMAN OOCYTE AND ITS FOLLICLE

    PubMed Central

    Hertig, Arthur T.; Adams, Eleanor C.

    1967-01-01

    Oocytes in primordial ("resting") follicles in adult human ovaries contain a complex paranuclear structure identified by light microscopists as Balbiani's vitelline body. By electron microscopy this structure is composed of a mass of mitochondria with associated endoplasmic reticulum, multiple compound aggregates which form a ring around the cytocentrum, and a single stack or coil of annulate lamellae either attached to the nuclear membrane or free in the cytoplasm. The compound aggregates contain vacuoles and finely divided electron-opaque material. Evidence is presented for the probable transport of this material between the oocyte and its environment. The cytocentrum contains a central aggregate of amorphous electron-opaque deposits which appear to become periodically aligned on fine fibrils to form the long coarse fibers at the periphery of the cytocentrum. The apparent prevalence of annulate lamellae attached or adjacent to the nuclear membrane of oocytes in ovaries removed during the mid-follicular (estrogenic) phase of the cycle indicates the need for further study of a possible hormonal influence on the resting oocyte. By light microscopy phosphatases were not found within the oocyte, but adenosine-monophosphatase activity is present in the cortical cells surrounding primordial follicles, and also at the periphery of each primitive follicle cell, most prominently at the oocyte side. Glucose-6-phosphate dehydrogenase activity is present within the oocyte cytoplasm. PMID:4292010

  10. Molecular properties of a novel, hydrophilic cation-binding protein associated with the plasma membrane.

    PubMed

    Ide, Yuki; Nagasaki, Nahoko; Tomioka, Rie; Suito, Momoe; Kamiya, Takehiro; Maeshima, Masayoshi

    2007-01-01

    A new type of protein was found in Arabidopsis thaliana, PCaP1, which is rich in glutamate and lysine residues. The protein bound (45)Ca(2+) even in the presence of a high concentration of Mg(2+). Real-time polymerase chain reaction and histochemical analysis of promoter-beta-glucuronidase fusions revealed that PCaP1 was expressed in most organs. The PCaP1 protein was detected immunochemically in these organs. Treatment of Arabidopsis seedlings with Cu(2+), sorbitol, or flagellin oligopeptide enhanced the transcription. On the other hand, other sugars, abscisic acid, gibberellic acid, dehydration, and low temperature had little or no effect on PCaP1 transcript abundance. The transient expression of PCaP1 fused to green fluorescent protein in Arabidopsis cells and the subcellular fractionation of tissue homogenate showed that PCaP1 protein is localized to the plasma membrane, although PCaP1 has no predicted transmembrane domain. PCaP1 was associated with the plasma membrane under natural conditions and was released from the membrane at high concentrations of Ca(2+) or Mg(2+) in vitro. These results suggest that the hydrophilic protein PCaP1 binds Ca(2+) and other cations and is stably associated with the plasma membrane.

  11. An acid phosphatase in the plasma membranes of human astrocytoma showing marked specificity toward phosphotyrosine protein.

    PubMed

    Leis, J F; Kaplan, N O

    1982-11-01

    The plasma membrane from the human tumor astrocytoma contains an active acid phosphatase activity based on hydrolysis of p-nitrophenyl phosphate. Other acid phosphatase substrates--beta-glycerophosphate, O-phosphorylcholine, and 5'-AMP--are not hydrolyzed significantly. The phosphatase activity is tartrate insensitive and is stimulated by Triton X-100 and EDTA. Of the three known phosphoamino acids, only free O-phosphotyrosine is hydrolyzed by the membrane phosphatase activity. Other acid phosphatases tested from potato, wheat germ, milk, and bovine prostate did not show this degree of specificity. The plasma membrane activity also dephosphorylated phosphotyrosine histone at a much greater rate than did the other acid phosphatases. pH profiles for free O-phosphotyrosine and phosphotyrosine histone showed a shift toward physiological pH, indicating possible physiological significance. Phosphotyrosine histone dephosphorylation activity was nearly 10 times greater than that seen for phosphoserine histone dephosphorylation, and Km values were much lower for phosphotyrosine histone dephosphorylation (0.5 microM vs. 10 microM). Fluoride and zinc significantly inhibited phosphoserine histone dephosphorylation. Vanadate, on the other hand, was a potent inhibitor of phosphotyrosine histone dephosphorylation (50% inhibition at 0.5 microM) but not of phosphoserine histone. ATP stimulated phosphotyrosine histone dephosphorylation (160-250%) but inhibited phosphoserine histone dephosphorylation (95%). These results suggest the existence of a highly specific phosphotyrosine protein phosphatase activity associated with the plasma membrane of human astrocytoma.

  12. SUMOylation determines turnover and localization of nephrin at the plasma membrane.

    PubMed

    Tossidou, Irini; Himmelseher, Erik; Teng, Beina; Haller, Hermann; Schiffer, Mario

    2014-12-01

    Podocyte effacement and the reformation of foot processes and slit diaphragms can be induced within minutes experimentally. Therefore, it seems likely that the slit diaphragm proteins underlie orchestrated recycling mechanisms under the control of posttranslational modifiers. One of these modifiers, SUMO (small ubiquitin-like modifier), is an ubiquitin-like protein with a 20% corresponding identity to ubiquitin. Modification by SUMOs to proteins on lysine residues can block the ubiquitination of the same site leading to the stabilization of the target protein. Here we found in vitro and in vivo that nephrin is a substrate modified by SUMO proteins thereby increasing its steady-state level and expression at the plasma membrane. A conversion of lysines to arginines at positions 1114 and 1224 of the intracellular tail of murine nephrin led to decreased stability of nephrin, decreased expression at the plasma membrane, and decreased PI3K/AKT signaling. Furthermore, treatment of podocytes with the SUMOylation inhibitor ginkgolic acid led to reduced membrane expression of nephrin. Similarly, the conversion of lysine to arginine at position 1100 of human nephrin caused decreased stability and expression at the plasma membrane. As SUMOylation is a reversible process, our results suggest that SUMOylation participates in the tight orchestration of nephrin turnover at the slit diaphragm.

  13. Receptor dimer stabilization by hierarchical plasma membrane microcompartments regulates cytokine signaling.

    PubMed

    You, Changjiang; Marquez-Lago, Tatiana T; Richter, Christian Paolo; Wilmes, Stephan; Moraga, Ignacio; Garcia, K Christopher; Leier, André; Piehler, Jacob

    2016-12-01

    The interaction dynamics of signaling complexes is emerging as a key determinant that regulates the specificity of cellular responses. We present a combined experimental and computational study that quantifies the consequences of plasma membrane microcompartmentalization for the dynamics of type I interferon receptor complexes. By using long-term dual-color quantum dot (QD) tracking, we found that the lifetime of individual ligand-induced receptor heterodimers depends on the integrity of the membrane skeleton (MSK), which also proved important for efficient downstream signaling. By pair correlation tracking and localization microscopy as well as by fast QD tracking, we identified a secondary confinement within ~300-nm-sized zones. A quantitative spatial stochastic diffusion-reaction model, entirely parameterized on the basis of experimental data, predicts that transient receptor confinement by the MSK meshwork allows for rapid reassociation of dissociated receptor dimers. Moreover, the experimentally observed apparent stabilization of receptor dimers in the plasma membrane was reproduced by simulations of a refined, hierarchical compartment model. Our simulations further revealed that the two-dimensional association rate constant is a key parameter for controlling the extent of MSK-mediated stabilization of protein complexes, thus ensuring the specificity of this effect. Together, experimental evidence and simulations support the hypothesis that passive receptor confinement by MSK-based microcompartmentalization promotes maintenance of signaling complexes in the plasma membrane.

  14. Oxidized Phospholipids Inhibit the Formation of Cholesterol-Dependent Plasma Membrane Nanoplatforms

    PubMed Central

    Brameshuber, Mario; Sevcsik, Eva; Rossboth, Benedikt K.; Manner, Christina; Deigner, Hans-Peter; Peksel, Begüm; Péter, Mária; Török, Zsolt; Hermetter, Albin; Schütz, Gerhard J.

    2016-01-01

    We previously developed a single-molecule microscopy method termed TOCCSL (thinning out clusters while conserving stoichiometry of labeling), which allows for direct imaging of stable nanoscopic platforms with raft-like properties diffusing in the plasma membrane. As a consensus raft marker, we chose monomeric GFP linked via a glycosylphosphatidylinositol (GPI) anchor to the cell membrane (mGFP-GPI). With this probe, we previously observed cholesterol-dependent homo-association to nanoplatforms diffusing in the plasma membrane of live CHO cells. Here, we report the release of this homo-association upon addition of 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) or 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine, two oxidized phospholipids (oxPLs) that are typically present in oxidatively modified low-density lipoprotein. We found a dose-response relationship for mGFP-GPI nanoplatform disintegration upon addition of POVPC, correlating with the signal of the apoptosis marker Annexin V-Cy3. Similar concentrations of lysolipid showed no effect, indicating that the observed phenomena were not linked to properties of the lipid bilayer itself. Inhibition of acid sphingomyelinase by NB-19 before addition of POVPC completely abolished nanoplatform disintegration by oxPLs. In conclusion, we were able to determine how oxidized lipid species disrupt mGFP-GPI nanoplatforms in the plasma membrane. Our results favor an indirect mechanism involving acid sphingomyelinase activity rather than a direct interaction of oxPLs with nanoplatform constituents. PMID:26745423

  15. Receptor dimer stabilization by hierarchical plasma membrane microcompartments regulates cytokine signaling

    PubMed Central

    You, Changjiang; Marquez-Lago, Tatiana T.; Richter, Christian Paolo; Wilmes, Stephan; Moraga, Ignacio; Garcia, K. Christopher; Leier, André; Piehler, Jacob

    2016-01-01

    The interaction dynamics of signaling complexes is emerging as a key determinant that regulates the specificity of cellular responses. We present a combined experimental and computational study that quantifies the consequences of plasma membrane microcompartmentalization for the dynamics of type I interferon receptor complexes. By using long-term dual-color quantum dot (QD) tracking, we found that the lifetime of individual ligand-induced receptor heterodimers depends on the integrity of the membrane skeleton (MSK), which also proved important for efficient downstream signaling. By pair correlation tracking and localization microscopy as well as by fast QD tracking, we identified a secondary confinement within ~300-nm-sized zones. A quantitative spatial stochastic diffusion-reaction model, entirely parameterized on the basis of experimental data, predicts that transient receptor confinement by the MSK meshwork allows for rapid reassociation of dissociated receptor dimers. Moreover, the experimentally observed apparent stabilization of receptor dimers in the plasma membrane was reproduced by simulations of a refined, hierarchical compartment model. Our simulations further revealed that the two-dimensional association rate constant is a key parameter for controlling the extent of MSK-mediated stabilization of protein complexes, thus ensuring the specificity of this effect. Together, experimental evidence and simulations support the hypothesis that passive receptor confinement by MSK-based microcompartmentalization promotes maintenance of signaling complexes in the plasma membrane. PMID:27957535

  16. Direct interaction with filamins modulates the stability and plasma membrane expression of CFTR

    PubMed Central

    Thelin, William R.; Chen, Yun; Gentzsch, Martina; Kreda, Silvia M.; Sallee, Jennifer L.; Scarlett, Cameron O.; Borchers, Christoph H.; Jacobson, Ken; Stutts, M. Jackson; Milgram, Sharon L.

    2007-01-01

    The role of the cystic fibrosis transmembrane conductance regulator (CFTR) as a cAMP-dependent chloride channel on the apical membrane of epithelia is well established. However, the processes by which CFTR is regulated on the cell surface are not clear. Here we report the identification of a protein-protein interaction between CFTR and the cytoskeletal filamin proteins. Using proteomic approaches, we identified filamins as proteins that associate with the extreme CFTR N terminus. Furthermore, we identified a disease-causing missense mutation in CFTR, serine 13 to phenylalanine (S13F), which disrupted this interaction. In cells, filamins tethered plasma membrane CFTR to the underlying actin network. This interaction stabilized CFTR at the cell surface and regulated the plasma membrane dynamics and confinement of the channel. In the absence of filamin binding, CFTR was internalized from the cell surface, where it prematurely accumulated in lysosomes and was ultimately degraded. Our data demonstrate what we believe to be a previously unrecognized role for the CFTR N terminus in the regulation of the plasma membrane stability and metabolic stability of CFTR. In addition, we elucidate the molecular defect associated with the S13F mutation. PMID:17235394

  17. Citrinin-induced fluidization of the plasma membrane of the fission yeast Schizosaccharomyces pombe.

    PubMed

    Blaskó, Ágnes; Mike, Nóra; Gróf, Pál; Gazdag, Zoltán; Czibulya, Zsuzsanna; Nagy, Lívia; Kunsági-Máté, Sándor; Pesti, Miklós

    2013-09-01

    Citrinin (CTN) is a toxic fungal metabolite that is a hazardous contaminant of foods and feeds. In the present study, its acute toxicity and effects on the plasma membrane of Schizosaccharomyces pombe were investigated. The minimum inhibitory concentration of CTN against the yeast cells proved to be 500 μM. Treatment with 0, 250, 500 or 1000 μM CTN for 60 min resulted in a 0%, 2%, 21% or 100% decrease, respectively, in the survival rate of the cell population. Treatment of cells with 0, 100, 500 or 1000 μM CTN for 20 min induced decrease in the phase-transition temperature of the 5-doxylstearic acid-labeled plasma membrane to 16.51, 16.04, 14.18 or 13.98°C, respectively as measured by electron paramagnetic resonance spectroscopy. This perturbation was accompanied by the efflux of essential K⁺ from the cells. The existence of an interaction between CTN and glutathione was detected for the first time by spectrofluorometry. Our observations may suggest a direct interaction of CTN with the free sulfhydryl groups of the integral proteins of the plasma membrane, leading to dose-dependent membrane fluidization. The change in fluidity disturbed the ionic homeostasis, contributing to the death of the cells, which is a novel aspect of CTN cytotoxicity.

  18. In vitro fusion between Saccharomyces cerevisiae secretory vesicles and cytoplasmic-side-out plasma membrane vesicles.

    PubMed Central

    Arrastua, Lorena; San Sebastian, Eider; Quincoces, Ana F; Antony, Claude; Ugalde, Unai

    2003-01-01

    The final step in the secretory pathway, which is the fusion event between secretory vesicles and the plasma membrane, was reconstructed using highly purified secretory vesicles and cytoplasmic-side-out plasma membrane vesicles from the yeast Saccharomyces cerevisiae. Both organelle preparations were obtained from a sec 6-4 temperature-sensitive mutant. Fusion was monitored by means of a fluorescence assay based on the dequenching of the lipophilic fluorescent probe octadecylrhodamine B-chloride (R18). The probe was incorporated into the membrane of secretory vesicles, and it diluted in unlabelled cytoplasmic-side-out plasma membrane vesicles as the fusion process took place. The obtained experimental dequenching curves were found by mathematical analysis to consist of two independent but simultaneous processes. Whereas one of them reflected the fusion process between both vesicle populations as confirmed by its dependence on the assay conditions, the other represented a non-specific transfer of the probe. The fusion process may now be examined in detail using the preparation, validation and analytical methods developed in this study. PMID:12435271

  19. Expressing and characterizing mechanosensitive channels in Xenopus oocytes.

    PubMed

    Maksaev, Grigory; Haswell, Elizabeth S

    2015-01-01

    The oocytes of the African clawed frog (Xenopus laevis) comprise one of the most widely used membrane protein expression systems. While frequently used for studies of transporters and ion channels, the application of this system to the study of mechanosensitive ion channels has been overlooked, perhaps due to a relative abundance of native expression systems. Recent advances, however, have illustrated the advantages of the oocyte system for studying plant and bacterial mechanosensitive channels. Here we describe in detail the methods used for heterologous expression and characterization of bacterial and plant mechanosensitive channels in Xenopus oocytes.

  20. Characterization of plasma membrane proteins from ovarian cancer cells using mass spectrometry.

    PubMed

    Springer, David L; Auberry, Deanna L; Ahram, Mamoun; Adkins, Joshua N; Feldhaus, Jane M; Wahl, Jon H; Wunschel, David S; Rodland, Karin D

    To determine how the repertoire of plasma membrane proteins change with disease state, specifically related to cancer, several methods for preparation of plasma membrane proteins were evaluated. Cultured cells derived from stage IV ovarian tumors were grown to 90% confluence and harvested in buffer containing CHAPS detergent. This preparation was centrifuged at low speed to remove insoluble cellular debris resulting in a crude homogenate. Glycosylated proteins in the crude homogenate were selectively enriched using lectin affinity chromatography. The crude homogenate and the lectin purified sample were prepared for mass spectrometric evaluation. The general procedure for protein identification began with trypsin digestion of protein fractions followed by separation by reversed phase liquid chromatography that was coupled directly to a conventional tandem mass spectrometer (i.e. LCQ ion trap). Mass and fragmentation data for the peptides were searched against a human proteome data base using the informatics program SEQUEST. Using this procedure 398 proteins were identified with high confidence, including receptors, membrane-associated ligands, proteases, phosphatases, as well as structural and adhesion proteins. Results indicate that lectin chromatography provides a select subset of proteins and that the number and quality of the identifications improve as does the confidence of the protein identifications for this subset. These results represent the first step in development of methods to separate and successfully identify plasma membrane proteins from advanced ovarian cancer cells. Further characterization of plasma membrane proteins will contribute to our understanding of the mechanisms underlying progression of this deadly disease and may lead to new targeted interventions as well as new biomarkers for diagnosis.

  1. Plasma membrane calcium channels in cancer: Alterations and consequences for cell proliferation and migration.

    PubMed

    Déliot, Nadine; Constantin, Bruno

    2015-10-01

    The study of calcium channels in molecular mechanisms of cancer transformation is still a novel area of research. Several studies, mostly conducted on cancer cell lines, however support the idea that a diversity of plasma membrane channels participates in the remodeling of Ca2+ homeostasis, which regulates various cancer hallmarks such as uncontrolled multiplication and increase in migration and invasion abilities. However few is still understood concerning the intracellular signaling cascades mobilized by calcium influx participating to cancer cell behavior. This review intends to gather some of these pathways dependent on plasma membrane calcium channels and described in prostate, breast and lung cancer cell lines. In these cancer cell types, the calcium channels involved in calcium signaling pathways promoting cancer behaviors are mostly non-voltage activated calcium channels and belong to the TRP superfamily (TRPC, TPRPV and TRPM families) and the Orai family. TRP and Orai channels are part of many signaling cascades involving the activation of transmembrane receptors by extracellular ligand from the tumor environment. TRPV can sense changes in the physical and chemical environment of cancer cells and TRPM7 are stretch activated and sensitive to cholesterol. Changes in activation and or expression of plasma-membrane calcium channels affect calcium-dependent signaling processes relevant to tumorigenesis. The studies cited in this review suggest that an increase in plasma membrane calcium channel expression and/or activity sustain an elevated calcium entry (constitutive or under the control of extracellular signals) promoting higher cell proliferation and migration in most cases. A variety of non-voltage-operated calcium channels display change expression and/or activity in a same cancer type and cooperate to the same process relevant to cancer cell behavior, or can be involved in a different sequence of events during the tumorigenesis. This article is part of a

  2. Imaging of mobile long-lived nanoplatforms in the live cell plasma membrane.

    PubMed

    Brameshuber, Mario; Weghuber, Julian; Ruprecht, Verena; Gombos, Imre; Horváth, Ibolya; Vigh, László; Eckerstorfer, Paul; Kiss, Endre; Stockinger, Hannes; Schütz, Gerhard J

    2010-12-31

    The plasma membrane has been hypothesized to contain nanoscopic lipid platforms, which are discussed in the context of "lipid rafts" or "membrane rafts." Based on biochemical and cell biological studies, rafts are believed to play a crucial role in many signaling processes. However, there is currently not much information on their size, shape, stability, surface density, composition, and heterogeneity. We present here a method that allows for the first time the direct imaging of nanoscopic long-lived platforms with raft-like properties diffusing in the live cell plasma membrane. Our method senses these platforms by their property to assemble a characteristic set of fluorescent marker proteins or lipids on a time scale of seconds. A special photobleaching protocol was used to reduce the surface density of labeled mobile platforms down to the level of well isolated diffraction-limited spots without altering the single spot brightness. The statistical distribution of probe molecules per platform was determined by single molecule brightness analysis. For demonstration, we used the consensus raft marker glycosylphosphatidylinositol-anchored monomeric GFP and the fluorescent lipid analog BODIPY-G(M1), which preferentially partitions into liquid-ordered phases. For both markers, we found cholesterol-dependent homo-association in the plasma membrane of living CHO and Jurkat T cells in the resting state, thereby demonstrating the existence of small, mobile, long-lived platforms containing these probes. We further applied the technology to address structural changes in the plasma membrane during fever-type heat shock: at elevated temperatures, the glycosylphosphatidylinositol-anchored monomeric GFP homo-association disappeared, accompanied by an increase in the expression of the small heat shock protein Hsp27.

  3. Characterization of Plasma Membrane Proteins from Ovarian Cancer Cells Using Mass Spectrometry

    SciTech Connect

    Springer, David L.; Auberry, Deanna L.; Ahram, Mamoun; Adkins, Joshua N.; Feldhaus, Jane M.; Wahl, Jon H.; Wunsch, David M.; Rodland, Karin D.

    2003-01-01

    To determine how the repertoire of plasma membrane proteins change with disease state, specifically related to cancer, several methods for preparation of plasma membrane proteins were evaluated. Cultured cells derived from stage IV ovarian tumors were grown to 90% confluence and harvested in buffer containing CHAPS detergent. This preparation was centrifuged at low speed to remove insoluble cellular debris resulting in a crude homogenate. Glycosylated proteins in the crude homogenate were selectively enriched using lectin affinity chromatography. The crude homogenate and the lectin purified sample were prepared for mass spectrometric evaluation. The general procedure for protein identification began with trypsin digestion of protein fractions followed by separation by reversed phase liquid chromatography that was coupled directly to a conventional tandem mass spectrometer (i.e. LCQ ion trap). Mass and fragmentation data for the peptides were searched against a human proteome data base using the informatics program SEQUEST. Using this procedure 398 proteins were identified with high confidence, including receptors, membrane-associated ligands, proteases, phosphatases, as well as structural and adhesion proteins. Results indicate that lectin chromatography provides a select subset of proteins and that the number and quality of the identifications improve as does the confidence of the protein identifications for this subset. These results represent the first step in development of methods to separate and successfully identify plasma membrane proteins from advanced ovarian cancer cells. Further characterization of plasma membrane proteins will contribute to our understanding of the mechanisms underlying progression of this deadly disease and may lead to new targeted interventions as well as new biomarkers for diagnosis.

  4. Characterization of Plasma Membrane Proteins from Ovarian Cancer Cells Using Mass Spectrometry

    DOE PAGES

    Springer, David L.; Auberry, Deanna L.; Ahram, Mamoun; ...

    2004-01-01

    To determine how the repertoire of plasma membrane proteins change with disease state, specifically related to cancer, several methods for preparation of plasma membrane proteins were evaluated. Cultured cells derived from stage IV ovarian tumors were grown to 90% confluence and harvested in buffer containing CHAPS detergent. This preparation was centrifuged at low speed to remove insoluble cellular debris resulting in a crude homogenate. Glycosylated proteins in the crude homogenate were selectively enriched using lectin affinity chromatography. The crude homogenate and the lectin purified sample were prepared for mass spectrometric evaluation. The general procedure for protein identification began with trypsinmore » digestion of protein fractions followed by separation by reversed phase liquid chromatography that was coupled directly to a conventional tandem mass spectrometer (i.e. LCQ ion trap). Mass and fragmentation data for the peptides were searched against a human proteome data base using the informatics program SEQUEST. Using this procedure 398 proteins were identified with high confidence, including receptors, membrane-associated ligands, proteases, phosphatases, as well as structural and adhesion proteins. Results indicate that lectin chromatography provides a select subset of proteins and that the number and quality of the identifications improve as does the confidence of the protein identifications for this subset. These results represent the first step in development of methods to separate and successfully identify plasma membrane proteins from advanced ovarian cancer cells. Further characterization of plasma membrane proteins will contribute to our understanding of the mechanisms underlying progression of this deadly disease and may lead to new targeted interventions as well as new biomarkers for diagnosis.« less

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

    PubMed

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

    2015-03-01

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

  6. Outer Hair Cell Lateral Wall Structure Constrains the Mobility of Plasma Membrane Proteins

    PubMed Central

    Yamashita, Tetsuji; Hakizimana, Pierre; Wu, Siva; Hassan, Ahmed; Jacob, Stefan; Temirov, Jamshid; Fang, Jie; Mellado-Lagarde, Marcia; Gursky, Richard; Horner, Linda; Leibiger, Barbara; Leijon, Sara; Centonze, Victoria E.; Berggren, Per-Olof; Frase, Sharon; Auer, Manfred; Brownell, William E.; Fridberger, Anders; Zuo, Jian

    2015-01-01

    Nature’s fastest motors are the cochlear outer hair cells (OHCs). These sensory cells use a membrane protein, Slc26a5 (prestin), to generate mechanical force at high frequencies, which is essential for explaining the exquisite hearing sensitivity of mammalian ears. Previous studies suggest that Slc26a5 continuously diffuses within the membrane, but how can a freely moving motor protein effectively convey forces critical for hearing? To provide direct evidence in OHCs for freely moving Slc26a5 molecules, we created a knockin mouse where Slc26a5 is fused with YFP. These mice and four other strains expressing fluorescently labeled membrane proteins were used to examine their lateral diffusion in the OHC lateral wall. All five proteins showed minimal diffusion, but did move after pharmacological disruption of membrane-associated structures with a cholesterol-depleting agent and salicylate. Thus, our results demonstrate that OHC lateral wall structure constrains the mobility of plasma membrane proteins and that the integrity of such membrane-associated structures are critical for Slc26a5’s active and structural roles. The structural constraint of membrane proteins may exemplify convergent evolution of cellular motors across species. Our findings also suggest a possible mechanism for disorders of cholesterol metabolism with hearing loss such as Niemann-Pick Type C diseases. PMID:26352669

  7. Molecular recognition based iron removal from human plasma with imprinted membranes.

    PubMed

    Yavuz, H; Andaç, M; Uzun, L; Say, R; Denizli, A

    2006-09-01

    The aim of this study is to prepare ion-imprinted poly(2-hydroxyethyl methacrylate) (HEMA) based membranes which can be used for the selective removal of Fe3+ ions from Fe3+-overdosed human plasma. N-methacryloyl-(L)-glutamic acid (MAGA) was chosen as the ion-complexing monomer. In the first step, Fe3+ was complexed with MAGA and then, the Fe3+-imprinted poly(HEMA-MAGA) membranes were prepared by UV-initiated photo-polymerization of HEMA and MAGA-Fe3+ complex in the presence of an initiator (benzoyl peroxide). After that, the template (i.e., Fe3+ ions) was removed by using 0.1 M EDTA solution at room temperature. The specific surface area of the Fe3+-imprinted poly(HEMA-MAGA) membranes was found to be 49.2 m2/g and the swelling ratio was 92%. According to the elemental analysis results, the polymeric membranes contained 145.7 micromol MAGA/g polymer. The maximum adsorption capacity was 164.2 micromol Fe3+/g membrane. The relative selectivity coefficients of ion-imprinted membranes for Fe3+/Zn2+ and Fe3+/Cr3+ were 12.6 and 62.5 times greater than the non-imprinted matrix, respectively. The Fe3+-imprinted poly(HEMA-MAGA) membranes could be used many times without decreasing their Fe3+ adsorption capacities significantly.

  8. A plasma-membrane linker for the phosphoinositide-specific phospholipase C in tobacco plants.

    PubMed

    Nakamura, Kimiyo; Sano, Hiroshi

    2009-01-01

    We previously screened genes that were transcriptionally activated during the early stage of wound response in tobacco plants (Nicotiana tabacum), and isolated a particular clone, which encoded a membrane-located protein, designated as NtC7. Upon overexpression in tobacco plants, NtC7 conferred a marked tolerance to osmotic stress, suggesting it to be involved in maintenance of osmotic adjustments. In this study, we searched for proteins which interact with NtC7 by the yeast two-hybrid screening, and isolated a clone encoding phosphoinositide-specific phospholipase C, designated as NtPI-PLC. Physical interaction between NtC7 and C2 domain of NtPI-PLC was confirmed by the pull-down assay. Expression of fused protein to green-fluorescence protein in onion epidermal cell layers indicated both proteins to predominantly localize to the plasma membrane. Their interaction in planta was shown by the bimolecular fluorescence complementation, which exhibited a clear fluorescence of reconstituted yellow fluorescence protein. Transcripts of NtC7 and NtPI-PLC were markedly increased 30 to 60 min after wounding. PI-PLC is one of key enzymes in metabolism of inositol phospholipids, which function in signal transduction and also in response to stresses including osmotic changes. It was shown to localize to plasma-membrane and, to a lesser extent, to cytosol. However, molecular mechanism of membrane localization has remained to be determined, because of the apparent lack of domains for membrane association. The present results suggest that one of such mechanisms is tethering NtPI-PLC to the plasma membrane through interaction with NtC7, which possesses a transmembrane domain at the C-terminus.

  9. Elevation of plasma membrane permeability by laser irradiation of selectively bound nanoparticles.

    PubMed

    Yao, Cuiping; Rahmanzadeh, Ramtin; Endl, Elmar; Zhang, Zhenxi; Gerdes, Johannes; Hüttmann, Gereon

    2005-01-01

    Irradiation of nanoabsorbers with pico- and nanosecond laser pulses could result in thermal effects with a spatial confinement of less than 50 nm. Therefore absorbing nanoparticles could be used to create controlled cellular effects. We describe a combination of laser irradiation with nanoparticles, which changes the plasma membrane permeability. We demonstrate that the system enables molecules to penetrate impermeable cell membranes. Laser light at 532 nm is used to irradiate conjugates of colloidal gold, which are delivered by antibodies to the plasma membrane of the Hodgkin's disease cell line L428 and/or the human large-cell anaplastic lymphoma cell line Karpas 299. After irradiation, membrane permeability is evaluated by fluorescence microscopy and flow cytometry using propidium iodide (PI) and fluorescein isothiocyanate (FITC) dextran. The fraction of transiently permeabilized and then resealed cells is affected by the laser parameter, the gold concentration, and the membrane protein of the different cell lines to which the nanoparticles are bound. Furthermore, a dependence on particle size is found for these interactions in the different cell lines. The results suggest that after optimization, this method could be used for gene transfection and gene therapy.

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

    PubMed Central

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

    2015-01-01

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

  11. Plasma zinc status and membrane lipid composition in genetically diabetic mice (db/db)

    SciTech Connect

    Burke, J.P.; Fenton, M.R.

    1986-03-05

    Sex and age matched diabetic C57BL/Ks-db+/db+ mice (db/db) were sacrificed at eight weeks of age. Plasma samples were collected and zinc levels determined. Livers were excised and mitochondrial and microsomal membranes prepared. Aliquots of membrane fractions were subjected to lipid extraction and cholesterol (Cl), phospholipid (PL) and fatty acid analysis (FA) performed. Plasma zinc levels in db/db mice were elevated 25% compared to m/m controls (148.8+/-8.1 ..mu..g/dl vs. 118.9+/-14.9 ..mu..g/dl). Cholesterol and PL levels remained unchanged in both mitochondrial and microsomal membranes. Analysis of PL composition from db/db mitochondria by two dimensional thin layer chromatography revealed no change in the percentage of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) but a 40% decrease in cardiolipin. Slight increases were observed in the percentage of phosphatidylserine and phosphatidylinositol (PS+PI) in microsomes isolated from db/db mice. Fatty acid analysis of microsomal PC and PE showed a decrease of 28% in the 18:1/18:0 ratio as well as a 21% decrease in the ratio of 20:4/18:2 in db/db animals. Analysis of succinate dehydrogenase (mitochondrial) and glucose-6-phosphatase (microsomal) revealed significant decreases in activity in livers of db/db mice. The altered zinc metabolism as well as the changes in membrane lipid composition suggest that this may be a model to study the role of zinc in membrane structure.

  12. Glucocorticoid interactions with ethanol effects on synaptic plasma membranes: influence on [125I]calmodulin binding.

    PubMed

    Sze, P Y

    1996-02-01

    Ca(++)-dependent binding of calmodulin (CaM) to brain synaptic plasma membranes is known to be inhibited by ethanol and stimulated by glucocorticoids. These opposite neurochemical actions between ethanol and the steroids in vitro are consistent with glucocorticoid antagonism of ethanol-induced sedation reported to occur in vivo. The present study was undertaken to characterize the interactions of corticosterone with ethanol effects on [125I]CaM binding in synaptic plasma membranes. From the shift of concentration-response curves when corticosterone and ethanol were present in combination, the interaction between steroid stimulation and ethanol inhibition occurred in an additive relationship over the range of their effective concentrations. From Scatchard analyses, ethanol-induced decrease in membrane affinity for [125I]CaM was antagonized by steroid-induced increase in the membrane affinity, indicating that the convergent event in their interaction was the alteration of membrane affinity for CaM. Glucocorticoid antagonism of ethanol inhibition of [125I]CaM binding exhibited a high degree of steroid specificity; steroids with glucocorticoid activity including cortisol, dexamethasone and triamcinolone were effective, whereas gonadal steroids and excitatory neuroactive steroid metabolites were ineffective. The demonstration that glucocorticoids antagonized the inhibition of CaM binding by ethanol provides support for the hypothesis that these steroids are among the endogenous factors that modulate neuronal sensitivity to ethanol.

  13. From the endoplasmic reticulum to the plasma membrane: mechanisms of CFTR folding and trafficking.

    PubMed

    Farinha, Carlos M; Canato, Sara

    2017-01-01

    CFTR biogenesis starts with its co-translational insertion into the membrane of endoplasmic reticulum and folding of the cytosolic domains, towards the acquisition of a fully folded compact native structure. Efficiency of this process is assessed by the ER quality control system that allows the exit of folded proteins but targets unfolded/misfolded CFTR to degradation. If allowed to leave the ER, CFTR is modified at the Golgi and reaches the post-Golgi compartments to be delivered to the plasma membrane where it functions as a cAMP- and phosphorylation-regulated chloride/bicarbonate channel. CFTR residence at the membrane is a balance of membrane delivery, endocytosis, and recycling. Several adaptors, motor, and scaffold proteins contribute to the regulation of CFTR stability and are involved in continuously assessing its structure through peripheral quality control systems. Regulation of CFTR biogenesis and traffic (and its dysregulation by mutations, such as the most common F508del) determine its overall activity and thus contribute to the fine modulation of chloride secretion and hydration of epithelial surfaces. This review covers old and recent knowledge on CFTR folding and trafficking from its synthesis to the regulation of its stability at the plasma membrane and highlights how several of these steps can be modulated to promote the rescue of mutant CFTR.

  14. Membrane damage and active but nonculturable state in liquid cultures of Escherichia coli treated with an atmospheric pressure plasma jet.

    PubMed

    Dolezalova, Eva; Lukes, Petr

    2015-06-01

    Electrical discharge plasmas can efficiently inactivate various microorganisms. Inactivation mechanisms caused by plasma, however, are not fully understood because of the complexity of both the plasma and biological systems. We investigated plasma-induced inactivation of Escherichia coli in water and mechanisms by which plasma affects bacterial cell membrane integrity. Atmospheric pressure argon plasma jet generated at ambient air in direct contact with bacterial suspension was used as a plasma source. We determined significantly lower counts of E. coli after treatment by plasma when they were assayed using a conventional cultivation technique than using a fluorescence-based LIVE/DEAD staining method, which indicated that bacteria may have entered the viable-but-nonculturable state (VBNC). We did not achieve resuscitation of these non-culturable cells, however, we detected their metabolic activity through the analysis of cellular mRNA, which suggests that cells may have been rather in the active-but-nonculturable state (ABNC). We hypothesize that peroxidation of cell membrane lipids by the reactive species produced by plasma was an important pathway of bacterial inactivation. Amount of malondialdehyde and membrane permeability of E. coli to propidium iodide increased with increasing bacterial inactivation by plasma. Membrane damage was also demonstrated by detection of free DNA in plasma-treated water.

  15. Aqueous two-phase partition applied to the isolation of plasma membranes and Golgi apparatus from cultured mammalian cells

    NASA Technical Reports Server (NTRS)

    Morre, D. M.; Morre, D. J.

    2000-01-01

    Partitioning in dextran-poly(ethylene)glycol (PEG) aqueous-aqueous phase systems represents a mature technology with many applications to separations of cells and to the preparation of membranes from mammalian cells. Most applications to membrane isolation and purification have focused on plasma membranes, plasma membrane domains and separation of right side-out and inside-out plasma membrane vesicles. The method exploits a combination of membrane properties, including charge and hydrophobicity. Purification is based upon differential distributions of the constituents in a sample between the two principal compartments of the two phases (upper and lower) and at the interface. The order of affinity of animal cell membranes for the upper phase is: endoplasmic reticulum plasma membranes. Salt concentrations and temperature affect partitioning behavior and must be precisely standardized. In some cases, it is more fortuitous to combine aqueous two-phase partition with other procedures to obtain a more highly purified preparation. A procedure is described for preparation of Golgi apparatus from transformed mammalian cells that combines aqueous two-phase partition and centrifugation. Also described is a periodic NADH oxidase, a new enzyme marker for right side-out plasma membrane vesicles not requiring detergent disruptions for measurement of activity.

  16. Charged anaesthetics alter LM-fibroblast plasma-membrane enzymes by selective fluidization of inner or outer membrane leaflets.

    PubMed

    Sweet, W D; Schroeder, F

    1986-10-15

    The functional consequences of the differences in lipid composition and structure between the two leaflets of the plasma membrane were investigated. Fluorescence of 1,6-diphenylhexa-1,3,5-triene(DPH), quenching, and differential polarized phase fluorimetry demonstrated selective fluidization by local anaesthetics of individual leaflets in isolated LM-cell plasma membranes. As measured by decreased limiting anisotropy of DPH fluorescence, cationic (prilocaine) and anionic (phenobarbital and pentobarbital) amphipaths preferentially fluidized the cytofacial and exofacial leaflets respectively. Unlike prilocaine, procaine, also a cation, fluidized both leaflets of these membranes equally. Pentobarbital stimulated 5'-nucleotidase between 0.1 and 5 mM and inhibited at higher concentrations, whereas phenobarbital only inhibited, at higher concentrations. Cationic drugs were ineffective. Two maxima of (Na+ + K+)-ATPase activation were obtained with both anionic drugs. Only one activation maximum was obtained with both cationic drugs. The maximum in activity below 1 mM for all four drugs clustered about a single limiting anisotropy value in the cytofacial leaflet, whereas there was no correlation between activity and limiting anisotropy in the exofacial leaflets. Therefore, although phenobarbital and pentobarbital below 1 mM fluidized the exofacial leaflet more than the cytofacial leaflet, the smaller fluidization in the cytofacial leaflet was functionally significant for (Na+ + K+)-ATPase. Mg2+-ATPase was stimulated at 1 mM-phenobarbital, unaffected by pentobarbital and slightly stimulated by both cationic drugs at concentrations fluidizing both leaflets. Thus the activity of (Na+ + K+)-ATPase was highly sensitive to selective fluidization of the leaflet containing its active site, whereas the other enzymes examined were little affected by fluidization of either leaflet.

  17. Plasma Modified Polypropylene Membranes as the Lithium-Ion Battery Separators

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

    To reduce the thermal shrinkage of the polymeric separators and improve the safety of the Li-ion batteries, plasma treatment and plasma enhanced vapor chemical deposition (PECVD) of SiOx-like are carried out on polypropylene (PP) separators, respectively. Critical parameters for separator properties, such as the thermal shrinkage rate, porosity, wettability, and mechanical strength, are evaluated on the plasma treated PP membranes. O2 plasma treatment is found to remarkably improve the wettability, porosity and electrolyte uptake. PECVD SiOx-like c