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Sample records for basolateral membrane vesicles

  1. Sugar uptake by intestinal basolateral membrane vesicles.

    PubMed

    Wright, E M; van Os, C H; Mircheff, A K

    1980-03-27

    A high yield of membrane vesicles was prepared from the basolateral surface of rat intestinal cells using an N2 cavitation bomb and density gradient centrifugation. The membranes were enriched 10-fold and were free of significatn contamination by brush border membranes and mitochondria. The rate of D-E114C]glucose and L-E13H]glucose uptake into the vesicle was measured using a rapid filtration technique. D-Glucose equilibrated within the vesicles with a half-time 1/25th that for L-glucose. The stereospecific uptake exhibited saturation kinetics with a Km of approx. 44 mM and a V of approx. 110 nmol . mg-1 min-1 at 10 degrees C. The activation energy for the process was 14 kcal . mol-1 below 15 degrees C and it approached 3 kcal . mol-1 above 22 degrees C. Carrier-mediated uptake was eliminated in the presence of 1 mM HgCl2 and 0.5 mM phloretin. The rate of transport was unaffected by the absence or presence of sodium concentration gradients. Competition studies demonstrated that all sugars with the D-glucose pyranose ring chair conformation shared the transport system, and that, with the possible exception of the -OH group at carbon No. 1, there were no specific requirements for an equatorial -OH group at any position in the pyranose ring. In the case of alpha-methyl-D-glucoside its inability to share the D-glucose transport system may be due to steric hindrance posed by the -OCH3 group rather than by a specific requirement for a free hydroxyl group at the position in the ring. It is concluded that sugars are transported across the basolateral membrane of the intestinal epithelium by a facilitated diffusion system reminiscent of that in human red blood cells. PMID:6245688

  2. Sugar uptake by intestinal basolateral membrane vesicles.

    PubMed

    Wright, E M; van Os, C H; Mircheff, A K

    1980-03-27

    A high yield of membrane vesicles was prepared from the basolateral surface of rat intestinal cells using an N2 cavitation bomb and density gradient centrifugation. The membranes were enriched 10-fold and were free of significatn contamination by brush border membranes and mitochondria. The rate of D-E114C]glucose and L-E13H]glucose uptake into the vesicle was measured using a rapid filtration technique. D-Glucose equilibrated within the vesicles with a half-time 1/25th that for L-glucose. The stereospecific uptake exhibited saturation kinetics with a Km of approx. 44 mM and a V of approx. 110 nmol . mg-1 min-1 at 10 degrees C. The activation energy for the process was 14 kcal . mol-1 below 15 degrees C and it approached 3 kcal . mol-1 above 22 degrees C. Carrier-mediated uptake was eliminated in the presence of 1 mM HgCl2 and 0.5 mM phloretin. The rate of transport was unaffected by the absence or presence of sodium concentration gradients. Competition studies demonstrated that all sugars with the D-glucose pyranose ring chair conformation shared the transport system, and that, with the possible exception of the -OH group at carbon No. 1, there were no specific requirements for an equatorial -OH group at any position in the pyranose ring. In the case of alpha-methyl-D-glucoside its inability to share the D-glucose transport system may be due to steric hindrance posed by the -OCH3 group rather than by a specific requirement for a free hydroxyl group at the position in the ring. It is concluded that sugars are transported across the basolateral membrane of the intestinal epithelium by a facilitated diffusion system reminiscent of that in human red blood cells.

  3. Phosphate transport by rat intestinal basolateral-membrane vesicles.

    PubMed Central

    Ghishan, F K; Kikuchi, K; Arab, N

    1987-01-01

    The characteristics of phosphate transport across intestinal basolateral membranes of the rat were determined by using enriched preparations in which uphill Na+-dependent D-glucose transport could not be demonstrated, but ATP-dependent Ca2+ transport was present. Phosphate transport was saturable, Na+-dependent and exhibited Michaelis-Menten kinetics. Vmax. was 51.1 +/- 4.2 pmol/10 s per mg of protein and Km was 14 +/- 3.9 microM. The transport process was electroneutral. Tracer-exchange experiments and counter-transport studies confirmed the presence of a Na+-Pi carrier at the basolateral membrane. The presence of inside-positive membrane potential did not enhance phosphate uptake, indicating that the Na+ effect is secondary to the presence of the Na+-Pi carrier rather than an induction of positive membrane potential. The stoichiometry of this carrier at pH 7.4 was 2 Na+:1 phosphate, as shown by direct studies utilizing the static-head method. These studies are the first to determine the presence of a phosphate carrier at the basolateral membrane. PMID:3663094

  4. Calcium uptake by brush-border and basolateral membrane vesicles in chick duodenum

    SciTech Connect

    Takito, J.; Shinki, T.; Sasaki, T.; Suda, T. )

    1990-01-01

    Calcium uptake was compared between duodenal brush-border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) isolated from vitamin D-deficient chicks and those injected with 625 ng of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3). The uptake by BBMV in the 1 alpha,25-(OH)2D3-treated birds attained a maximum (280% of the control) at 12 h and was maintained at an elevated level (210%) at 24 h after the injection of the vitamin. In contrast, ATP-dependent calcium uptake by BLMV reached a maximum (185% of the control) at 6 h and decreased to the control level at 24 h. The kinetic analysis revealed that 1 alpha,25(OH)2D3 increased Vmax values without any changes in apparent Km values in both BBMV and BLMV. The activity of ATP-dependent calcium uptake was localized exclusively in the basolateral membrane, and the activity was inhibited by vanadate (IC50, 1 microM), but not by oligomycin, theophylline, calmodulin, trifluoperazine, or calbindin D28K. These results indicate that calcium transport through both the brush-border and basolateral membranes is involved in the 1 alpha,25(OH)2D3-dependent intestinal calcium absorption. The initiation of calcium absorption by 1 alpha,25(OH)2D3 appears to be due to an increase in the rate of calcium efflux at the basolateral membrane rather than the rate at the brush-border membrane.

  5. A carrier-mediated transport for folate in basolateral membrane vesicles of rat small intestine.

    PubMed Central

    Said, H M; Redha, R

    1987-01-01

    The mechanism of exit of folate from the enterocyte, i.e. transport across the basolateral membrane, is not known. In this study we examined, using basolateral membrane vesicles, the transport of folic acid across the basolateral membrane of rat intestine. Uptake of folic acid by these vesicles represents transport of the substrate into the intravesicular compartment and not binding to the membrane surface. The rate of folic acid transport was linear for the first 1 min of incubation but decreased thereafter, reaching equilibrium after 5 min of incubation. The transport of folic acid was: (1) saturable as a function of concentration with an apparent Km of 0.6 +/- 0.17 microM and Vmax. of 1.01 +/- 0.11 pmol/30 s per mg of protein; (2) inhibited in a competitive manner by the structural analogues 5-methyltetrahydrofolate and methotrexate (Ki = 2 and 1.4 microM, respectively); (4) electroneutral; (5) Na+-independent; (6) sensitive to the effect of the anion exchange inhibitor 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS). These data indicate the existence of a carrier-mediated transport system for folic acid in rat intestinal basolateral membrane and demonstrate that the transport process is electroneutral, Na+-independent and sensitive to the effect of anion exchange inhibition. PMID:3689340

  6. Proton-stimulated Cl-HCO/sub 3/ antiport by basolateral membrane vesicles of lobster hepatopancreas

    SciTech Connect

    Ahearn, G.A.; Grover, M.L.; Tsuji, R.T.; Clay, L.P.

    1987-05-01

    Purified epithelial basolateral membrane vesicles were prepared from lobster hepatopancreas by sorbitol gradient centrifugation. Na+-K+-adenosinetriphosphatase, alkaline phosphatase, and cytochrome-c oxidase enzyme activities in the final membrane preparation were enriched 9.6-, 1.4-, and 0.4-fold, respectively, compared with their activities in the original tissue homogenate. Vesicle osmotic reactivity was demonstrated using 60-min equilibrium /sup 36/Cl uptake experiments at a variety of transmembrane osmotic gradients. /sup 36/Cl uptake into vesicles preloaded with HCO/sub 3/ was significantly greater than into vesicles lacking HCO/sub 3/. This exchange process was stimulated by a transmembrane proton gradient (internal pH greater than external pH). Proton-gradient-dependent Cl-HCO/sub 3/ exchange was potential sensitive and stimulated by an electrically negative vesicle interior. /sup 36/Cl influx (4-s exposures) into HCO/sub 3/-loaded vesicles occurred by the combination of 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid sensitive, carrier-mediated transfer and apparent diffusion. /sup 36/Cl influx was a hyperbolic function of both internal (HCO/sub 3/) and internal (Cl). The two internal anions displayed a 100-fold difference in apparent affinity constants with HCO/sub 3/ being strongly preferred. /sup 36/Cl influx was stimulated more by preloaded monovalent than by divalent anions. Na was an inhibitor of proton-dependent anion antiport, whereas K had no effect. A model for HCl-HCO/sub 3/ antiport is suggested that employs combined transmembrane concentration gradients of Cl and HCO/sub 3/ to power anion exchange and transfer protons against a concentration gradient.

  7. NaCl reflection coefficients in proximal tubule apical and basolateral membrane vesicles. Measurement by induced osmosis and solvent drag.

    PubMed

    Pearce, D; Verkman, A S

    1989-06-01

    Two independent methods, induced osmosis and solvent drag, were used to determine the reflection coefficients for NaCl (sigma NaCl) in brush border and basolateral membrane vesicles isolated from rabbit proximal tubule. In the induced osmosis method, vesicles loaded with sucrose were subjected to varying inward NaCl gradients in a stopped-flow apparatus. sigma NaCl was determined from the osmolality of the NaCl solution required to cause no initial osmotic water flux as measured by light scattering (null point). By this method sigma NaCl was greater than 0.92 for both apical and basolateral membranes with best estimates of 1.0. sigma NaCl was determined by the solvent drag method using the Cl-sensitive fluorescent indicator, 6-methoxy-N-[3-sulfopropyl]quinolinium (SPQ), to detect the drag of Cl into vesicles by inward osmotic water movement caused by an outward osmotic gradient. sigma NaCl was determined by comparing experimental data with theoretical curves generated using the coupled flux equations of Kedem and Katchalsky. By this method we found that sigma NaCl was greater than 0.96 for apical and greater than 0.98 for basolateral membrane vesicles, with best estimates of 1.0 for both membranes. These results demonstrate that sigma NaCl for proximal tubule apical and basolateral membranes are near unity. Taken together with previous results, these data suggest that proximal tubule water channels are long narrow pores that exclude NaCl. PMID:2765660

  8. NaCl reflection coefficients in proximal tubule apical and basolateral membrane vesicles. Measurement by induced osmosis and solvent drag.

    PubMed

    Pearce, D; Verkman, A S

    1989-06-01

    Two independent methods, induced osmosis and solvent drag, were used to determine the reflection coefficients for NaCl (sigma NaCl) in brush border and basolateral membrane vesicles isolated from rabbit proximal tubule. In the induced osmosis method, vesicles loaded with sucrose were subjected to varying inward NaCl gradients in a stopped-flow apparatus. sigma NaCl was determined from the osmolality of the NaCl solution required to cause no initial osmotic water flux as measured by light scattering (null point). By this method sigma NaCl was greater than 0.92 for both apical and basolateral membranes with best estimates of 1.0. sigma NaCl was determined by the solvent drag method using the Cl-sensitive fluorescent indicator, 6-methoxy-N-[3-sulfopropyl]quinolinium (SPQ), to detect the drag of Cl into vesicles by inward osmotic water movement caused by an outward osmotic gradient. sigma NaCl was determined by comparing experimental data with theoretical curves generated using the coupled flux equations of Kedem and Katchalsky. By this method we found that sigma NaCl was greater than 0.96 for apical and greater than 0.98 for basolateral membrane vesicles, with best estimates of 1.0 for both membranes. These results demonstrate that sigma NaCl for proximal tubule apical and basolateral membranes are near unity. Taken together with previous results, these data suggest that proximal tubule water channels are long narrow pores that exclude NaCl.

  9. Characterization of calcium transport by basolateral membrane vesicles of human small intestine

    SciTech Connect

    Kikuchi, Kazuhiro; Kikuchi, Toshiko; Ghishan, F.K. )

    1988-10-01

    The present studies investigated the mechanism of Ca{sup 2+} transport across basolateral membrane vesicles (BLMVs) prepared from human small intestine. Ca{sup 2+} uptake represented transport into the intravesicular space as evident by osmolality study and by the demonstration of Ca{sup 2+} efflux from the intravesicular space by Ca{sup 2+} ionophore A23187. Ca{sup 2+} uptake was stimulated by Mg{sup 2+}-ATP. Kinetic parameters for ATP-dependent Ca{sup 2+} uptake revealed a Michaelis constant (K{sub m}) of 0.02{plus minus}0.01 {mu}M and a maximum rate of uptake (V{sub max}) of 1.00{plus minus}0.03 nmol{center dot}mg protein{sup {minus}1}{center dot}min{sup {minus}1}. Ca{sup 2+} uptake in the presence of Mg{sup 2+} was inhibited by 75%. The K{sub m} of ATP concentration required for half-maximal Ca{sup 2+} uptake was 0.50{plus minus}0.1 mM. Basolateral membranes depleted of calmodulin by EDTA osmotic shock decreased ATP-dependent Ca{sup 2+} uptake by 65%. Trifluoperazine, an anticalmodulin drug, inhibited ATP-dependent Ca{sup 2+} uptake by 50%, while no inhibition was noted in calmodulin-depleted membranes. Efflux of Ca{sup 2+} in the BLMVs was stimulated by trans-Na{sup +}. Na{sup +}-dependent Ca{sup 2+} uptake was saturable with respect to Ca{sup 2+} concentration and exhibited a K{sub m} of 0.09{plus minus}0.03 {mu}M and a V{sub max} of 1.08{plus minus}0.01 nmol{center dot}mg protein{sup {minus}1}{center dot}min{sup {minus}1}. These results are consistent with the existence of a Na{sup +}-Ca{sup 2+} exchange system and ATP and Mg{sup 2+}-dependent, calmodulin-regulated Ca{sup 2+}, transport mechanism in BLMVs of human enterocytes.

  10. Electrogenic sulfate uptake by crustacean hepatopancreatic basolateral membrane vesicles. [Homarus americanus

    SciTech Connect

    Cattey, M.A.; Gerencser, G.A.; Aheam, G.A. Univ. of Florida, Gainesville )

    1990-02-26

    Basolateral membrane vesicles (BLMV) were isolated from Atlantic lobster (Homarus americanus) hepatopancreas and purified by discontinuous sucrose gradient centrifugation. BLMV prepared in this fashion were osmotically reactive exhibiting linear dependence of vesicular {sup 35}SO{sub 4}{sup {minus}2} uptake to increasing external osmotic pressure with negligible non-specific isotope binding. Under short circuited conditions (valinomycin/K{sup +}) BLMV responded to either a HCO{sub 3}{sup {minus}} gradient directed out or equilibrated HCO{sub 3}{sup {minus}} (10 mM) by displaying short term accumulation of sulfate above that of equilibrium. Uptake of divalent anion was unaffected by an inwardly directed transmembrane Na{sup +} or tetramethylammonium{sup +} gradient. {sup 35}SO{sub 4}{sup {minus}2}/HCO{sub 3}{sup {minus}} exchange in the presence of valinomycin was stimulated by transient inside positive K{sup +} diffusion potentials and inhibited by transient inside negative K{sup +} diffusion potentials. The role of electrogenic anion exchange by hepatopancreas BLMV in transcellular sulfate transport is discussed.

  11. Sodium-bicarbonate cotransport occurs in rat kidney cortical membranes but not in rat small intestinal basolateral membranes.

    PubMed Central

    Hagenbuch, B; Stange, G; Murer, H

    1987-01-01

    Basolateral membrane vesicles were isolated from rat kidney cortex and small intestinal enterocytes. Both membrane preparations show ATP-dependent calcium uptake and cytochalasin B-sensitive D-glucose transport. In renal membranes, sodium influx is stimulated by bicarbonate; bicarbonate-dependent sodium flux is membrane-potential-dependent and inhibited by 4,4'-di-isothiocyanato-2, 2'-stilbenedisulphanic acid ('DIDS'). Small intestinal basolateral membranes do not show bicarbonate-dependent sodium fluxes. PMID:2825641

  12. Calcium transport by rat duodenal villus and crypt basolateral membranes

    SciTech Connect

    Walters, J.R.F.; Weiser, M.M.

    1987-02-01

    Rat duodenal cells were isolated sequentially to give fractions enriched for villus and crypt cells. From each of these fractions, basolateral-enriched membrane vesicles were prepared and ATP-dependent calcium uptake was studied. Calcium uptake was sensitive to temperature, was inhibited by vanadate and by A23187, and was lower in vitamin D-deficient animals. In normal animals, (UVCa)-transport was approximately twofold greater in villus-tip than in crypt cell-fraction basolateral membranes though the affinity of the uptake for calcium was similar (K/sub m/ = 0.3 M). In vitamin D-deficient animals, the crypt-to-villus gradient was reduced, and in all fractions, calcium transport was similar to or lower than that in the crypts of normal animals. Six hours after vitamin D-deficient animals were repleted with 1,25-dihydroxycholecalciferol, a significant increase in calcium transport by everted gut sacs was present; however, basolateral calcium transport was significantly increased in only the mid-villus fractions, and no change was seen in the villus-tip fractions. Thus vitamin D appears necessary for the development of increased basolateral membrane calcium pump activity in duodenal villus cells, but not all cells in vitamin D-deficient rats are able to respond to 1,25-dihydroxycholecalciferol.

  13. Tyrosine motifs are required for prestin basolateral membrane targeting

    PubMed Central

    Zhang, Yifan; Moeini-Naghani, Iman; Bai, JunPing; Santos-Sacchi, Joseph; Navaratnam, Dhasakumar S.

    2015-01-01

    ABSTRACT Prestin is targeted to the lateral wall of outer hair cells (OHCs) where its electromotility is critical for cochlear amplification. Using MDCK cells as a model system for polarized epithelial sorting, we demonstrate that prestin uses tyrosine residues, in a YXXΦ motif, to target the basolateral surface. Both Y520 and Y667 are important for basolateral targeting of prestin. Mutation of these residues to glutamine or alanine resulted in retention within the Golgi and delayed egress from the Golgi in Y667Q. Basolateral targeting is restored upon mutation to phenylalanine suggesting the importance of a phenol ring in the tyrosine side chain. We also demonstrate that prestin targeting to the basolateral surface is dependent on AP1B (μ1B), and that prestin uses transferrin containing early endosomes in its passage from the Golgi to the basolateral plasma membrane. The presence of AP1B (μ1B) in OHCs, and parallels between prestin targeting to the basolateral surface of OHCs and polarized epithelial cells suggest that outer hair cells resemble polarized epithelia rather than neurons in this important phenotypic measure. PMID:25596279

  14. Endocytosis of albumin and thyroglobulin at the basolateral membrane of thyrocytes organized in follicles.

    PubMed

    Gire, V; Kostrouch, Z; Bernier-Valentin, F; Rabilloud, R; Munari-Silem, Y; Rousset, B

    1996-02-01

    Serum proteins such as albumin are present inside thyroid follicles in both normal and pathological situations. To analyze the mechanism of entry of these proteins, we investigated the ability of polarized thyrocytes to internalize soluble molecules at their basolateral pole. Experiments were conducted on in vitro reconstituted thyroid follicles using BSA and pig thyroglobulin (Tg) coupled to gold particles for electron microscopy, conjugated to fluorescein for conventional and confocal fluorescence microscopy, or radioiodinated for biochemical measurements. Incubations were carried out at 37 C. BSA and Tg coupled to gold particles were rapidly internalized from the culture medium and sequentially found in small vesicles and early endosomes and in late endosomes and lysosomes. Fluorescence microscope analyses revealed that the majority of cells forming reconstituted thyroid follicles are capable of internalizing BSA and Tg, but that Tg was more efficiently endocytosed than BSA. Using radioiodinated ligands, it was observed that the endocytosis of Tg was 10 times higher than that of BSA. The internalization of [125I]Tg was inhibited by increasing concentrations of unlabeled Tg. In contrast, endocytosis of 125I-labeled BSA was independent of the unlabeled BSA concentration. Experiments performed at 4 C indicated the presence of a basolateral membrane binding activity for [125I]Tg; the Tg concentration that reduced the binding of labeled Tg by 50% ranged from 4-6 microM. These data are evidence of a process of internalization of soluble molecules at the basolateral pole of thyrocytes, with BSA being internalized by fluid phase endocytosis and Tg by selective endocytosis. Our findings explain how serum albumin can enter thyroid follicles and disclose a new cellular handling and transport pathway of Tg. We propose that selective uptake of Tg operating on molecules secreted at the basolateral surface of thyrocytes could control the amount of Tg released in the

  15. Biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation

    SciTech Connect

    Bartles, J.R.; Feracci, H.M.; Stieger, B.; Hubbard, A.L.

    1987-09-01

    We have used pulse-chase metabolic radiolabeling with L-(/sup 35/S)methionine in conjunction with subcellular fractionation and specific protein immunoprecipitation techniques to compare the posttranslational transport pathways taken by endogenous domain-specific integral proteins of the rat hepatocyte plasma membrane in vivo. Our results suggest that both apical (HA 4, dipeptidylpeptidase IV, and aminopeptidase N) and basolateral (CE 9 and the asialoglycoprotein receptor (ASGP-R)) proteins reach the hepatocyte plasma membrane with similar kinetics. The mature molecular mass form of each of these proteins reaches its maximum specific radioactivity in a purified hepatocyte plasma membrane fraction after only 45 min of chase. However, at this time, the mature radiolabeled apical proteins are not associated with vesicles derived from the apical domain of the hepatocyte plasma membrane, but instead are associated with vesicles which, by several criteria, appear to be basolateral plasma membrane. These vesicles: (a) fractionate like basolateral plasma membrane in sucrose density gradients and in free-flow electrophoresis; (b) can be separated from the bulk of the likely organellar contaminants, including membranes derived from the late Golgi cisternae, transtubular network, and endosomes; (c) contain the proven basolateral constituents CE 9 and the ASGP-R, as judged by vesicle immunoadsorption using fixed Staphylococcus aureus cells and anti-ASGP-R antibodies; and (d) are oriented with their ectoplasmic surfaces facing outward, based on the results of vesicle immunoadsorption experiments using antibodies specific for the ectoplasmic domain of the ASGP-R. Only at times of chase greater than 45 min do significant amounts of the mature radiolabeled apical proteins arrive at the apical domain, and they do so at different rates.

  16. Hepatic taurine transport: a Na/sup +/-dependent carrier on the basolateral plasma membrane

    SciTech Connect

    Bucuvalas, J.C.; Goodrich, A.L.; Suchy, F.J.

    1987-09-01

    Highly purified rat basolateral liver plasma membrane vesicles were used examine the mechanism and the driving forces for hepatic uptake of the ..beta..-amino acid, taurine. An inwardly directed 100 mM NaCl gradient stimulated the initial rate of taurine uptake and energized a transient twofold accumulation of taurine above equilibrium (overshoot). In contrast, uptake was slower and no overshoot was detected in the presence of a KCl gradient. A negative intravesicular electrical potential generated by the presence of permeant anions or an outwardly directed K/sup +/ gradient with valinomycin increased Na/sup +/-stimulated taurine uptake. External Cl/sup -/ stimulated Na/sup +/-dependent taurine uptake independent of effects on the transmembrane electrical potential difference. Na/sup +/-dependent taurine uptake showed a sigmoidal dependence on extravesicular Na/sup +/ concentration, suggesting multiple Na/sup +/ ions are involved in the translocation of each taurine molecule. Na/sup +/-dependent taurine uptake demonstrated Michaelis-Menten kinetics with a maximum velocity of 0.537 nmol x mg protein/sup -1/ x min/sup -1/ and an apparent K/sub m/ of 174 ..mu..M. (/sup 3/H)taurine uptake was inhibited by the presence of excess unlabeled taurine, ..beta..-alanine, or hypotaurine but not by L-glutamine or L-alanine. In summary, using basolateral liver plasma membrane vesicles, the authors have shown that hepatic uptake of taurine occurs by a carrier-mediated, secondary active transport process specific for ..beta..-amino acids. Uptake is electrogenic, stimulated by external Cl/sup -/, and requires multiple Na/sup +/ ions for the translocation of each taurine molecule.

  17. Vesicle trafficking and cell surface membrane patchiness.

    PubMed

    Tang, Q; Edidin, M

    2001-07-01

    Membrane proteins and lipids often appear to be distributed in patches on the cell surface. These patches are often assumed to be membrane domains, arising from specific molecular associations. However, a computer simulation (Gheber and Edidin, 1999) shows that membrane patchiness may result from a combination of vesicle trafficking and dynamic barriers to lateral mobility. The simulation predicts that the steady-state patches of proteins and lipids seen on the cell surface will decay if vesicle trafficking is inhibited. To test this prediction, we compared the apparent sizes and intensities of patches of class I HLA molecules, integral membrane proteins, before and after inhibiting endocytic vesicle traffic from the cell surface, either by incubation in hypertonic medium or by expression of a dominant-negative mutant dynamin. As predicted by the simulation, the apparent sizes of HLA patches increased, whereas their intensities decreased after endocytosis and vesicle trafficking were inhibited. PMID:11423406

  18. Fabrication of a Dual Substrate Display to Test Roles of Cell Adhesion Proteins in Vesicle Targeting to Plasma Membrane Domains

    PubMed Central

    Hunt, Stephen J.; Nelson, W. James

    2009-01-01

    While much is known of the molecular machinery involved in protein sorting during exocytosis, less is known about the spatial regulation of exocytosis at the plasma membrane (PM). This study outlines a novel method, Dual Substrate Display, used to formally test the hypothesis that E-cadherin-mediated adhesion directs basolateral vesicle exocytosis to specific sites at the PM. We show that vesicles containing the basolateral marker protein VSV-G preferentially target to sites of adhesion to E-cadherin rather than collagen VI or a control peptide. These results support the hypothesis that E-cadherin adhesion initiates signaling at the PM resulting in targeted sites for exocytosis. PMID:17803993

  19. Toroidal membrane vesicles in spherical confinement.

    PubMed

    Bouzar, Lila; Menas, Ferhat; Müller, Martin Michael

    2015-09-01

    We investigate the morphology of a toroidal fluid membrane vesicle confined inside a spherical container. The equilibrium shapes are assembled in a geometrical phase diagram as a function of scaled area and reduced volume of the membrane. For small area the vesicle can adopt its free form. When increasing the area, the membrane cannot avoid contact and touches the confining sphere along a circular contact line, which extends to a zone of contact for higher area. The elastic energies of the equilibrium shapes are compared to those of their confined counterparts of spherical topology to predict under which conditions a topology change is favored energetically.

  20. Toroidal membrane vesicles in spherical confinement

    NASA Astrophysics Data System (ADS)

    Bouzar, Lila; Menas, Ferhat; Müller, Martin Michael

    2015-09-01

    We investigate the morphology of a toroidal fluid membrane vesicle confined inside a spherical container. The equilibrium shapes are assembled in a geometrical phase diagram as a function of scaled area and reduced volume of the membrane. For small area the vesicle can adopt its free form. When increasing the area, the membrane cannot avoid contact and touches the confining sphere along a circular contact line, which extends to a zone of contact for higher area. The elastic energies of the equilibrium shapes are compared to those of their confined counterparts of spherical topology to predict under which conditions a topology change is favored energetically.

  1. Polypeptide vesicles with densely packed multilayer membranes.

    PubMed

    Song, Ziyuan; Kim, Hojun; Ba, Xiaochu; Baumgartner, Ryan; Lee, Jung Seok; Tang, Haoyu; Leal, Cecilia; Cheng, Jianjun

    2015-05-28

    Multilamellar membranes are important building blocks for constructing self-assembled structures with improved barrier properties, such as multilamellar lipid vesicles. Polymeric vesicles (polymersomes) have attracted growing interest, but multilamellar polymersomes are much less explored. Here, we report the formation of polypeptide vesicles with unprecedented densely packed multilayer membrane structures with poly(ethylene glycol)-block-poly(γ-(4,5-dimethoxy-2-nitrobenzyl)-l-glutamate) (PEG-b-PL), an amphiphilic diblock rod-coil copolymer containing a short PEG block and a short hydrophobic rod-like polypeptide segment. The polypeptide rods undergo smectic ordering with PEG buried between the hydrophobic polypeptide layers. The size of both blocks and the rigidity of the hydrophobic polypeptide block are critical in determining the membrane structures. Increase of the PEG length in PEG-b-PL results in the formation of bilayer sheets, while using random-coil polypeptide block leads to the formation of large compound micelles. UV treatment causes ester bond cleavage of the polypeptide side chain, which induces helix-to-coil transition, change of copolymer amphiphilicity, and eventual disassembly of vesicles. These polypeptide vesicles with unique membrane structures provide a new insight into self-assembly structure control by precisely tuning the composition and conformation of polymeric amphiphiles.

  2. Phorbol ester-stimulated phosphorylation of basolateral membranes from canine kidney

    SciTech Connect

    Hammerman, M.R.; Rogers, S.; Morrissey, J.J.; Gavin, J.R. III

    1986-06-01

    To determine whether protein kinase C is present in the basolateral membrane of the renal proximal tubular cell, we performed experiments to ascertain whether specific binding of (/sup 3/H)phorbol 12,13-dibutyrate could be demonstrated in basolateral membranes isolated from canine kidney. Specific binding was demonstrable that was half maximal at between 10(-7) and 10(-8) M phorbol 12,13-dibutyrate. Binding was inhibited by 12-O-tetradecanoylphorbol-13-acetate (TPA) and other tumor-promoting phorbol esters, but not by inactive phorbol esters, including 4 alpha-phorbol. Incubation of basolateral membranes with TPA and phorbol 12,13-dibutyrate, but not with 4 alpha-phorbol, in the presence of submicromolar concentrations of free calcium, enhanced phosphorylation of several proteins demonstrable in autoradiograms of sodium dodecyl sulfate-polyacrylamide gels originating from membranes subsequently exposed to (gamma-32P)ATP for 30 s. Dephosphorylation of (/sup 32/P)phosphoproteins was observed in gels from membranes incubated with (gamma-32P)ATP over time. TPA-stimulated phosphorylation of one protein band with Mr 135,000 was quantitated and was found to increase as a function of (TPA). Half-maximal TPA-stimulated phosphorylation of this protein band occurred at slightly less than 10(-9) M TPA. Our findings are consistent with a role for protein kinase C-effected phosphorylation of basolateral membrane proteins in the mediation or modulation of hormonal actions in the proximal tubular cell.

  3. Sulfate transport by chick renal tubule brush-border and basolateral membranes

    SciTech Connect

    Renfro, J.L.; Clark, N.B.; Metts, R.E.; Lynch, M.A.

    1987-01-01

    Brush-border and basolateral membrane vesicles (BBMV and BLMV, respectively) were prepared from chick kidney by a calcium precipitation method and by centrifugation on an 8% Percoll self-generating gradient, respectively. In BBMV a 100-mM Na gluconate gradient, out>in, caused concentrative (/sup 35/S) sulfate uptake approximately fivefold greater at 1 min than at 60 min (equilibrium) whether or not the membranes were short-circuited with 100 mM K gluconate, in=out, plus 20 ..mu..g valinomycin/mg protein. A 48-mM HCO/sub 3//sup -/ gradient, in>out, stimulated a 2.5-fold higher uptake at 1 min than at 60 min, and short circuiting as above had no effect on the magnitude of this response. Imposition of a H/sup +/ gradient caused concentrative uptake fourfold higher at 1 min than at equilibrium. Short circuiting as above or addition of 0.1 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP) significantly inhibited the pH gradient effect. Creation of an inside positive electrical potential with 100 mM K gluconate, out>in, plus valinomycin, also caused concentrative sulfate uptake. Based on inhibitor/competitor effects, these are distinct sulfate transport processes. In chick BLMV, imposition of an HCO/sub 3//sup -/ gradient, in>out, produced concentrative sulfate uptake. 4-Acetamido-4'-isothiocyanostilbene 2,2'-disulfonic acid disodium at 0.1 mM was an effective inhibitor of BLMV bicarbonate-sulfate exchange.

  4. Ba2+-inhibitable /sup 86/Rb+ fluxes across membranes of vesicles from toad urinary bladder

    SciTech Connect

    Garty, H.; Civan, M.M.

    1987-01-01

    /sup 86/Rb+ fluxes have been measured in suspensions of vesicles prepared from the epithelium of toad urinary bladder. A readily measurable barium-sensitive, ouabain-insensitive component has been identified; the concentration of external Ba2+ required for half-maximal inhibition was 0.6 mM. The effects of externally added cations on /sup 86/Rb+ influx and efflux have established that this pathway is conductive, with a selectivity for K+, Rb+ and Cs+ over Na+ and Li+. The Rb+ uptake is inversely dependent on external pH, but not significantly affected by internal Ca2+ or external amiloride, quinine, quinidine or lidocaine. It is likely, albeit not yet certain, that the conductive Rb+ pathway is incorporated in basolateral vesicles oriented right-side-out. It is also not yet clear whether this pathway comprises the principle basolateral K+ channel in vivo, and that its properties have been unchanged during the preparative procedures. Subject to these caveats, the data suggest that the inhibition by quinidine of Na+ transport across toad bladder does not arise primarily from membrane depolarization produced by a direct blockage of the basolateral channels. It now seems more likely that the quinidine-induced elevation of intracellular Ca2+ activity directly blocks apical Na+ entry.

  5. Active and passive Na+ fluxes across the basolateral membrane of rabbit urinary bladder.

    PubMed

    Eaton, D C; Frace, A M; Silverthorn, S U

    1982-01-01

    The apical membrane of rabbit urinary bladder can be functionally removed by application of nystatin at high concentration if the mucosal surface of the tissue is bathed in a saline which mimics intracellular ion concentrations. Under these conditions, the tissue is as far as the movement of univalent ions no more than a sheet of basolateral membrane with some tight junctional membrane in parallel. In this manner the Na+ concentration at the inner surface of the basolateral membrane can be varied by altering the concentration in the mucosal bulk solution. When this was done both mucosal-to-serosal 22Na flux and net change in basolateral current were measured. The flux and the current could be further divided into the components of each that were either blocked by ouabain or insensitive to ouabain. Ouabain-insensitive mucosal-to-serosal Na+ flux was a linear function of mucosal Na+ concentration. Ouabain-sensitive Na+ flux and ouabain-sensitive, Na+-induced current both display a saturating relationship which cannot be accounted for by the presence of unstirred layers. If the interaction of Na+ with the basolateral transport process is assumed to involve the interaction of some number of Na+ ions, n, with a maximal flux, MMAX, then the data can be fit by assuming 3.2 equivalent sites for interaction and a value for MMAX of 287.8 pM cm-2 sec-1 with an intracellular Na concentration of 2.0 mM Na+ at half-maximal saturation. By comparing these values with the ouabain-sensitive, Na+-induced current, we calculate a Na+ to K+ coupling ratio of 1.40 +/- 0.07 for the transport process.

  6. Growth hormone activates phospholipase C in proximal tubular basolateral membranes from canine kidney

    SciTech Connect

    Rogers, S.A.; Hammerman, M.R. )

    1989-08-01

    To delineate pathways for signal transduction by growth hormone (GH) in proximal tubule, the authors incubated basolateral membranes isolated from canine kidney with human growth hormone (hGH) or human prolactin (hPrl) and measured levels of inositol trisphosphate (InsP{sub 3}) in suspensions and of diacylglycerol extractable from the membranes. Incubation with hGH, but not hPrl, increased levels of InsP{sub 3} and diacylglycerol in a concentration-dependent manner. Half-maximal effects occurred between 0.1 and 1 nM hGH. Increased levels of InsP{sub 3} were measured after as little as 5 sec of incubation with 1 nM hGH, and increase was maximal after 15 sec. Increases were no longer detectable after 60 sec because of dephosphorylation of InsP{sub 3} in membrane suspensions. hGH did not affect rates of dephosphorylation. hGH-stimulated increases in InsP{sub 3} were detectable in membranes suspended in 0, 0.1, and 0.2 {mu}M calcium but not in 0.3 or 1.0 {mu}M calcium. {sup 125}I-labeled hGH-receptor complexes with M{sub r} values of 66,000 and 140,000 were identified in isolated basolateral membranes. The findings establish that GH activates phospholipase C in isolated canine renal proximal tubular basolateral membranes, potentially after binding to a specific receptor. This process could mediate signal transmission by GH across the plasma membrane of the proximal tubular cell and elsewhere.

  7. Bacterial Outer Membrane Vesicles and Vaccine Applications

    PubMed Central

    Acevedo, Reinaldo; Fernández, Sonsire; Zayas, Caridad; Acosta, Armando; Sarmiento, Maria Elena; Ferro, Valerie A.; Rosenqvist, Einar; Campa, Concepcion; Cardoso, Daniel; Garcia, Luis; Perez, Jose Luis

    2014-01-01

    Vaccines based on outer membrane vesicles (OMV) were developed more than 20 years ago against Neisseria meningitidis serogroup B. These nano-sized structures exhibit remarkable potential for immunomodulation of immune responses and delivery of meningococcal antigens or unrelated antigens incorporated into the vesicle structure. This paper reviews different applications in OMV Research and Development (R&D) and provides examples of OMV developed and evaluated at the Finlay Institute in Cuba. A Good Manufacturing Practice (GMP) process was developed at the Finlay Institute to produce OMV from N. meningitidis serogroup B (dOMVB) using detergent extraction. Subsequently, OMV from N. meningitidis, serogroup A (dOMVA), serogroup W (dOMVW), and serogroup X (dOMVX) were obtained using this process. More recently, the extraction process has also been applied effectively for obtaining OMV on a research scale from Vibrio cholerae (dOMVC), Bordetella pertussis (dOMVBP), Mycobacterium smegmatis (dOMVSM), and BCG (dOMVBCG). The immunogenicity of the OMV has been evaluated for specific antibody induction, and together with functional bactericidal and challenge assays in mice has shown their protective potential. dOMVB has been evaluated with non-neisserial antigens, including with a herpes virus type 2 glycoprotein, ovalbumin, and allergens. In conclusion, OMV are proving to be more versatile than first conceived and remain an important technology for development of vaccine candidates. PMID:24715891

  8. Exocyst Is Involved in Cystogenesis and Tubulogenesis and Acts by Modulating Synthesis and Delivery of Basolateral Plasma Membrane and Secretory Proteins

    PubMed Central

    Lipschutz, Joshua H.; Guo, Wei; O'Brien, Lucy E.; Nguyen, Yen H.; Novick, Peter; Mostov, Keith E.

    2000-01-01

    Epithelial cyst and tubule formation are critical processes that involve transient, highly choreographed changes in cell polarity. Factors controlling these changes in polarity are largely unknown. One candidate factor is the highly conserved eight-member protein complex called the exocyst. We show that during tubulogenesis in an in vitro model system the exocyst relocalized along growing tubules consistent with changes in cell polarity. In yeast, the exocyst subunit Sec10p is a crucial component linking polarized exocytic vesicles with the rest of the exocyst complex and, ultimately, the plasma membrane. When the exocyst subunit human Sec10 was exogenously expressed in epithelial Madin-Darby canine kidney cells, there was a selective increase in the synthesis and delivery of apical and basolateral secretory proteins and a basolateral plasma membrane protein, but not an apical plasma membrane protein. Overexpression of human Sec10 resulted in more efficient and rapid cyst formation and increased tubule formation upon stimulation with hepatocyte growth factor. We conclude that the exocyst plays a central role in the development of epithelial cysts and tubules. PMID:11102522

  9. Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles

    PubMed Central

    Kieselbach, Thomas; Zijnge, Vincent; Granström, Elisabeth; Oscarsson, Jan

    2015-01-01

    Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA) into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs). To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease. PMID:26381655

  10. Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles.

    PubMed

    Kieselbach, Thomas; Zijnge, Vincent; Granström, Elisabeth; Oscarsson, Jan

    2015-01-01

    Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA) into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs). To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease. PMID:26381655

  11. Membrane tubulation from giant lipid vesicles in alternating electric fields.

    PubMed

    Antonova, K; Vitkova, V; Meyer, C

    2016-01-01

    We report on the formation of tubular membrane protrusions from giant unilamellar vesicles in alternating electric fields. The construction of the experimental chamber permitted the application of external AC fields with strength of dozens of V/mm and kHz frequency during relatively long time periods (several minutes). Besides the vesicle electrodeformation from quasispherical to prolate ellipsoidal shape, the formation of long tubular membrane protrusions with length of up to several vesicle diameters, arising from the vesicular surface in the field direction, was registered and analyzed. The threshold electric field at which the electro-induced protrusions appeared was lower than the field strengths inducing membrane electroporation. PMID:26871107

  12. H/sup +/-translocating ATPases: advances using membrane vesicles

    SciTech Connect

    Sze, H.

    1985-01-01

    In this paper, two primary active transport systems (H/sup +/ -ATPases) in plant cells are examined using membrane vesicles as a simple experimental tool. One electrogenic, H/sup +/ -translocating ATPase is vanadate-sensitive and associated with the plasma membrane. Another electrogenic, H/sup +/ -translocating ATPases is anion-sensitive, and localized on the tonoplast (and perhaps other membranes). According to the working model, the plasma membrane and tonoplast-type H/sup +/ -ATPases are detectable in inside-out plasma membrane and right-side-out tonoplast vesicles. The direction of H/sup +/ pumping into these vesicles would be consistent with the results from intact cells where H/sup +/ are extruded from the cell across the plasma membrane and pumped into the vacuole from the cytoplasm. Understanding the properties of H/sup +/ -pumping ATPases using membrane vesicles has paved the way for studies to identify secondary active transport systems coupled to the proton electrochemical gradient. Redox-driven transport systems can also be studied directly using the isolated vesicles. As transport proteins are identified, the functional activities can be specifically studied after reconstitution of the purified protein(s) into phospholipid membrane vesicles. 154 references.

  13. Membrane trafficking: decoding vesicle identity with contrasting chemistries.

    PubMed

    Frost, Adam

    2011-10-11

    Proteins involved in membrane traffic must distinguish between different classes of vesicles. New work now shows that α-synuclein and ALPS motifs represent two extreme types of amphipathic helix that are tuned to detect both the curvature of transport vesicles as well as their bulk lipid content.

  14. Outer membrane vesicles as platform vaccine technology

    PubMed Central

    Stork, Michiel; van der Ley, Peter

    2015-01-01

    Abstract Outer membrane vesicles (OMVs) are released spontaneously during growth by many Gram‐negative bacteria. They present a range of surface antigens in a native conformation and have natural properties like immunogenicity, self‐adjuvation and uptake by immune cells which make them attractive for application as vaccines against pathogenic bacteria. In particular with Neisseria meningitidis, they have been investigated extensively and an OMV‐containing meningococcal vaccine has recently been approved by regulatory agencies. Genetic engineering of the OMV‐producing bacteria can be used to improve and expand their usefulness as vaccines. Recent work on meningitis B vaccines shows that OMVs can be modified, such as for lipopolysaccharide reactogenicity, to yield an OMV product that is safe and effective. The overexpression of crucial antigens or simultaneous expression of multiple antigenic variants as well as the expression of heterologous antigens enable expansion of their range of applications. In addition, modifications may increase the yield of OMV production and can be combined with specific production processes to obtain high amounts of well‐defined, stable and uniform OMV particle vaccine products. Further improvement can facilitate the development of OMVs as platform vaccine product for multiple applications. PMID:26912077

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

    PubMed

    Lin, Qingqing; London, Erwin

    2014-01-01

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

  16. Evidence for carrier-mediated chloride/bicarbonate exchange in canalicular rat liver plasma membrane vesicles

    SciTech Connect

    Meier, P.J.; Knickelbein, R.; Moseley, R.H.; Dobbins, J.W.; Boyer, J.L.

    1985-04-01

    To determine whether anion exchangers might play a role in hepatic bile formation, the authors looked for the presence of Cl/sup -/:OH/sup -/ and Cl/sup -/:HCO3/sup -/ exchange in highly purified canalicular (c) and basolateral (bl) rat liver plasma membrane (LPM) vesicles. In cLPM vesicles, a pH gradient stimulated /sup 36/Cl- uptake twofold above values obtained during pH-equilibrated conditions. When 50 mM HCO3/sup -/ was also present inside the vesicles, the same pH gradient resulted in Cl/sup -/ uptake to levels fourfold above pH- and HCO3--equilibrated controls and two- to threefold above Cl- equilibrium. Initial rates of both pH and HCO3/sup -/ gradient-stimulated Cl/sup -/ uptake were completely inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS). A valinomycin-induced K/sup +/ diffusion potential (inside positive) also stimulated Cl/sup -/ uptake in cLPM, but this conductive Cl- pathway was insensitive to DIDS. The DIDS-sensitive, pH and HCO3- gradient-stimulated Cl/sup -/ uptake demonstrated: saturation with Cl/sup -/; partial inhibition by bumetanide (26%), furosemide (33%), probenecid (37%), and 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene (49%); cis-inhibition by chloride and nitrate but not by sulfate and various organic anions, and independence from the membrane potential. These data demonstrate the presence of an electroneutral Cl/sup -/:OH/sup -/ and Cl/sup -/:HCO3/sup -/ exchanger in rat liver canalicular membranes that favors Cl/sup -/:HCO3/sup -/ exchange. In contrast, no evidence was found for the presence of a Cl/sup -/:HCO3/sup -/ (OH/sup -/) exchange system in blLPM vesicles.

  17. VAMP-1: a synaptic vesicle-associated integral membrane protein.

    PubMed Central

    Trimble, W S; Cowan, D M; Scheller, R H

    1988-01-01

    Several proteins are associated with, or are integral components of, the lipid bilayer that forms the delineating membrane of neuronal synaptic vesicles. To characterize these molecules, we used a polyclonal antiserum raised against purified cholinergic synaptic vesicles from Torpedo to screen a cDNA expression library constructed from mRNA of the electromotor nucleus. One clone encodes VAMP-1 (vesicle-associated membrane protein 1), a nervous-system-specific protein of 120 amino acids whose primary sequence can be divided into three domains: a proline-rich amino terminus, a highly charged internal region, and a hydrophobic carboxyl-terminal domain that is predicted to comprise a membrane anchor. Tryptic digestion of intact and lysed vesicles suggests that the protein faces the cytoplasm, where it may play a role in packaging, transport, or release of neurotransmitters. Images PMID:3380805

  18. Inward and outward membrane tubes pulled from giant vesicles

    NASA Astrophysics Data System (ADS)

    Dasgupta, Raktim; Dimova, Rumiana

    2014-07-01

    Membrane nanotubes are extruded from giant unilamellar lipid vesicles using a controlled hydrodynamic flow and membrane-attached beads manipulated via optical tweezers. Within a single experiment, the technique can be used to assess various important mechanical and rheological characteristics of the membrane such as the bending rigidity, tension and intermonolayer slip. The application of small flow velocities leads to the extrusion of tubes with sufficiently large diameters conveniently measurable under an optical microscope. For the first time, we show that by suitably controlling the medium flow, inward tubes inside the vesicles can be formed. This approach offers great potential for studying tubulation mechanisms in membrane systems, exhibiting positive as well as negative spontaneous curvatures and should offer a more realistic model for biomembranes because the vesicle membrane tension can adapt freely.

  19. Prokaryotic membrane vesicles: new insights on biogenesis and biological roles.

    PubMed

    Haurat, M Florencia; Elhenawy, Wael; Feldman, Mario F

    2015-02-01

    Biogenesis and trafficking of membrane vesicles are essential and well-studied processes in eukaryotes. In contrast, vesiculation in bacteria is not well understood. Outer membrane vesicles (OMVs) are produced in Gram-negative bacteria by blebbing of the outer membrane. In addition to the roles in pathogenesis, cell-to-cell communication and stress response, recent work has suggested that OMVs play important roles in immunomodulation and the establishment and balance of the gut microbiota. In this review we discuss the known and novel roles of OMVs and the different biogenesis models proposed, and address the evidence for cargo selection into OMVs. We also discuss the growing evidence for the existence of membrane vesicles in Gram-positive bacteria and Archaea. Due to their biological importance and promising applications in vaccinology, the biogenesis of OMVs is an important topic in microbiology.

  20. Active endocannabinoids are secreted on extracellular membrane vesicles.

    PubMed

    Gabrielli, Martina; Battista, Natalia; Riganti, Loredana; Prada, Ilaria; Antonucci, Flavia; Cantone, Laura; Matteoli, Michela; Maccarrone, Mauro; Verderio, Claudia

    2015-02-01

    Endocannabinoids primarily influence neuronal synaptic communication within the nervous system. To exert their function, endocannabinoids need to travel across the intercellular space. However, how hydrophobic endocannabinoids cross cell membranes and move extracellularly remains an unresolved problem. Here, we show that endocannabinoids are secreted through extracellular membrane vesicles produced by microglial cells. We demonstrate that microglial extracellular vesicles carry on their surface N-arachidonoylethanolamine (AEA), which is able to stimulate type-1 cannabinoid receptors (CB1), and inhibit presynaptic transmission, in target GABAergic neurons. This is the first demonstration of a functional role of extracellular vesicular transport of endocannabinoids.

  1. Structural Basis of Vesicle Formation at the Inner Nuclear Membrane.

    PubMed

    Hagen, Christoph; Dent, Kyle C; Zeev-Ben-Mordehai, Tzviya; Grange, Michael; Bosse, Jens B; Whittle, Cathy; Klupp, Barbara G; Siebert, C Alistair; Vasishtan, Daven; Bäuerlein, Felix J B; Cheleski, Juliana; Werner, Stephan; Guttmann, Peter; Rehbein, Stefan; Henzler, Katja; Demmerle, Justin; Adler, Barbara; Koszinowski, Ulrich; Schermelleh, Lothar; Schneider, Gerd; Enquist, Lynn W; Plitzko, Jürgen M; Mettenleiter, Thomas C; Grünewald, Kay

    2015-12-17

    Vesicular nucleo-cytoplasmic transport is becoming recognized as a general cellular mechanism for translocation of large cargoes across the nuclear envelope. Cargo is recruited, enveloped at the inner nuclear membrane (INM), and delivered by membrane fusion at the outer nuclear membrane. To understand the structural underpinning for this trafficking, we investigated nuclear egress of progeny herpesvirus capsids where capsid envelopment is mediated by two viral proteins, forming the nuclear egress complex (NEC). Using a multi-modal imaging approach, we visualized the NEC in situ forming coated vesicles of defined size. Cellular electron cryo-tomography revealed a protein layer showing two distinct hexagonal lattices at its membrane-proximal and membrane-distant faces, respectively. NEC coat architecture was determined by combining this information with integrative modeling using small-angle X-ray scattering data. The molecular arrangement of the NEC establishes the basic mechanism for budding and scission of tailored vesicles at the INM.

  2. Release of outer membrane vesicles from Bordetella pertussis.

    PubMed

    Hozbor, D; Rodriguez, M E; Fernández, J; Lagares, A; Guiso, N; Yantorno, O

    1999-05-01

    The aim of the study reported here was to investigate the production of Bordetella pertussis outer membrane vesicles (OMVs). Numerous vesicles released from cells grown in Stainer-Scholte liquid medium were observed. The formation of similar vesicle-like structures could also be artificially induced by sonication of concentrated bacterial suspensions. Immunoblot analysis showed that OMVs contain adenylate cyclase-hemolysin (AC-Hly), among other polypeptides, as well as the lipopolysaccharide (LPS). Experiments carried out employing purified AC-Hly and OMVs isolated from B. pertussis AC-Hly- showed that AC-Hly is an integral component of the vesicles. OMVs reported here contain several protective immunogens and might be considered a possible basic material for the development of acellular pertussis vaccines.

  3. Nanoparticle-triggered release from lipid membrane vesicles.

    PubMed

    Reimhult, Erik

    2015-12-25

    Superparamagnetic iron oxide nanoparticles are used in a rapidly expanding number of research and practical applications in biotechnology and biomedicine. We highlight how recent developments in iron oxide nanoparticle design and understanding of nanoparticle membrane interactions have led to applications in magnetically triggered, liposome delivery vehicles with controlled structure. Nanoscale vesicles actuated by incorporated nanoparticles allow for controlling location and timing of compound release, which enables e.g. use of more potent drugs in drug delivery as the interaction with the right target is ensured. This review emphasizes recent results on the connection between nanoparticle design, vesicle assembly and the stability and release properties of the vesicles. While focused on lipid vesicles magnetically actuated through iron oxide nanoparticles, these insights are of general interest for the design of capsule and cell delivery systems for biotechnology controlled by nanoparticles.

  4. Relative osmotic effects of raffinose, KCl, and NaCl across basolateral cell membrane.

    PubMed

    Welling, L W; Welling, D J; Ochs, T

    1990-10-01

    Lumen-collapsed segments of rabbit S2 proximal tubule were bathed in isotonic medium and then exposed acutely to a medium made hypertonic by the addition of raffinose, NaCl, KCl, Na gluconate, K gluconate, or choline Cl. The result was a rapid efflux of water and a shrinking of the tubule, which could be measured by video techniques within the first 0.1 s. After reequilibration in isotonic medium, each tubule was then exposed to a second hypertonic medium to provide a direct comparison between two different solutes, either NaCl vs. KCl or raffinose vs. any one of the other solutes. Because raffinose is impermeant across the basolateral cell membrane, the ratio of its effect to that of another solute is a measure of the reflection coefficient (sigma) of that other solute. The following results were obtained: sigma KCl = 0.70 +/- 0.02, sigma K gluconate = 0.97 +/- 0.07, sigma Na gluconate = 0.84 +/- 0.06, and sigma choline Cl = 0.75 +/- 0.06. We previously have reported sigma NaCl = 0.56 +/- 0.07. If sigma of each salt is considered to be the arithmetic average of its component parts, and if gluconate and choline are considered to be impermeant, we also obtain sigma Na+ = 0.68, sigma K+ = 0.94, and sigma Cl- = 0.50. PMID:2221098

  5. Structural lipid changes and Na(+)/K(+)-ATPase activity of gill cells' basolateral membranes during saltwater acclimation in sea lamprey (Petromyzon marinus, L.) juveniles.

    PubMed

    Lança, Maria João; Machado, Maria; Ferreira, Ana Filipa; Quintella, Bernardo Ruivo; de Almeida, Pedro Raposo

    2015-11-01

    Seawater acclimation is a critical period for anadromous species and a process yet to be understood in lampreys. Considering that changes in lipid composition of the gill cells' basolateral membranes may disrupt the major transporter Na(+)K(+)-ATPase, the goal of this study was to detect changes at this level during juvenile sea lamprey seawater acclimation. The results showed that saltwater acclimation has a direct effect on the fatty acid composition of gill cells basolateral membrane's phospholipids. When held in full-strength seawater, the fatty acid profile of basolateral membrane's phospholipids suffered a restructure by increasing either saturation or the ratio between oleic acid and eicosapentaenoic acid. Simultaneously, the activity of Na(+)K(+)-ATPase revealed a significant and positive correlation with basolateral membrane's cholesterol content in the presence of highest salinity. Our results pointed out for lipid adjustments involving the functional transporter present on the gill cell basolateral membranes to ensure the role played by branchial Na(+)K(+)-ATPase in ion transport during saltwater acclimation process. The responses observed contributed to the strategy adopted by gill cell's basolateral membranes to compensate for osmotic and ionic stressors, to ensure the success of the process of seawater acclimation associated with the downstream trophic migration of juvenile sea lamprey.

  6. Phospholipid flippases: building asymmetric membranes and transport vesicles

    PubMed Central

    Sebastian, Tessy T.; Baldridge, Ryan D.; Xu, Peng; Graham, Todd R.

    2012-01-01

    Phospholipid flippases in the type IV P-type ATPase family (P4-ATPases) are essential components of the Golgi, plasma membrane and endosomal system that play critical roles in membrane biogenesis. These pumps flip phospholipid across the bilayer to create an asymmetric membrane structure with substrate phospholipids, such as phosphatidylserine and phosphatidylethanolamine, enriched within the cytosolic leaflet. The P4-ATPases also help form transport vesicles that bud from Golgi and endosomal membranes, thereby impacting the sorting and localization of many different proteins in the secretory and endocytic pathways. At the organismal level, P4-ATPase deficiencies are linked to liver disease, obesity, diabetes, hearing loss, neurological deficits, immune deficiency and reduced fertility. Here, we review the biochemical, cellular and physiological functions of P4-ATPases, with an emphasis on their roles in vesicle-mediated protein transport. PMID:22234261

  7. Membrane Elasticity in Giant Vesicles with Fluid Phase Coexistence

    PubMed Central

    Baumgart, T.; Das, S.; Webb, W. W.; Jenkins, J. T.

    2005-01-01

    Biological membranes are known to contain compositional heterogeneities, often termed rafts, with distinguishable composition and function, and these heterogeneities participate in vigorous transport processes. Membrane lipid phase coexistence is expected to modulate these processes through the differing mechanical properties of the bulk domains and line tension at phase boundaries. In this contribution, we compare the predictions from a shape theory derived for vesicles with fluid phase coexistence to the geometry of giant unilamellar vesicles with coexisting liquid-disordered (Ld) and liquid-ordered (Lo) phases. We find a bending modulus for the Lo phase higher than that of the Ld phase and a saddle-splay (Gauss) modulus difference with the Gauss modulus of the Lo phase being more negative than the Ld phase. The Gauss modulus critically influences membrane processes that change topology, such as vesicle fission or fusion, and could therefore be of significant biological relevance in heterogeneous membranes. Our observations of experimental vesicle geometries being modulated by Gaussian curvature moduli differences confirm the prediction by the theory of Juelicher and Lipowsky. PMID:15894634

  8. Tetraspan vesicle membrane proteins: synthesis, subcellular localization, and functional properties.

    PubMed

    Hübner, Kirsten; Windoffer, Reinhard; Hutter, Harald; Leube, Rudolf E

    2002-01-01

    Tetraspan vesicle membrane proteins (TVPs) are characterized by four transmembrane regions and cytoplasmically located end domains. They are ubiquitous and abundant components of vesicles in most, if not all, cells of multicellular organisms. TVP-containing vesicles shuttle between various membranous compartments and are localized in biosynthetic and endocytotic pathways. Based on gene organization and amino acid sequence similarities TVPs can be grouped into three distinct families that are referred to as physins, gyrins, and secretory carrier-associated membrane proteins (SCAMPs). In mammals synaptophysin, synaptoporin, pantophysin, and mitsugumin29 constitute the physins, synaptogyrin 1-4 the gyrins, and SCAMP1-5 the SCAMPs. Members of each family are cell-type-specifically synthesized resulting in unique patterns of TVP coexpression and subcellular colocalization. TVP orthologs have been identified in most multicellular organisms, including diverse animal and plant species, but have not been detected in unicellular organisms. They are subject to protein modification, most notably to phosphorylation, and are part of multimeric complexes. Experimental evidence is reviewed showing that TVPs contribute to vesicle trafficking and membrane morphogenesis. PMID:11893164

  9. Sodium channels in membrane vesicles from cultured toad bladder cells

    SciTech Connect

    Asher, C.; Moran, A.; Rossier, B.C.; Garty, H. Ben Gurion Univ., Beer-Sheva Institut de Pharmacologie de l'Universite de Lausanne )

    1988-04-01

    Electrical potential-driven {sup 22}Na{sup +} fluxes were measured in membrane vesicles prepared from TBM-18(cl23) cells (a clone of the established cell line TB-M). Fifty to seventy percent of the tracer uptake in vesicles derived from cells that were cultivated on a porous support were blocked by the diuretic amiloride. The amiloride inhibition constant was <0.1 {mu}M, indicating that this flux is mediated by the apical Na{sup +}-specific channels. Vesicles prepared from cells that were not grown on a porous support exhibited much smaller amiloride-sensitive fluxes. Two Ca{sup 2+}-dependent processes that down-regulated the channel conductance and were previously identified in native epithelia were found in the cultured cells as well. Vesicles isolated from cells that were preincubated with 5 {times} 10{sup {minus}7} M aldosterone for 16-20 h exhibited higher amiloride-sensitive conductance than vesicles derived from control, steroid-depleted cells. Thus membrane derived from TBM-18(cl23) cells can be used to characterize the epithelial Na{sup +} channel and its hormonal regulation.

  10. Depletion of membrane skeleton in red blood cell vesicles.

    PubMed Central

    Iglic, A; Svetina, S; Zeks, B

    1995-01-01

    A possible physical interpretation of the partial detachment of the membrane skeleton in the budding region of the cell membrane and consequent depletion of the membrane skeleton in red blood cell vesicles is given. The red blood cell membrane is considered to consist of the bilayer part and the membrane skeleton. The skeleton is, under normal conditions, bound to the bilayer over its whole area. It is shown that, when in such conditions it is in the expanded state, some cell shape changes can induce its partial detachment. The partial detachment of the skeleton from the bilayer is energetically favorable if the consequent decrease of the skeleton expansion energy is larger than the corresponding increase of the bilayer-skeleton binding energy. The effect of shape on the skeleton detachment is analyzed theoretically for a series of the pear class shapes, having decreasing neck diameter and ending with a parent-daughter pair of spheres. The partial detachment of the skeleton is promoted by narrowing of the cell neck, by increasing the lateral tension in the skeleton and its area expansivity modulus, and by diminishing the attraction forces between the skeleton and the bilayer. If the radius of the daughter vesicle is sufficiently small relative to the radius of the parent cell, the daughter vesicle can exist either completely underlaid with the skeleton or completely depleted of the skeleton. PMID:7669905

  11. Redox-reactive membrane vesicles produced by Shewanella.

    PubMed

    Gorby, Y; McLean, J; Korenevsky, A; Rosso, K; El-Naggar, M Y; Beveridge, T J

    2008-06-01

    This manuscript is dedicated to our friend, mentor, and coauthor Dr Terry Beveridge, who devoted his scientific career to advancing fundamental aspects of microbial ultrastructure using innovative electron microscopic approaches. During his graduate studies with Professor Robert Murray, Terry provided some of the first glimpses and structural evaluations of the regular surface arrays (S-layers) of Gram-negative bacteria (Beveridge & Murray, 1974, 1975, 1976a). Beginning with his early electron microscopic assessments of metal binding by cell walls from Gram-positive bacteria (Beveridge & Murray, 1976b, 1980) and continuing with more than 30 years of pioneering research on microbe-mineral interactions (Hoyle & Beveridge, 1983, 1984; Ferris et al., 1986; Gorby et al., 1988; Beveridge, 1989; Mullen et al., 1989; Urrutia Mera et al., 1992; Mera & Beveridge, 1993; Brown et al., 1994; Konhauser et al., 1994; Beveridge et al., 1997; Newman et al., 1997; Lower et al., 2001; Glasauer et al., 2002; Baesman et al., 2007), Terry helped to shape the developing field of biogeochemistry. Terry and his associates are also widely regarded for their research defining the structure and function of outer membrane vesicles from Gram-negative bacteria that facilitate processes ranging from the delivery of pathogenic enzymes to the possible exchange of genetic information. The current report represents the confluence of two of Terry's thematic research streams by demonstrating that membrane vesicles produced by dissimilatory metal-reducing bacteria from the genus Shewanella catalyze the enzymatic transformation and precipitation of heavy metals and radionuclides. Under low-shear conditions, membrane vesicles are commonly tethered to intact cells by electrically conductive filaments known as bacterial nanowires. The functional role of membrane vesicles and associated nanowires is not known, but the potential for mineralized vesicles that morphologically resemble nanofossils to serve as

  12. Corneal endothelial cells possess an elaborate multipolar shape to maximize the basolateral to apical membrane area

    PubMed Central

    Harrison, Theresa A.; He, Zhiguo; Boggs, Kristin; Thuret, Gilles; Liu, Hong-Xiang

    2016-01-01

    Purpose The corneal endothelium is widely believed to consist of geometrically regular cells interconnected by junctional complexes. However, while en face visualization of the endothelial apical surface reveals characteristic polygonal borders, the overall form of the component cells has rarely been observed. Methods To visualize the shape of individual endothelial cells within the native monolayer, two independent Cre/LoxP-based cell labeling approaches were used. In the first, a P0-Cre mouse driver strain was bred to an R26-tdTomato reporter line to map neural crest–derived endothelial cells with cytosolic red fluorescent protein. In the second, HPRT-Cre induction of small numbers of green and red fluorescent protein–filled cells within a background of unlabeled cells was achieved using a dual-color reporter system, mosaic analysis with double markers (MADM). Selective imaging of the endothelial lateral membranes at different apicobasal levels was accomplished after staining with antibodies to ZO-1 and the neural cell adhesion molecule (NCAM). Results When viewed in their entirety in whole-mount preparations, fluorescent protein–filled cells appear star-shaped, extending multiple dendritic processes that radiate outward in the plane of the monolayer. Examination of rare cases where cells expressing different fluorescent proteins lie directly adjacent to one another reveals that these long processes undergo extensive interdigitation. The resulting overlap allows individual cells to extend over a greater area than if the cell boundaries were mutually exclusive. Anti-NCAM staining of these interlocking peripheral cell extensions reveals an elaborate system of lateral membrane folds that, when viewed in optical sections, increase in complexity from the apical to the basal pole. This not only produces a substantial increase in the basolateral, relative to the apical, membrane but also greatly extends the paracellular pathway as a highly convoluted space

  13. Bending elasticity of vesicle membranes studied by Monte Carlo simulations of vesicle thermal shape fluctuations.

    PubMed

    Penič, Samo; Iglič, Aleš; Bivas, Isak; Fošnarič, Miha

    2015-07-01

    The membrane bending stiffness of nearly spherical lipid vesicles can be deduced from the analysis of their thermal shape fluctuations. The theoretical basis of this analysis [Milner and Safran, Phys. Rev. A: At., Mol., Opt. Phys., 1987, 36, 4371-4379] uses the mean field approximation. In this work we apply Monte Carlo simulations and estimate the error in the determination of the bending stiffness due to the approximations applied in the theory. It is less than 10%. The method presented in this work can also be used to determine the changes of the bending stiffness of biological membranes due to their chemical and/or structural modifications.

  14. Synaptobrevin Transmembrane Domain Influences Exocytosis by Perturbing Vesicle Membrane Curvature

    PubMed Central

    Chang, Che-Wei; Jackson, Meyer B.

    2015-01-01

    Membrane fusion requires that nearly flat lipid bilayers deform into shapes with very high curvature. This makes membrane bending a critical force in determining fusion mechanisms. A lipid bilayer will bend spontaneously when material is distributed asymmetrically between its two monolayers, and its spontaneous curvature (C0) will influence the stability of curved fusion intermediates. Prior work on Ca2+-triggered exocytosis revealed that fusion pore lifetime (τ) varies with vesicle content (Q), and showed that this relation reflects membrane bending energetics. Lipids that alter C0 change the dependence of τ on Q. These results suggested that the greater stability of an initial exocytotic fusion pore associated with larger vesicles reflects the need to bend more membrane during fusion pore dilation. In this study, we explored the possibility of manipulating C0 by mutating the transmembrane domain (TMD) of the vesicle membrane protein synaptobrevin 2 (syb2). Amperometric measurements of exocytosis in mouse chromaffin cells revealed that syb2 TMD mutations altered the relation between τ and Q. The effects of these mutations showed a striking periodicity, changing sign as the structural perturbation moved through the inner and outer leaflets. Some glycine and charge mutations also influenced the dependence of τ on Q in a manner consistent with expected changes in C0. These results suggest that side chains in the syb2 TMD influence the kinetics of exocytosis by perturbing the packing of the surrounding lipids. The present results support the view that membrane bending occurs during fusion pore expansion rather than during fusion pore formation. This supports the view of an initial fusion pore through two relatively flat membranes formed by protein. PMID:26153704

  15. Taurine transport in renal brush-border-membrane vesicles.

    PubMed Central

    Rozen, R; Tenenhouse, H S; Scriver, C R

    1979-01-01

    Taurine transport in isolated brush-border-membrane vesicles from rat kidney is concentrative and it is driven by the Na+ gradient and transmembrane potential difference; binding is not a significant component of net uptake. The Na+-dependent component of net uptake is saturable with an apparent Km of 17 microM. The taurine-transport mechanism is selective for beta-amino compounds. PMID:486101

  16. Direct determination of the driving forces for taurocholate uptake into rat liver plasma membrane vesicles.

    PubMed Central

    Duffy, M C; Blitzer, B L; Boyer, J L

    1983-01-01

    To determine directly the driving forces for bile acid entry into the hepatocyte, the uptake of [3H]taurocholic acid into rat liver plasma membrane vesicles was studied. The membrane preparation contained predominantly right-side-out vesicles, and was highly enriched in plasma membrane marker enzymes. The uptake of taurocholate at equilibrium was inversely related to medium osmolarity, indicating transport into an osmotically sensitive space. In the presence of an inwardly directed sodium gradient (NaCl or sodium gluconate), the initial rate of uptake was rapid and taurocholate was transiently accumulated at a concentration twice that at equilibrium (overshoot). Other inwardly directed cation gradients (K+, Li+, choline+) or the presence of sodium in the absence of a gradient (Na+ equilibrated) resulted in a slower initial uptake rate and did not sustain an overshoot. Bile acids inhibited sodium-dependent taurocholate uptake, whereas bromsulphthalein inhibited both sodium-dependent and sodium-independent uptake and D-glucose had no effect on uptake. Uptake was temperature dependent, with maximal overshoots occurring at 25 degrees C. Imposition of a proton gradient across the vesicle (pHo less than pHi) in the absence of a sodium gradient failed to enhance taurocholate uptake, indicating that double ion exchange (Na+-H+, OH- -anion) is unlikely. Creation of a negative intravesicular potential by altering accompanying anions or by valinomycin-induced K+-diffusion potentials did not enhance taurocholate uptake, suggesting an electroneutral transport mechanism. The kinetics of taurocholate uptake demonstrated saturability with a Michaelis constant at 52 microM and maximum velocity of 4.5 nmol X mg-1 X protein X min-1. These studies provide definitive evidence for a sodium gradient-dependent, carrier-mediated, electrically neutral transport mechanism for hepatic taurocholate uptake. These findings are consistent with a model for bile secretion in which the basolateral

  17. Origin of life: LUCA and extracellular membrane vesicles (EMVs)

    NASA Astrophysics Data System (ADS)

    Gill, S.; Forterre, P.

    2016-01-01

    Cells from the three domains of life produce extracellular membrane vesicles (EMVs), suggesting that EMV production is an important aspect of cellular physiology. EMVs have been implicated in many aspects of cellular life in all domains, including stress response, toxicity against competing strains, pathogenicity, detoxification and resistance against viral attack. These EMVs represent an important mode of inter-cellular communication by serving as vehicles for transfer of DNA, RNA, proteins and lipids between cells. Here, we review recent progress in the understanding of EMV biology and their various roles. We focus on the role of membrane vesicles in early cellular evolution and how they would have helped shape the nature of the last universal common ancestor. A membrane-protected micro-environment would have been a key to the survival of spontaneous molecular systems and efficient metabolic reactions. Interestingly, the morphology of EMVs is strongly reminiscent of the morphology of some virions. It is thus tempting to make a link between the origin of the first protocell via the formation of vesicles and the origin of viruses.

  18. Numerical computations of the dynamics of fluidic membranes and vesicles.

    PubMed

    Barrett, John W; Garcke, Harald; Nürnberg, Robert

    2015-11-01

    Vesicles and many biological membranes are made of two monolayers of lipid molecules and form closed lipid bilayers. The dynamical behavior of vesicles is very complex and a variety of forms and shapes appear. Lipid bilayers can be considered as a surface fluid and hence the governing equations for the evolution include the surface (Navier-)Stokes equations, which in particular take the membrane viscosity into account. The evolution is driven by forces stemming from the curvature elasticity of the membrane. In addition, the surface fluid equations are coupled to bulk (Navier-)Stokes equations. We introduce a parametric finite-element method to solve this complex free boundary problem and present the first three-dimensional numerical computations based on the full (Navier-)Stokes system for several different scenarios. For example, the effects of the membrane viscosity, spontaneous curvature, and area difference elasticity (ADE) are studied. In particular, it turns out, that even in the case of no viscosity contrast between the bulk fluids, the tank treading to tumbling transition can be obtained by increasing the membrane viscosity. Besides the classical tank treading and tumbling motions, another mode (called the transition mode in this paper, but originally called the vacillating-breathing mode and subsequently also called trembling, transition, and swinging mode) separating these classical modes appears and is studied by us numerically. We also study how features of equilibrium shapes in the ADE and spontaneous curvature models, like budding behavior or starfish forms, behave in a shear flow. PMID:26651720

  19. Protective role of E. coli outer membrane vesicles against antibiotics.

    PubMed

    Kulkarni, Heramb M; Nagaraj, R; Jagannadham, Medicharla V

    2015-12-01

    The outer membrane vesicles (OMVs) from bacteria are known to posses both defensive and protective functions and thus participate in community related functions. In the present study, outer membrane vesicles have been shown to protect the producer bacterium and two other bacterial species from the growth inhibitory effects of some antibiotics. The OMVs isolated from E. coli MG1655 protected the bacteria against membrane-active antibiotics colistin, melittin. The OMVs of E. coli MG1655 could also protect P. aeruginosa NCTC6751 and A. radiodioresistens MMC5 against these membrane-active antibiotics. However, OMVs could not protect any of these bacteria against the other antibiotics ciprofloxacin, streptomycin and trimethoprim. Hence, OMVs appears to protect the bacterial community against membrane-active antibiotics and not other antibiotics, which have different mechanism of actions. The OMVs of E. coli MG1655 sequester the antibiotic colistin, whereas their protein components degrade the antimicrobial peptide melittin. Proteomic analysis of OMVs revealed the presence of proteases and peptidases which appear to be involved in this process. Thus, the protection of bacteria by OMVs against antibiotics is situation dependent and the mechanism differs for different situations. These studies suggest that OMVs of bacteria form a common defense for the bacterial community against specific antibiotics. PMID:26640046

  20. Protective role of E. coli outer membrane vesicles against antibiotics.

    PubMed

    Kulkarni, Heramb M; Nagaraj, R; Jagannadham, Medicharla V

    2015-12-01

    The outer membrane vesicles (OMVs) from bacteria are known to posses both defensive and protective functions and thus participate in community related functions. In the present study, outer membrane vesicles have been shown to protect the producer bacterium and two other bacterial species from the growth inhibitory effects of some antibiotics. The OMVs isolated from E. coli MG1655 protected the bacteria against membrane-active antibiotics colistin, melittin. The OMVs of E. coli MG1655 could also protect P. aeruginosa NCTC6751 and A. radiodioresistens MMC5 against these membrane-active antibiotics. However, OMVs could not protect any of these bacteria against the other antibiotics ciprofloxacin, streptomycin and trimethoprim. Hence, OMVs appears to protect the bacterial community against membrane-active antibiotics and not other antibiotics, which have different mechanism of actions. The OMVs of E. coli MG1655 sequester the antibiotic colistin, whereas their protein components degrade the antimicrobial peptide melittin. Proteomic analysis of OMVs revealed the presence of proteases and peptidases which appear to be involved in this process. Thus, the protection of bacteria by OMVs against antibiotics is situation dependent and the mechanism differs for different situations. These studies suggest that OMVs of bacteria form a common defense for the bacterial community against specific antibiotics.

  1. Numerical computations of the dynamics of fluidic membranes and vesicles

    NASA Astrophysics Data System (ADS)

    Barrett, John W.; Garcke, Harald; Nürnberg, Robert

    2015-11-01

    Vesicles and many biological membranes are made of two monolayers of lipid molecules and form closed lipid bilayers. The dynamical behavior of vesicles is very complex and a variety of forms and shapes appear. Lipid bilayers can be considered as a surface fluid and hence the governing equations for the evolution include the surface (Navier-)Stokes equations, which in particular take the membrane viscosity into account. The evolution is driven by forces stemming from the curvature elasticity of the membrane. In addition, the surface fluid equations are coupled to bulk (Navier-)Stokes equations. We introduce a parametric finite-element method to solve this complex free boundary problem and present the first three-dimensional numerical computations based on the full (Navier-)Stokes system for several different scenarios. For example, the effects of the membrane viscosity, spontaneous curvature, and area difference elasticity (ADE) are studied. In particular, it turns out, that even in the case of no viscosity contrast between the bulk fluids, the tank treading to tumbling transition can be obtained by increasing the membrane viscosity. Besides the classical tank treading and tumbling motions, another mode (called the transition mode in this paper, but originally called the vacillating-breathing mode and subsequently also called trembling, transition, and swinging mode) separating these classical modes appears and is studied by us numerically. We also study how features of equilibrium shapes in the ADE and spontaneous curvature models, like budding behavior or starfish forms, behave in a shear flow.

  2. Orientation of Membrane Vesicles from Escherichia coli as Detected by Freeze-Cleave Electron Microscopy

    PubMed Central

    Altendorf, K. H.; Staehelin, L. A.

    1974-01-01

    The application of freeze-cleave electron microscopy to whole cells of Escherichia coli revealed that the particles exposed on the resulting two inner membrane faces are asymmetrically distributed. This method can therefore be used to determine the orientation of membrane vesicles from E. coli. Membrane vesicles freshly prepared in potassium phosphate buffer (K+-vesicles) by osmotic lysis of spheroplasts consisted almost entirely of right-side-out vesicles. Their size suggested that each cell gives rise to one vesicle. When the membrane vesicles were subjected to one cycle of freezing and thawing, the number of inside-out vesicles rose to about 25%. However, due to the small size of most of the inside-out vesicles, these contribute only 2 to 3% of the total membrane surface area of the preparation. The inside-out vesicles appear to arise from infoldings of the membrane of right-side-out vesicles. They also accumulate within the latter, thus producing multivesicular membrane sacs. Na+-vesicles (vesicles prepared in sodium phosphate buffer) subjected to freezing and thawing appeared to lose structural rigidity more than did K+-vesicles. In contrast to the membrane vesicles prepared by the osmotic lysis of spheroplasts, those obtained by breaking intact cells by a single passage through a French pressure cell were uniformly very small (only 40 to 110 nm in diameter); approximately 60 to 80% were inside-out. To reconcile the polarity of the membrane vesicles with the enzymic activities of such preparations, we propose that “dislocation” of membrane proteins occurs during osmotic lysis of spheroplasts. Images PMID:4590489

  3. Methods of isolation and purification of outer membrane vesicles from gram-negative bacteria.

    PubMed

    Klimentová, Jana; Stulík, Jiří

    2015-01-01

    Outer membrane vesicles secreted by gram-negative bacteria play an important role in bacterial physiology as well as in virulence and host-pathogen interaction. Isolated vesicles of some bacteria have also been studied for their immunomodulatory potential in the vaccine development. However, the production of vesicles in sufficient amount, purity and reproducibility remains a critical challenge for subsequent analyses in most bacteria. In the present review methods of production, isolation, purification and quantification of outer membrane vesicles are summarized and discussed.

  4. Transport of gibberellin a(1) in cowpea membrane vesicles.

    PubMed

    O'neill, S D; Keith, B; Rappaport, L

    1986-04-01

    The permeability properties of gibberellin A(1) (GA(1)) were examined in membrane vesicles isolated from cowpea hypocotyls. The rate of GA(1) uptake was progressively greater as pH decreased, indicating that the neutral molecule is more permeable than anionic GA(1). Membrane vesicles used in this study possessed a tonoplast-type H(+)-translocating ATPase as assayed by MgATP-dependent quenching of acridine orange fluorescence and methylamine uptake. However, GA(1) uptake was not stimulated by MgATP. At concentrations in excess of 1 micromolar, GA(1), GA(5), and GA, collapsed both MgATP-generated and artifically imposed pH gradients, apparently by shuttling H(+) across the membrane as neutral GA. The relatively high permeability of neutral GA and the potentially detrimental effects of GA in uncoupling pH gradients across intracellular membranes supports the view that GA(1) accumulation and compartmentation must occur by conversion of GA(1) to more polar metabolites.

  5. Na sup + -glycine cotransport in canalicular liver plasma membrane vesicles

    SciTech Connect

    Moseley, R.H.; Ballatori, N.; Murphy, S.M. Yale Univ. School of Medicine, New Haven, CT Univ. of Rochester School of Medicine and Dentistry, NY )

    1988-08-01

    By use of purified rat canalicular liver plasma membrane (cLPM) vesicles, the present study determined the driving forces for glycine transport across this membrane domain. Initial rates of ({sup 3}H)glycine uptake in cLPM vesicles were stimulated by an inwardly directed Na{sup +} gradient but not by a K{sup +} gradient. Na{sup +} gradient-dependent uptake of glycine demonstrated cation specificity for Na{sup +}, dependence on extravesicular Cl{sup {minus}}, stimulation by an intravesicular-negative membrane potential, and inhibition by dissipation of the Na{sup +} gradient with gramicidin D. Na{sup +} gradient-dependent glycine cotransport also demonstrated greater sensitivity to inhibition by sarcosine than 2-(methylamino)-isobutyric acid. Accelerated exchange diffusion of ({sup 3}H)glycine was demonstrated in the presence of Na{sup +} when cLPM vesicles were preloaded with glycine but not with L-alanine or L-proline. Substrate velocity analysis of net Na{sup +}-dependent ({sup 3}H)glycine uptake over the range of amino acid concentrations from 5 {mu}M to 5 mM demonstrated two saturable transport systems, one of high capacity and low affinity and one of low capacity and comparatively high affinity. These results indicate that, in addition to previously described neutral and anionic amino acid transport systems, Na{sup +} gradient-dependent glycine transport mechanisms are present on the canalicular domain of the liver plasma membrane. These canalicular reabsorptive mechanisms may serve to reclaim some of the glycine generated within the canalicular lumen from the intrabiliary hydrolysis of glutathione.

  6. Involvement of a di-leucine motif in targeting of ABCC1 to the basolateral plasma membrane of polarized epithelial cells.

    PubMed

    Emi, Yoshikazu; Harada, Yasue; Sakaguchi, Masao

    2013-11-01

    Localization of ATP-binding cassette transporter isoform C1 (ABCC1) to the basolateral membrane of polarized cells is crucial for export of a variety of cellular metabolites; however, the mechanism regulating basolateral targeting of the transporter is poorly understood. Here we describe identification of a basolateral targeting signal in the first cytoplasmic loop domain (CLD1) of human ABCC1. Comparison of the CLD1 amino acid sequences from ABCC1 to ABCC2 revealed that ABCC1 possesses a characteristic sequence, E(295)EVEALI(301), which is comprised of a cluster of acidic glutamate residues followed by a di-leucine motif. This characteristic sequence is highly conserved among vertebrate ABCC1 orthologs and is positioned at a site that is structurally equivalent to the apical targeting signal previously described in ABCC2. Alanine scanning mutagenesis of this sequence in full-length human ABCC1 showed that both L(300) and I(301) residues were required for basolateral targeting of ABCC1 in polarized HepG2 and MDCK cells. Conversely, E(295), E(296), and E(298) residues were not required for basolateral localization of the transporter. Therefore, a di-leucine motif within the CLD1 is a basolateral targeting determinant of ABCC1.

  7. A trypsin-sensitive receptor on membrane vesicles is required for nuclear envelope formation in vitro

    PubMed Central

    1988-01-01

    The reformation of functioning organelles at the end of mitosis presents a problem in vesicle targeting. Using extracts made from Xenopus laevis frog eggs, we have studied in vitro the vesicles that reform the nuclear envelope. In the in vitro assay, nuclear envelope growth is linear with time. Furthermore, the final surface area of the nuclear envelopes formed is directly dependent upon the amount of membrane vesicles added to the assay. Egg membrane vesicles could be fractionated into two populations, only one of which was competent for nuclear envelope assembly. We found that vesicles active in nuclear envelope assembly contained markers (BiP and alpha-glucosidase II) characteristic of the endoplasmic reticulum (ER), but that the majority of ER-derived vesicles do not contribute to nuclear envelope size. This functional distinction between nuclear vesicles and ER-derived vesicles implies that nuclear vesicles are unique and possess at least one factor required for envelope assembly that is lacking in other vesicles. Consistent with this, treatment of vesicles with trypsin destroyed their ability to form a nuclear envelope; electron microscopic studies indicate that the trypsin-sensitive proteins is required for vesicles to bind to chromatin. However, the protease- sensitive component(s) is resistant to treatments that disrupt protein- protein interactions, such as high salt, EDTA, or low ionic strength solutions. We propose that an integral membrane protein, or protein tightly associated with the membrane, is critical for nuclear vesicle targeting or function. PMID:3392106

  8. Bacterial Outer Membrane Vesicles Induce Plant Immune Responses.

    PubMed

    Bahar, Ofir; Mordukhovich, Gideon; Luu, Dee Dee; Schwessinger, Benjamin; Daudi, Arsalan; Jehle, Anna Kristina; Felix, Georg; Ronald, Pamela C

    2016-05-01

    Gram-negative bacteria continuously pinch off portions of their outer membrane, releasing membrane vesicles. These outer membrane vesicles (OMVs) are involved in multiple processes including cell-to-cell communication, biofilm formation, stress tolerance, horizontal gene transfer, and virulence. OMVs are also known modulators of the mammalian immune response. Despite the well-documented role of OMVs in mammalian-bacterial communication, their interaction with plants is not well studied. To examine whether OMVs of plant pathogens modulate the plant immune response, we purified OMVs from four different plant pathogens and used them to treat Arabidopsis thaliana. OMVs rapidly induced a reactive oxygen species burst, medium alkalinization, and defense gene expression in A. thaliana leaf discs, cell cultures, and seedlings, respectively. Western blot analysis revealed that EF-Tu is present in OMVs and that it serves as an elicitor of the plant immune response in this form. Our results further show that the immune coreceptors BAK1 and SOBIR1 mediate OMV perception and response. Taken together, our results demonstrate that plants can detect and respond to OMV-associated molecules by activation of their immune system, revealing a new facet of plant-bacterial interactions. PMID:26926999

  9. Bacterial Outer Membrane Vesicles Induce Plant Immune Responses.

    PubMed

    Bahar, Ofir; Mordukhovich, Gideon; Luu, Dee Dee; Schwessinger, Benjamin; Daudi, Arsalan; Jehle, Anna Kristina; Felix, Georg; Ronald, Pamela C

    2016-05-01

    Gram-negative bacteria continuously pinch off portions of their outer membrane, releasing membrane vesicles. These outer membrane vesicles (OMVs) are involved in multiple processes including cell-to-cell communication, biofilm formation, stress tolerance, horizontal gene transfer, and virulence. OMVs are also known modulators of the mammalian immune response. Despite the well-documented role of OMVs in mammalian-bacterial communication, their interaction with plants is not well studied. To examine whether OMVs of plant pathogens modulate the plant immune response, we purified OMVs from four different plant pathogens and used them to treat Arabidopsis thaliana. OMVs rapidly induced a reactive oxygen species burst, medium alkalinization, and defense gene expression in A. thaliana leaf discs, cell cultures, and seedlings, respectively. Western blot analysis revealed that EF-Tu is present in OMVs and that it serves as an elicitor of the plant immune response in this form. Our results further show that the immune coreceptors BAK1 and SOBIR1 mediate OMV perception and response. Taken together, our results demonstrate that plants can detect and respond to OMV-associated molecules by activation of their immune system, revealing a new facet of plant-bacterial interactions.

  10. Comparative Analysis of Membrane Vesicles from Three Piscirickettsia salmonis Isolates Reveals Differences in Vesicle Characteristics

    PubMed Central

    Tandberg, Julia I.; Lagos, Leidy X.; Langlete, Petter; Berger, Eva; Rishovd, Anne-Lise; Roos, Norbert; Varkey, Deepa; Paulsen, Ian T.; Winther-Larsen, Hanne C.

    2016-01-01

    Membrane vesicles (MVs) are spherical particles naturally released from the membrane of Gram-negative bacteria. Bacterial MV production is associated with a range of phenotypes including biofilm formation, horizontal gene transfer, toxin delivery, modulation of host immune responses and virulence. This study reports comparative profiling of MVs from bacterial strains isolated from three widely disperse geographical areas. Mass spectrometry identified 119, 159 and 142 proteins in MVs from three different strains of Piscirickettsia salmonis isolated from salmonids in Chile (LF-89), Norway (NVI 5692) and Canada (NVI 5892), respectively. MV comparison revealed several strain-specific differences related to higher virulence capability for LF-89 MVs, both in vivo and in vitro, and stronger similarities between the NVI 5692 and NVI 5892 MV proteome. The MVs were similar in size and appearance as analyzed by electron microscopy and dynamic light scattering. The MVs from all three strains were internalized by both commercial and primary immune cell cultures, which suggest a potential role of the MVs in the bacterium’s utilization of leukocytes. When MVs were injected into an adult zebrafish infection model, an upregulation of several pro-inflammatory genes were observed in spleen and kidney, indicating a modulating effect on the immune system. The present study is the first comparative analysis of P. salmonis derived MVs, highlighting strain-specific vesicle characteristics. The results further illustrate that the MV proteome from one bacterial strain is not representative of all bacterial strains within one species. PMID:27764198

  11. SULFATE/BICARBONATE ANTIPORT BY LOBSTER HEPATOPANCREATIC BASOLATERAL MEMBRANE VESICLES. (R823068)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  12. SULFATE-OXALATE EXCHANGE IN LOBSTER HEPATOPANCRETIC BASOLATERAL MEMBRANE VESICLES. (R823068)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  13. Association of the fusion protein NSF with clathrin-coated vesicle membranes.

    PubMed Central

    Steel, G J; Tagaya, M; Woodman, P G

    1996-01-01

    N-ethylmaleimide-sensitive fusion protein (NSF) is a component of intracellular transport reactions. In order to understand the role of NSF during the fusion of endocytic transport vesicles with the endosome, we have investigated the binding of NSF to purified clathrin-coated vesicle components. First, we have examined whether detergent-solubilized coated vesicle membranes will support formation of NSF-containing 'fusion complexes'. Our results show that these membranes are substantially enriched in components capable of driving formation of these complexes, when compared with membranes from other sources. Secondly, we have analysed coated vesicle preparations for their NSF content. Coated vesicle preparations contain significant amounts of NSF. This was shown to be associated with coated vesicles rather than contaminating membranes by a number of criteria, and was found to be bound in an ATP-independent manner. These findings are discussed in the light of current models for vesicle fusion. Images PMID:8631296

  14. Characterization and Immunogenicity of Outer Membrane Vesicles from Brucella abortus.

    PubMed

    Kaur, Gagandeep; Singh, Satparkash; Sunil Kumar, B V; Mahajan, Kanika; Verma, Ramneek

    2016-01-01

    Bovine brucellosis is a worldwide spread zoonotic disease. The objectives of this study were characterization of outer membrane vesicles from B. abortus and to evaluate their immunogenicity in mice. For this purpose, OMVs were derived from B. abortus strain 99 using ultracentrifugation method. Isolated OMVs were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transmission electron microscopy which revealed spherical 20-300 nm structures rich in proteins. OMVs also showed immuno-reactivity with mice antisera in Western blot. Further, indirect ELISA showed specific and high-titer immune responses against the antigens present in OMVs suggesting their potential for a safe acellular vaccine candidate.

  15. Magnetic field alignable domains in phospholipid vesicle membranes containing lanthanides.

    PubMed

    Beck, Paul; Liebi, Marianne; Kohlbrecher, Joachim; Ishikawa, Takashi; Rüegger, Heinz; Zepik, Helmut; Fischer, Peter; Walde, Peter; Windhab, Erich

    2010-01-14

    Magnetic fields were applied as a structuring force on phospholipid-based vesicular systems, using paramagnetic lanthanide ions as magnetic handles anchored to the vesicle membrane. Different vesicle formulations were investigated using small angle neutron scattering (SANS) in a magnetic field of up to 8 T, cryo-transmission electron microscopy (cryo-TEM), (31)P NMR spectroscopy, dynamic light scattering (DLS), and permeability measurements with a fluorescent water-soluble marker (calcein). The investigated vesicle formulations consisted usually of 80 mol % of the phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 20 mol % of a chelator lipid (DMPE-DTPA; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-diethylenetriaminepentaacetate) with complexed lanthanide ions (Tm(3+), Dy(3+), or La(3+)), and the total lipid concentration was 15 mM. Vesicles containing the paramagnetic lanthanide Tm(3+) or Dy(3+) exhibited a temperature-dependent response to magnetic fields, which can be explained by considering the formation of lipid domains, which upon reaching a critical size become alignable in a magnetic field. The features of this "magnetic field alignable domain model" are as follows: with decreasing temperature (from 30 to 2.5 degrees C) solid domains, consisting mainly of the higher melting phospholipid (DMPE-DTPA.lanthanide), begin to form and grow in size. The domains assemble the large magnetic moments conferred by the lanthanides and orient in magnetic fields. The direction of alignment depends on the type of lanthanide used. The domains orient with their normal parallel to the magnetic field with thulium (Tm(3+)) and perpendicular with dysprosium (Dy(3+)). No magnetic field alignable domains were observed if DMPE-DTPA is replaced either by POPE-DTPA (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine-diethylenetriamine-pentaacetate) or by DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine).

  16. LPS Remodeling Triggers Formation of Outer Membrane Vesicles in Salmonella

    PubMed Central

    Elhenawy, Wael; Bording-Jorgensen, Michael; Valguarnera, Ezequiel; Haurat, M. Florencia; Wine, Eytan

    2016-01-01

    ABSTRACT Outer membrane vesicles (OMV) are proposed to mediate multiple functions during pathogenesis and symbiosis. However, the mechanisms responsible for OMV formation remain poorly understood. It has been shown in eukaryotic membranes that lipids with an inverted-cone shape favor the formation of positive membrane curvatures. Based on these studies, we formulated the hypothesis that lipid A deacylation might impose shape modifications that result in the curvature of the outer membrane (OM) and subsequent OMV formation. We tested the effect of lipid A remodeling on OMV biogenesis employing Salmonella enterica serovar Typhimurium as a model organism. Expression of the lipid A deacylase PagL resulted in increased vesiculation, without inducing an envelope stress response. Mass spectrometry analysis revealed profound differences in the patterns of lipid A in OM and OMV, with accumulation of deacylated lipid A forms exclusively in OMV. OMV biogenesis by intracellular bacteria upon macrophage infection was drastically reduced in a pagL mutant strain. We propose a novel mechanism for OMV biogenesis requiring lipid A deacylation in the context of a multifactorial process that involves the orchestrated remodeling of the outer membrane. PMID:27406567

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

    PubMed Central

    Prada, Ilaria; Meldolesi, Jacopo

    2016-01-01

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

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

    PubMed

    Prada, Ilaria; Meldolesi, Jacopo

    2016-01-01

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

  19. Focus on Extracellular Vesicles: Physiological Role and Signalling Properties of Extracellular Membrane Vesicles

    PubMed Central

    Iraci, Nunzio; Leonardi, Tommaso; Gessler, Florian; Vega, Beatriz; Pluchino, Stefano

    2016-01-01

    Extracellular vesicles (EVs) are a heterogeneous population of secreted membrane vesicles, with distinct biogenesis routes, biophysical properties and different functions both in physiological conditions and in disease. The release of EVs is a widespread biological process, which is conserved across species. In recent years, numerous studies have demonstrated that several bioactive molecules are trafficked with(in) EVs, such as microRNAs, mRNAs, proteins and lipids. The understanding of their final impact on the biology of specific target cells remains matter of intense debate in the field. Also, EVs have attracted great interest as potential novel cell-free therapeutics. Here we describe the proposed physiological and pathological functions of EVs, with a particular focus on their molecular content. Also, we discuss the advances in the knowledge of the mechanisms regulating the secretion of EV-associated molecules and the specific pathways activated upon interaction with the target cell, highlighting the role of EVs in the context of the immune system and as mediators of the intercellular signalling in the brain. PMID:26861302

  20. Hydrostatic pressures developed by osmotically swelling vesicles bound to planar membranes

    PubMed Central

    1989-01-01

    When phospholipid vesicles bound to a planar membrane are osmotically swollen, they develop a hydrostatic pressure (delta P) and fuse with the membrane. We have calculated the steady-state delta P, from the equations of irreversible thermodynamics governing water and solute flows, for two general methods of osmotic swelling. In the first method, vesicles are swollen by adding a solute to the vesicle- containing compartment to make it hyperosmotic. delta P is determined by the vesicle membrane's permeabilities to solute and water. If the vesicle membrane is devoid of open channels, then delta P is zero. When the vesicle membrane contains open channels, then delta P peaks at a channel density unique to the solute permeability properties of both the channel and the membrane. The solute enters the vesicle through the channels but leaks out through the region of vesicle-planar membrane contact. delta P is largest for channels having high permeabilities to the solute and for solutes with low membrane permeabilities in the contact region. The model predicts the following order of solutes producing pressures of decreasing magnitude: KCl greater than urea greater than formamide greater than or equal to ethylene glycol. Differences between osmoticants quantitatively depend on the solute permeability of the channel and the density of channels in the vesicle membrane. The order of effectiveness is the same as that experimentally observed for solutes promoting fusion. Therefore, delta P drives fusion. When channels with small permeabilities are used, coupling between solute and water flows within the channel has a significant effect on delta P. In the second method, an impermeant solute bathing the vesicles is isosmotically replaced by a solute which permeates the channels in the vesicle membrane. delta P resulting from this method is much less sensitive to the permeabilities of the channel and membrane to the solute. delta P approaches the theoretical limit set by the

  1. Cellular Asymmetric Catalysis by UDP-glucuronosyltransferase 1A8 Shows Functional Localization to the Basolateral Plasma Membrane*

    PubMed Central

    Ziegler, Kerstin; Tumova, Sarka; Kerimi, Asimina; Williamson, Gary

    2015-01-01

    UDP-glucuronosyltransferases (UGTs) are highly expressed in liver, intestine and kidney, and catalyze the glucuronic acid conjugation of both endogenous compounds and xenobiotics. Using recombinant human UGT isoforms, we show that glucuronic acid conjugation of the model substrate, (−)-epicatechin, is catalyzed mainly by UGT1A8 and UGT1A9. In HepG2 cells, pretreatment with polyunsaturated fatty acids increased substrate glucuronidation. In the intestinal Caco-2/HT29-MTX co-culture model, overall relative glucuronidation rates were much higher than in HepG2 cells, and (−)-epicatechin was much more readily conjugated when applied to the basolateral side of the cell monolayer. Under these conditions, 95% of the conjugated product was effluxed back to the site of application, and none of the other phase 2-derived metabolites followed this distribution pattern. HT29-MTX cells contained >1000-fold higher levels of UGT1A8 mRNA than Caco-2 or HepG2 cells. Gene expression of UGT1A8 increased after treatment of cells with docosahexaenoic acid, as did UGT1A protein levels. Immunofluorescence staining and Western blotting showed the presence of UGT1A in the basal and lateral parts of the plasma membrane of HT29-MTX cells. These results suggest that some of the UGT1A8 enzyme is not residing in the endoplasmic reticulum but spans the plasma membrane, resulting in increased accessibility to compounds outside the cell. This facilitates more efficient conjugation of substrate and is additionally coupled with rapid efflux by functionally associated basolateral transporters. This novel molecular strategy allows the cell to carry out conjugation without the xenobiotic entering into the interior of the cell. PMID:25586184

  2. DNA Inversion Regulates Outer Membrane Vesicle Production in Bacteroides fragilis

    PubMed Central

    Nakayama-Imaohji, Haruyuki; Hirota, Katsuhiko; Yamasaki, Hisashi; Yoneda, Saori; Nariya, Hirofumi; Suzuki, Motoo; Secher, Thomas; Miyake, Yoichiro; Oswald, Eric; Hayashi, Tetsuya; Kuwahara, Tomomi

    2016-01-01

    Phase changes in Bacteroides fragilis, a member of the human colonic microbiota, mediate variations in a vast array of cell surface molecules, such as capsular polysaccharides and outer membrane proteins through DNA inversion. The results of the present study show that outer membrane vesicle (OMV) formation in this anaerobe is also controlled by DNA inversions at two distantly localized promoters, IVp-I and IVp-II that are associated with extracellular polysaccharide biosynthesis and the expression of outer membrane proteins. These promoter inversions are mediated by a single tyrosine recombinase encoded by BF2766 (orthologous to tsr19 in strain NCTC9343) in B. fragilis YCH46, which is located near IVp-I. A series of BF2766 mutants were constructed in which the two promoters were locked in different configurations (IVp-I/IVp-II = ON/ON, OFF/OFF, ON/OFF or OFF/ON). ON/ON B. fragilis mutants exhibited hypervesiculating, whereas the other mutants formed only a trace amount of OMVs. The hypervesiculating ON/ON mutants showed higher resistance to treatment with bile, LL-37, and human β-defensin 2. Incubation of wild-type cells with 5% bile increased the population of cells with the ON/ON genotype. These results indicate that B. fragilis regulates the formation of OMVs through DNA inversions at two distantly related promoter regions in response to membrane stress, although the mechanism underlying the interplay between the two regions controlled by the invertible promoters remains unknown. PMID:26859882

  3. Specificity of siderophore receptors in membrane vesicles of Bacillus megaterium.

    PubMed Central

    Aswell, J E; Haydon, A H; Turner, H R; Dawkins, C A; Arceneaux, J E

    1977-01-01

    Membrane vesicles of Bacillus megaterium strains SK11 and Ard1 bound the ferrischizokinen and ferriferrioxamine B siderhores (iron transport cofactors). An approximately equimolar uptake of both labels of [3H, 59Fe]ferrischizokinen indicated binding of the intact chelate. Binding reached equilibrium in 2 to 5 min, was temperature independent, and was unaltered by the addition of several energy sources. A 91% dissociation of bound [Fe]ferrischizokinen was achieved in 60 s by the addition of excess ferrischizokinen. Ferriaerobactin, a siderophore which is structurally related to ferrischizokinen, caused no detectable release of bound [59Fe]ferrischizokinen. Of several other ferrigydroxamates tested, only ferriferrichrome A achieved the release (11%) of [Fe]ferrischizokinen. Rapid dissociation (92%) of bound [59Fe]ferriferrioxamine B by the addition of ferriferrioxamine B was observed, and a 67% release of [59Fe]ferriferrioxamine B was caused by ferriA2265, its structural relative. Ferrischizokinen, ferriferrichrome A, and ferrirhodotorulic acid produced a 6, 25, and 29% dissociation, respectively, of [59Fe]ferriferrioxamine B; ferriaerobactin caused no dissociation. [59Fe]ferriaerobactin was bound by the membranes, but its dissociation was not effected by unlabeled ferriaerobactin, suggesting no specific receptors for this chelate. The respective binding affinity constants and maximal binding capacities of membrane vesicles of strain SK11 were 2 x 10(7) M-1 and 280 pmol per mg of protein for ferrischizokinen and 7 x 10(7) M-1 and 37 pmol per mg of protein for ferriferrioxamine B. These values in strain Ard1 were, respectively, 1.4 x 10(7) M-1 and 186 pmol per mg of protein for ferrischizokinen and 11 x 10(7) M-1 and 23 pmol per mg of protein for ferriferrioxamine B. Separate, specific binding sites (receptors) for ferrischizokinen and ferriferrioxamine B exist on the vesicles. The ferrischizokinen receptors have a lower affinity but a higher binding capacity

  4. Cell-sized asymmetric lipid vesicles facilitate the investigation of asymmetric membranes

    NASA Astrophysics Data System (ADS)

    Kamiya, Koki; Kawano, Ryuji; Osaki, Toshihisa; Akiyoshi, Kazunari; Takeuchi, Shoji

    2016-09-01

    Asymmetric lipid giant vesicles have been used to model the biochemical reactions in cell membranes. However, methods for producing asymmetric giant vesicles lead to the inclusion of an organic solvent layer that affects the mechanical and physical characteristics of the membrane. Here we describe the formation of asymmetric giant vesicles that include little organic solvent, and use them to investigate the dynamic responses of lipid molecules in the vesicle membrane. We formed the giant vesicles via the inhomogeneous break-up of a lipid microtube generated by applying a jet flow to an asymmetric planar lipid bilayer. The asymmetric giant vesicles showed a lipid flip-flop behaviour in the membrane, superficially similar to the lipid flip-flop activity observed in apoptotic cells. In vitro synthesis of membrane proteins into the asymmetric giant vesicles revealed that the lipid asymmetry in bilayer membranes improves the reconstitution ratio of membrane proteins. Our asymmetric giant vesicles will be useful in elucidating lipid–lipid and lipid–membrane protein interactions involved in the regulation of cellular functions.

  5. Cell-sized asymmetric lipid vesicles facilitate the investigation of asymmetric membranes.

    PubMed

    Kamiya, Koki; Kawano, Ryuji; Osaki, Toshihisa; Akiyoshi, Kazunari; Takeuchi, Shoji

    2016-09-01

    Asymmetric lipid giant vesicles have been used to model the biochemical reactions in cell membranes. However, methods for producing asymmetric giant vesicles lead to the inclusion of an organic solvent layer that affects the mechanical and physical characteristics of the membrane. Here we describe the formation of asymmetric giant vesicles that include little organic solvent, and use them to investigate the dynamic responses of lipid molecules in the vesicle membrane. We formed the giant vesicles via the inhomogeneous break-up of a lipid microtube generated by applying a jet flow to an asymmetric planar lipid bilayer. The asymmetric giant vesicles showed a lipid flip-flop behaviour in the membrane, superficially similar to the lipid flip-flop activity observed in apoptotic cells. In vitro synthesis of membrane proteins into the asymmetric giant vesicles revealed that the lipid asymmetry in bilayer membranes improves the reconstitution ratio of membrane proteins. Our asymmetric giant vesicles will be useful in elucidating lipid-lipid and lipid-membrane protein interactions involved in the regulation of cellular functions. PMID:27554415

  6. Cell-sized asymmetric lipid vesicles facilitate the investigation of asymmetric membranes

    NASA Astrophysics Data System (ADS)

    Kamiya, Koki; Kawano, Ryuji; Osaki, Toshihisa; Akiyoshi, Kazunari; Takeuchi, Shoji

    2016-09-01

    Asymmetric lipid giant vesicles have been used to model the biochemical reactions in cell membranes. However, methods for producing asymmetric giant vesicles lead to the inclusion of an organic solvent layer that affects the mechanical and physical characteristics of the membrane. Here we describe the formation of asymmetric giant vesicles that include little organic solvent, and use them to investigate the dynamic responses of lipid molecules in the vesicle membrane. We formed the giant vesicles via the inhomogeneous break-up of a lipid microtube generated by applying a jet flow to an asymmetric planar lipid bilayer. The asymmetric giant vesicles showed a lipid flip-flop behaviour in the membrane, superficially similar to the lipid flip-flop activity observed in apoptotic cells. In vitro synthesis of membrane proteins into the asymmetric giant vesicles revealed that the lipid asymmetry in bilayer membranes improves the reconstitution ratio of membrane proteins. Our asymmetric giant vesicles will be useful in elucidating lipid-lipid and lipid-membrane protein interactions involved in the regulation of cellular functions.

  7. Silicone nanocapsules templated inside the membranes of catanionic vesicles.

    PubMed

    Kepczyński, Mariusz; Lewandowska, Joanna; Romek, Marek; Zapotoczny, Szczepan; Ganachaud, François; Nowakowska, Maria

    2007-06-19

    A simple and effective way to synthesize hollow silicone resin particles of controlled diameter is presented. The synthesis utilizes catanionic vesicles as templates for the polycondensation/polymerization processes of 1,3,5,7-tetramethylcyclotetrasiloxane (D4H) within their membranes. Two different surfactant systems were used to form the vesicular templates: mixtures of dodecyltrimethylammonium bromide (DTAB) and sodium dodecylbenzenesulfonate (SDBS) in the cationic (the DTAB/SDBS system) or anionic (the SDBS/DTAB system) rich region of the phase diagram. The templates obtained from these surfactant mixtures form spontaneously unilamellar vesicles in aqueous solution. The vesicular templates swell upon addition of D4H, thus increasing their size. The silicone resin was obtained in acid- or base-catalyzed polycondensation and ring-opening polymerization processes of D4H. In the case of the DTAB/SDBS system the formation of a densely cross-linked silicone material with SiO3/2 units allowed the nanocapsules to retain the vesicular shape after removal of the template, whereas in the SDBS/DTAB system, the polymer produces capsules which are too smooth to support surfactant lysis. The morphology of the silicone nanocapsules was analyzed using transmission electron microscopy (TEM) and, in some cases, atomic force microscopy (AFM). TEM and AFM reveal discrete hollow particles with a small amount of linked or aggregated hollow silica shells.

  8. Presence of membranous vesicles in cat seminal plasma: ultrastructural characteristics, protein profile and enzymatic activity.

    PubMed

    Polisca, A; Troisi, A; Minelli, A; Bellezza, I; Fontbonne, A; Zelli, R

    2015-02-01

    This study sought to verify the presence of membranous vesicles in cat seminal plasma by means of transmission electron microscopy and to identify protein profile and some of the enzymatic activities associated with these particles. The transmission electron microscopy observations showed the existence of different sized vesicular membranous structures of more or less spherical shape. These vesicles were surrounded by single-, double- or multiple-layered laminar membranes. The vesicle diameters ranged from 16.3 to 387.4 nm, with a mean of 116.5 ± 70.7 nm. Enzyme activity determinations showed the presence of dipeptilpeptidase IV, aminopeptidase, alkaline and acid phosphatase. To our knowledge, this is the first report that identifies and characterizes the membranous vesicles in cat seminal plasma. However, further studies are necessary to identify the exact site of production of these membranous vesicles in the cat male genital tract and to determine their specific roles in the reproductive events of this species.

  9. Gram-negative outer membrane vesicles: beyond the cell surface

    PubMed Central

    MASHBURN-WARREN, L.; MCLEAN, R. J. C.; WHITELEY, M.

    2011-01-01

    Considerable interest has recently mounted regarding the biological roles of Gram-negative outer membrane vesicles (MVs). The first discovery of MVs was made over four decades ago, and it is now clear that most Gram-negative bacteria produce MVs, with Pseudomonas aeruginosa and Escherichia coli as the most extensively studied. Much of our knowledge of the biological roles of MVs and mechanism of MV formation is due to T.J. Beveridge and colleagues. Beveridge pioneered the field of MV research not only by enhancing our understanding of MV function, but also through the application of a wide variety of physical, chemical, and genetic techniques to complement his elegant electron microscopy investigations. Here we review the contributions of Beveridge’s group to our understanding of MV biology. PMID:18459967

  10. Membrane vesicles: A simplified system for studying auxin transport

    SciTech Connect

    Goldsmith, M.H.M.

    1989-01-01

    Indoleacetic acid (IAA), the auxin responsible for regulation of growth, is transported polarly in plants. Several different models have been suggested to account for IAA transport by cells and its accumulation by membrane vesicles. One model sees diffusion of IAA driven by a pH gradient. The anion of a lipophilic weak acid like IAA or butyrate accumulates in an alkaline compartment in accord with the size of the pH gradient The accumulation of IAA may be diminished by the permeability of its lipophilic anion. This anion leak may be blocked by NPA. With anion efflux blocked, a gradient of two pH units would support an IAA accumulation of less than 50-fold at equilibrium (2) Another model sees diffusion of IAA in parallel with a saturable symport (IAA[sup [minus

  11. Temperate bacteriophages collected by outer membrane vesicles in Komagataeibacter intermedius.

    PubMed

    Kharina, Alla; Podolich, Olga; Faidiuk, Iuliia; Zaika, Sergiy; Haidak, Andriy; Kukharenko, Olga; Zaets, Iryna; Tovkach, Fedor; Reva, Oleg; Kremenskoy, Maxim; Kozyrovska, Natalia

    2015-04-01

    The acetic acid bacteria have mainly relevance for bacterial cellulose production and fermented bio-products manufacture. The purpose of this study was to identify temperate bacteriophages in a cellulose-producing bacterial strain Komagataeibacter intermedius IMBG180. Prophages from K. intermedius IMBG180 were induced with mitomycin C and nalidixic acid. Transmission electron microscopy analysis exhibited tailed bacteriophages belonging to Myoviridae. A PCR assay targeting the capsid gene of the myoviruses proved phylogenetic position of induced phages. Nalidixic acid was poor inducer of prophages, however, it induced the OMV-like particles release. Size of OMVs depended on an antibiotic applied for phage induction and varied in the range of 30-80 and 120-200 nm. Inside some of them, tails of phages have been visible. Under conditions, inducing prophages, OMVs acted as the collectors of formed phage particles, using outer membrane receptors for phage detection (in this case, outer membrane siderophore receptor), and fulfilled therefore "a cleaning," as well as defensive functions, preventing bacteriophage spread outside population. This is the first description of myoviruses affiliated to K. intermedius, as well as outer membrane vesicles interaction with phages within this host.

  12. Interaction of general anesthetics with phospholipid vesicles and biological membranes.

    PubMed

    Vanderkooi, J M; Landesberg, R; Selick, H; McDonald, G G

    1977-01-01

    Low concentrations of general anesthetics, including halothane, ethrane, trilene, diethyl ether and chloroform are observed to shift the phase transitions of phospholipid vesicles to lower temperatures, and from these data partition coefficients for the anesthetic between lipid and water can be calculated. In contrast to the anesthetics, high concentrations of ethanol are required to shift the phase transition of lipids and glycerol causes no effect. Above the phase transition general anesthetics alter nuclear magnetic resonance spectra of phospholipid dispersions and increase the rotational and lateral diffusion rates of fluorescent probes located in the hydrocarbon core of the bilayer, indicating that they induce disorder in the structure. In red blood cell membranes and sarcoplasmic reticulum fragments, the rotational diffusion rate of 1-phenyl-6-phenylhexatriene is increased in the presence of general anesthetics. The 220 MHz nuclear magnetic resonance spectra of sarcoplasmic reticulum reveal some resolved lines from the lecithin fatty acid protons; addition of general anesthetic increases the contribution of these peaks. The data from the NMR and fluorescence techniques lead to the conclusion that general anesthetics increase the pool size of melted lipids in the bimolecular phospholipid layers of biological membranes; this would account for the ability of general anesthetics to increase passive diffusion rates of various substances in membranes.

  13. 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

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

    PubMed

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

    2016-01-01

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

  15. Selective flow-induced vesicle rupture to sort by membrane mechanical properties

    PubMed Central

    Pommella, Angelo; Brooks, Nicholas J.; Seddon, John M.; Garbin, Valeria

    2015-01-01

    Vesicle and cell rupture caused by large viscous stresses in ultrasonication is central to biomedical and bioprocessing applications. The flow-induced opening of lipid membranes can be exploited to deliver drugs into cells, or to recover products from cells, provided that it can be obtained in a controlled fashion. Here we demonstrate that differences in lipid membrane and vesicle properties can enable selective flow-induced vesicle break-up. We obtained vesicle populations with different membrane properties by using different lipids (SOPC, DOPC, or POPC) and lipid:cholesterol mixtures (SOPC:chol and DOPC:chol). We subjected vesicles to large deformations in the acoustic microstreaming flow generated by ultrasound-driven microbubbles. By simultaneously deforming vesicles with different properties in the same flow, we determined the conditions in which rupture is selective with respect to the membrane stretching elasticity. We also investigated the effect of vesicle radius and excess area on the threshold for rupture, and identified conditions for robust selectivity based solely on the mechanical properties of the membrane. Our work should enable new sorting mechanisms based on the difference in membrane composition and mechanical properties between different vesicles, capsules, or cells. PMID:26302783

  16. Brownian Dynamics of Electrostatically Adhering Small Vesicles to a Membrane Surface Induces Domains and Probes Viscosity.

    PubMed

    Tabaei, Seyed R; Gillissen, Jurriaan J J; Kim, Min Chul; Ho, James C S; Liedberg, Bo; Parikh, Atul N; Cho, Nam-Joon

    2016-05-31

    Using single-particle tracking, we investigate the interaction of small unilamellar vesicles (SUVs) that are electrostatically tethered to the freestanding membrane of a giant unilamellar vesicle (GUV). We find that the surface mobility of the GUV-riding SUVs is Brownian, insensitive to the bulk viscosity, vesicle size, and vesicle fluidity but strongly altered by the viscosity of the underlying membrane. Analyzing the diffusional behavior of SUVs within the Saffman-Delbrück model for the dynamics of membrane inclusions supports the notion that the mobility of the small vesicles is coupled to that of dynamically induced lipid clusters within the target GUV membrane. The reversible binding also offers a nonperturbative means for measuring the viscosity of biomembranes, which is an important parameter in cell physiology and function.

  17. Intracellular calcium store filling by an L-type calcium current in the basolateral amygdala at subthreshold membrane potentials.

    PubMed

    Power, John M; Sah, Pankaj

    2005-01-15

    The long-term changes that underlie learning and memory are activated by rises in intracellular Ca2+ that activate a number of signalling pathways and trigger changes in gene transcription. Ca2+ rises due to influx via L-type voltage-dependent Ca2+ channels (L-VDCCs) and release from intracellular Ca2+ stores have been consistently implicated in the biochemical cascades that underlie the final changes in memory formation. Here, we show that pyramidal neurones in the basolateral amygdala express an L-VDCC that is active at resting membrane potentials. Subthreshold depolarization of neurones either by current injection or summating synaptic potentials led to a sustained rise in cytosolic Ca2+ that was blocked by the dihydropyridine nicardipine. Activation of metabotropic receptors released Ca2+ from intracellular Ca2+ stores. At hyperpolarized potentials, metabotropic-evoked store release ran down with repeated stimulation. Depolarization of cells to -50 mV, or maintaining them at the resting membrane potential, restored release from intracellular Ca2+ stores, an effect that was blocked by nicardipine. These results show that Ca2+ influx via a low-voltage-activated L-type Ca2+ current refills inositol 1,4,5-trisphosphate (IP(3))-sensitive intracellular Ca2+ stores, and maintains Ca2+ release and wave generation by metabotropic receptor activation.

  18. Intracellular calcium store filling by an L-type calcium current in the basolateral amygdala at subthreshold membrane potentials

    PubMed Central

    Power, John M; Sah, Pankaj

    2005-01-01

    The long-term changes that underlie learning and memory are activated by rises in intracellular Ca2+ that activate a number of signalling pathways and trigger changes in gene transcription. Ca2+ rises due to influx via L-type voltage-dependent Ca2+ channels (L-VDCCs) and release from intracellular Ca2+ stores have been consistently implicated in the biochemical cascades that underlie the final changes in memory formation. Here, we show that pyramidal neurones in the basolateral amygdala express an L-VDCC that is active at resting membrane potentials. Subthreshold depolarization of neurones either by current injection or summating synaptic potentials led to a sustained rise in cytosolic Ca2+ that was blocked by the dihydropyridine nicardipine. Activation of metabotropic receptors released Ca2+ from intracellular Ca2+ stores. At hyperpolarized potentials, metabotropic-evoked store release ran down with repeated stimulation. Depolarization of cells to −50 mV, or maintaining them at the resting membrane potential, restored release from intracellular Ca2+ stores, an effect that was blocked by nicardipine. These results show that Ca2+ influx via a low-voltage-activated L-type Ca2+ current refills inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular Ca2+ stores, and maintains Ca2+ release and wave generation by metabotropic receptor activation. PMID:15550460

  19. Fusion of Sendai virus with vesicles of oligomerizable lipids: a microcalorimetric analysis of membrane fusion.

    PubMed

    Ravoo, B J; Weringa, W D; Engberts, J B

    2000-01-01

    Sendai virus fuses efficiently with small and large unilamellar vesicles of the lipid 1,2-di-n-hexadecyloxypropyl-4- (beta-nitrostyryl) phosphate (DHPBNS) at pH 7.4 and 37 degrees C, as shown by lipid mixing assays and electron microscopy. However, fusion is strongly inhibited by oligomerization of the head groups of DHPBNS in the bilayer vesicles. The enthalpy associated with fusion of Sendai virus with DHPBNS vesicles was measured by isothermal titration microcalorimetry, comparing titrations of Sendai virus into (i) solutions of DHPBNS vesicles (which fuse with the virus) and (ii) oligomerized DHPBNS vesicles (which do not fuse with the virus), respectively. The observed heat effect of fusion of Sendai virus with DHPBNS vesicles is strongly dependent on the buffer medium, reflecting a partial charge neutralization of the Sendai F and HN proteins upon insertion into the negatively-charged vesicle membrane. No buffer effect was observed for the titration of Sendai virus into oligomerized DHPBNS vesicles, indicating that inhibition of fusion is a result of inhibition of insertion of the fusion protein into the target membrane. Fusion of Sendai virus with DHPBNS vesicles is endothermic and entropy-driven. The positive enthalpy term is dominated by heat effects resulting from merging of the protein-rich viral envelope with the lipid vesicle bilayers rather than by the fusion of the viral with the vesicle bilayers per se.

  20. The lethal cargo of Myxococcus xanthus outer membrane vesicles.

    PubMed

    Berleman, James E; Allen, Simon; Danielewicz, Megan A; Remis, Jonathan P; Gorur, Amita; Cunha, Jack; Hadi, Masood Z; Zusman, David R; Northen, Trent R; Witkowska, H Ewa; Auer, Manfred

    2014-01-01

    Myxococcus xanthus is a bacterial micro-predator known for hunting other microbes in a wolf pack-like manner. Outer membrane vesicles (OMVs) are produced in large quantities by M. xanthus and have a highly organized structure in the extracellular milieu, sometimes occurring in chains that link neighboring cells within a biofilm. OMVs may be a vehicle for mediating wolf pack activity by delivering hydrolytic enzymes and antibiotics aimed at killing prey microbes. Here, both the protein and small molecule cargo of the OMV and membrane fractions of M. xanthus were characterized and compared. Our analysis indicates a number of proteins that are OMV-specific or OMV-enriched, including several with putative hydrolytic function. Secondary metabolite profiling of OMVs identifies 16 molecules, many associated with antibiotic activities. Several hydrolytic enzyme homologs were identified, including the protein encoded by MXAN_3564 (mepA), an M36 protease homolog. Genetic disruption of mepA leads to a significant reduction in extracellular protease activity suggesting MepA is part of the long-predicted (yet to date undetermined) extracellular protease suite of M. xanthus. PMID:25250022

  1. The lethal cargo of Myxococcus xanthus outer membrane vesicles

    PubMed Central

    Berleman, James E.; Allen, Simon; Danielewicz, Megan A.; Remis, Jonathan P.; Gorur, Amita; Cunha, Jack; Hadi, Masood Z.; Zusman, David R.; Northen, Trent R.; Witkowska, H. Ewa; Auer, Manfred

    2014-01-01

    Myxococcus xanthus is a bacterial micro-predator known for hunting other microbes in a wolf pack-like manner. Outer membrane vesicles (OMVs) are produced in large quantities by M. xanthus and have a highly organized structure in the extracellular milieu, sometimes occurring in chains that link neighboring cells within a biofilm. OMVs may be a vehicle for mediating wolf pack activity by delivering hydrolytic enzymes and antibiotics aimed at killing prey microbes. Here, both the protein and small molecule cargo of the OMV and membrane fractions of M. xanthus were characterized and compared. Our analysis indicates a number of proteins that are OMV-specific or OMV-enriched, including several with putative hydrolytic function. Secondary metabolite profiling of OMVs identifies 16 molecules, many associated with antibiotic activities. Several hydrolytic enzyme homologs were identified, including the protein encoded by MXAN_3564 (mepA), an M36 protease homolog. Genetic disruption of mepA leads to a significant reduction in extracellular protease activity suggesting MepA is part of the long-predicted (yet to date undetermined) extracellular protease suite of M. xanthus. PMID:25250022

  2. Outer membrane vesicles of Pasteurella multocida contain virulence factors

    PubMed Central

    Fernández-Rojas, Miguel A; Vaca, Sergio; Reyes-López, Magda; de la Garza, Mireya; Aguilar-Romero, Francisco; Zenteno, Edgar; Soriano-Vargas, Edgardo; Negrete-Abascal, Erasmo

    2014-01-01

    Pasteurella multocida (Pm) is a gram-negative bacterium able to infect different animal species, including human beings. This bacterium causes economic losses to the livestock industry because of its high morbidity and mortality in animals. In this work, we report the characterization of outer membrane vesicles (OMVs) released into the culture medium by different Pm serogroups. Purified OMVs in the range of 50–300 nm were observed by electron microscopy. Serum obtained from chickens infected with Pm recognized several proteins from Pm OMVs. Additionally, rabbit antiserum directed against a secreted protease from Actinobacillus pleuropneumoniae recognized a similar protein in the Pm OVMs, suggesting that OMVs from these bacterial species contain common immunogenic proteins. OmpA, a multifunctional protein, was identified in OMVs from different Pm serogroups, and its concentration was twofold higher in OMVs from Pm serogroups B and D than in OMVs from other serogroups. Three outer membrane proteins were also identified: OmpH, OmpW, and transferrin-binding protein. Three bands of 65, 110, and 250 kDa with proteolytic activity were detected in Pm OMVs of serogroups A and E. Additionally, β-lactamase activity was detected only in OMVs from Pm 12945 Ampr (serogroup A). Pm OMVs may be involved in different aspects of disease pathogenesis. PMID:25065983

  3. Size-dependent, stochastic nature of lipid exchange between nano-vesicles and model membranes

    NASA Astrophysics Data System (ADS)

    Tabaei, Seyed R.; Gillissen, Jurriaan J. J.; Vafaei, Setareh; Groves, Jay T.; Cho, Nam-Joon

    2016-07-01

    The interaction of nanoscale lipid vesicles with cell membranes is of fundamental importance for the design and development of vesicular drug delivery systems. Here, we introduce a novel approach to study vesicle-membrane interactions whereby we are able to probe the influence of nanoscale membrane properties on the dynamic adsorption, exchange, and detachment of vesicles. Using total internal reflection fluorescence (TIRF) microscopy, we monitor these processes in real-time upon the electrostatically tuned attachment of individual, sub-100 nm vesicles to a supported lipid bilayer. The observed exponential vesicle detachment rate depends strongly on the vesicle size, but not on the vesicle charge, which suggests that lipid exchange occurs during a single stochastic event, which is consistent with membrane stalk formation. The fluorescence microscopy assay developed in this work may enable measuring of the probability of stalk formation in a controlled manner, which is of fundamental importance in membrane biology, offering a new tool to understand nanoscale phenomena in the context of biological sciences.The interaction of nanoscale lipid vesicles with cell membranes is of fundamental importance for the design and development of vesicular drug delivery systems. Here, we introduce a novel approach to study vesicle-membrane interactions whereby we are able to probe the influence of nanoscale membrane properties on the dynamic adsorption, exchange, and detachment of vesicles. Using total internal reflection fluorescence (TIRF) microscopy, we monitor these processes in real-time upon the electrostatically tuned attachment of individual, sub-100 nm vesicles to a supported lipid bilayer. The observed exponential vesicle detachment rate depends strongly on the vesicle size, but not on the vesicle charge, which suggests that lipid exchange occurs during a single stochastic event, which is consistent with membrane stalk formation. The fluorescence microscopy assay developed

  4. Exploration of the shapes of double-walled vesicles with a confined inner membrane.

    PubMed

    Guo, Kunkun; Li, Jianfeng

    2011-07-20

    We investigate double-walled vesicles as a simple model system for multi-vesicular structures, where the inner membrane is confined within the outer membrane. Various shapes of double-walled vesicles in two dimensions are obtained by means of our recently-developed discrete space variation method, and the shapes of each layer are found to be interdependent. Confined within the outer membrane, an inner membrane with a larger surface area always shows a cristae shape. As previous simulations and theoretical analyses of a single-walled vesicle have been done before, the geometric properties of double-walled vesicles, including the mean square radius of gyration and volume within the vesicle membrane, are studied in detail as functions of the pressure and surface area. It is found that due to the inter-space restriction of each layer, double-walled vesicles exhibit different behaviors compared with the previously-observed scaling laws of single-walled vesicles. It is straightforward to extend this study to more complicated and realistic biological systems, such as those including electrostatic interactions between membranes and solvent, phase separation, and cooperative interactions between multicomponent membranes.

  5. Vesicle self-reproduction: The involvement of membrane hydraulic and solute permeabilities

    NASA Astrophysics Data System (ADS)

    Božič, B.; Svetina, S.

    2007-09-01

    Conditions for self-reproduction are sought for a growing vesicle with its growth defined by an exponential increase of vesicle membrane area and by adequate flow of the solution across the membrane. In the first step of the presumed vesicle self-reproduction process, the initially spherical vesicle must double its volume in the doubling time of the membrane area and, through the appropriate shape transformations, attain the shape of two equal spheres connected by an infinitesimally thin neck. The second step involves separation of the two spheres and relies on conditions that cause the neck to be broken. In this paper we consider the first step of this self-reproduction process for a vesicle suspended in a solution whose solute can permeate the vesicle membrane. It is shown that vesicle self-reproduction occurs only for certain combinations of the values of membrane hydraulic and solute permeabilities and the external solute concentration, these quantities being related to the mechanical properties of the membrane and the membrane area doubling time. The analysis includes also the relaxation of a perturbed system towards stationary self-reproduction behavior and the case where the final shape consists of two connected spheres of different radii.

  6. Outer-Inner Membrane Vesicles Naturally Secreted by Gram-Negative Pathogenic Bacteria

    PubMed Central

    Pérez-Cruz, Carla; Delgado, Lidia; López-Iglesias, Carmen; Mercade, Elena

    2015-01-01

    Outer-inner membrane vesicles (O-IMVs) were recently described as a new type of membrane vesicle secreted by the Antarctic bacterium Shewanella vesiculosa M7T. Their formation is characterized by the protrusion of both outer and plasma membranes, which pulls cytoplasmic components into the vesicles. To demonstrate that this is not a singular phenomenon in a bacterium occurring in an extreme environment, the identification of O-IMVs in pathogenic bacteria was undertaken. With this aim, a structural study by Transmission Electron Microscopy (TEM) and Cryo-transmission electron microscopy (Cryo-TEM) was carried out, confirming that O-IMVs are also secreted by Gram-negative pathogenic bacteria such as Neisseria gonorrhoeae, Pseudomonas aeruginosa PAO1 and Acinetobacter baumannii AB41, in which they represent between 0.23% and 1.2% of total vesicles produced. DNA and ATP, which are components solely found in the cell cytoplasm, were identified within membrane vesicles of these strains. The presence of DNA inside the O-IMVs produced by N. gonorrhoeae was confirmed by gold DNA immunolabeling with a specific monoclonal IgM against double-stranded DNA. A proteomic analysis of N. gonorrhoeae-derived membrane vesicles identified proteins from the cytoplasm and plasma membrane. This confirmation of O-IMV extends the hitherto uniform definition of membrane vesicles in Gram-negative bacteria and explains the presence of components in membrane vesicles such as DNA, cytoplasmic and inner membrane proteins, as well as ATP, detected for the first time. The production of these O-IMVs by pathogenic Gram-negative bacteria opens up new areas of study related to their involvement in lateral gene transfer, the transfer of cytoplasmic proteins, as well as the functionality and role of ATP detected in these new vesicles. PMID:25581302

  7. Structure formation of lipid membranes: Membrane self-assembly and vesicle opening-up to octopus-like micelles

    NASA Astrophysics Data System (ADS)

    Noguchi, Hiroshi

    2013-02-01

    We briefly review our recent studies on self-assembly and vesicle rupture of lipid membranes using coarse-grained molecular simulations. For single component membranes, lipid molecules self-assemble from random gas states to vesicles via disk-shaped clusters. Clusters aggregate into larger clusters, and subsequently the large disks close into vesicles. The size of vesicles are determined by kinetics than by thermodynamics. When a vesicle composed of lipid and detergent types of molecules is ruptured, a disk-shaped micelle called bicelle can be formed. When both surfactants have negligibly low critical micelle concentration, it is found that bicelles connected with worm-like micelles are also formed depending on the surfactant ratio and spontaneous curvature of the membrane monolayer.

  8. Enzyme modification of platinum microelectrodes for detection of cholesterol in vesicle lipid bilayer membranes.

    PubMed

    Devadoss, Anando; Palencsár, M Simona; Jiang, Dechen; Honkonen, Michael L; Burgess, James D

    2005-11-15

    Platinum microelectrodes are modified with a lipid bilayer membrane incorporating cholesterol oxidase. Details for electrode surface modification are presented along with characterization studies of electrode response to cholesterol solution and to cholesterol contained in the lipid bilayer membrane of vesicles. Ferrocyanide voltammetric experiments are used to track deposition of a submonolayer of a thiol-functionalized lipid on the platinum electrode surface, vesicle fusion for bilayer formation on the thiolipid-modified surface, and incorporation of cholesterol oxidase in the electrode-supported thiolipid/lipid bilayer membrane. The data are consistent with formation of a lipid bilayer structure on the electrode surface that contains defects. Experiments for detection of cholesterol solubilized in cyclodextrin solution show steady-state current responses that correlate with cholesterol concentration. Direct contact between the electrode and a vesicle lipid bilayer membrane shows a response that correlates with vesicle membrane cholesterol content. PMID:16285691

  9. Geometric conservation laws for cells or vesicles with membrane nanotubes or singular points.

    PubMed

    Yin, Yajun; Yin, Jie

    2006-07-12

    On the basis of the integral theorems about the mean curvature and Gauss curvature, geometric conservation laws for cells or vesicles are proved. These conservation laws may depict various special bionano structures discovered in experiments, such as the membrane nanotubes and singular points grown from the surfaces of cells or vesicles. Potential applications of the conservation laws to lipid nanotube junctions that interconnect cells or vesicles are discussed.

  10. An AFM-based pit-measuring method for indirect measurements of cell-surface membrane vesicles

    SciTech Connect

    Zhang, Xiaojun; Chen, Yuan; Chen, Yong

    2014-03-28

    Highlights: • Air drying induced the transformation of cell-surface membrane vesicles into pits. • An AFM-based pit-measuring method was developed to measure cell-surface vesicles. • Our method detected at least two populations of cell-surface membrane vesicles. - Abstract: Circulating membrane vesicles, which are shed from many cell types, have multiple functions and have been correlated with many diseases. Although circulating membrane vesicles have been extensively characterized, the status of cell-surface membrane vesicles prior to their release is less understood due to the lack of effective measurement methods. Recently, as a powerful, micro- or nano-scale imaging tool, atomic force microscopy (AFM) has been applied in measuring circulating membrane vesicles. However, it seems very difficult for AFM to directly image/identify and measure cell-bound membrane vesicles due to the similarity of surface morphology between membrane vesicles and cell surfaces. Therefore, until now no AFM studies on cell-surface membrane vesicles have been reported. In this study, we found that air drying can induce the transformation of most cell-surface membrane vesicles into pits that are more readily detectable by AFM. Based on this, we developed an AFM-based pit-measuring method and, for the first time, used AFM to indirectly measure cell-surface membrane vesicles on cultured endothelial cells. Using this approach, we observed and quantitatively measured at least two populations of cell-surface membrane vesicles, a nanoscale population (<500 nm in diameter peaking at ∼250 nm) and a microscale population (from 500 nm to ∼2 μm peaking at ∼0.8 μm), whereas confocal microscopy only detected the microscale population. The AFM-based pit-measuring method is potentially useful for studying cell-surface membrane vesicles and for investigating the mechanisms of membrane vesicle formation/release.

  11. Membrane vesicle production by Chlamydia trachomatis as an adaptive response

    PubMed Central

    Frohlich, Kyla M.; Hua, Ziyu; Quayle, Alison J.; Wang, Jin; Lewis, Maria E.; Chou, Chau-wen; Luo, Miao; Buckner, Lyndsey R.; Shen, Li

    2014-01-01

    Bacteria have evolved specific adaptive responses to cope with changing environments. These adaptations include stress response phenotypes with dynamic modifications of the bacterial cell envelope and generation of membrane vesicles (MVs). The obligate intracellular bacterium, Chlamydia trachomatis, typically has a biphasic lifestyle, but can enter into an altered growth state typified by morphologically aberrant chlamydial forms, termed persistent growth forms, when induced by stress in vitro. How C. trachomatis can adapt to a persistent growth state in host epithelial cells in vivo is not well understood, but is an important question, since it extends the host-bacterial relationship in vitro and has thus been indicated as a survival mechanism in chronic chlamydial infections. Here, we review recent findings on the mechanistic aspects of bacterial adaptation to stress with a focus on how C. trachomatis remodels its envelope, produces MVs, and the potential important consequences of MV production with respect to host-pathogen interactions. Emerging data suggest that the generation of MVs may be an important mechanism for C. trachomatis intracellular survival of stress, and thus may aid in the establishment of a chronic infection in human genital epithelial cells. PMID:24959424

  12. Sulfate transport in apical membrane vesicles isolated from tracheal epithelium

    SciTech Connect

    Elgavish, A.; DiBona, D.R.; Norton, P.; Meezan, E.

    1987-09-01

    Sulfate uptake in apical membrane vesicles isolated from bovine tracheal epithelium is shown to occur into an osmotically sensitive intravesicular space, via a carrier-mediated system. This conclusion is based on three lines of evidence: 1) saturation kinetics: 2) substrate specificity; and 3) inhibition by the anion transport inhibitors SITS and DIDS. The affinity of the transport system is highest in low ionic strength media and decreases in the presence of gluconate. Chloride appears to cis-inhibit sulfate uptake and to trans-stimulate sulfate efflux. Cis-inhibition and trans-stimulation studies with a variety of anions indicate that this exchange system may be shared by HCO/sub 3//sup -/, S/sub 2/O/sub 3//sup 2 -/, SeO/sub 4//sup 2 -/, and MoO/sub 4//sup 2 -/ but not by H/sub 2/PO/sub 4//sup -/ or HAsO/sub 4//sup 2/. Studies indicate that protons may play two distinct roles in sulfate transport in this system. These studies show that the carrier-mediated system can function in the absence of chloride. The overshoot observed in the presence of a proton gradient indicates that under those conditions the mechanism of transport may be a SO/sub 4//sup 2 -/-OH/sup -/ exchange.

  13. Glycoengineered Outer Membrane Vesicles: A Novel Platform for Bacterial Vaccines

    PubMed Central

    Price, Nancy L.; Goyette-Desjardins, Guillaume; Nothaft, Harald; Valguarnera, Ezequiel; Szymanski, Christine M.; Segura, Mariela; Feldman, Mario F.

    2016-01-01

    The World Health Organization has indicated that we are entering into a post-antibiotic era in which infections that were routinely and successfully treated with antibiotics can now be lethal due to the global dissemination of multidrug resistant strains. Conjugate vaccines are an effective way to create a long-lasting immune response against bacteria. However, these vaccines present many drawbacks such as slow development, high price, and batch-to-batch inconsistencies. Alternate approaches for vaccine development are urgently needed. Here we present a new vaccine consisting of glycoengineered outer membrane vesicles (geOMVs). This platform exploits the fact that the initial steps in the biosynthesis of most bacterial glycans are similar. Therefore, it is possible to easily engineer non-pathogenic Escherichia coli lab strains to produce geOMVs displaying the glycan of the pathogen of interest. In this work we demonstrate the versatility of this platform by showing the efficacy of geOMVs as vaccines against Streptococcus pneumoniae in mice, and against Campylobacter jejuni in chicken. This cost-effective platform could be employed to generate vaccines to prevent infections caused by a wide variety of microbial agents in human and animals. PMID:27103188

  14. Outer membrane vesicles - offensive weapons or good Samaritans?

    PubMed

    Olsen, Ingar; Amano, Atsuo

    2015-01-01

    Outer membrane vesicles (OMVs) from Gram-negative bacteria were first considered as artifacts and were followed with disbelief and bad reputation. Later, their existence was accepted and they became characterized as bacterial bombs, virulence bullets, and even decoys. Today, we know that OMVs also can be involved in cell-cell signaling/communication and be mediators of immune regulation and cause disease protection. Furthermore, OMVs represent a distinct bacterial secretion pathway selecting and protecting their cargo, and they can even be good Samaritans providing nutrients to the gut microbiota maintaining commensal homeostasis beneficial to the host. The versatility in functions of these nanostructures is remarkable and includes both defense and offense. The broad spectrum of usability does not stop with that, as it now seems that OMVs can be used as vaccines and adjuvants or vehicles engineered for drug treatment of emerging and new diseases not only caused by bacteria but also by virus. They may even represent new ways of selective drug treatment.

  15. Vibrio fischeri-derived outer membrane vesicles trigger host development

    PubMed Central

    Aschtgen, Marie-Stephanie; Wetzel, Keith; Goldman, William; McFall-Ngai, Margaret; Ruby, Edward

    2016-01-01

    Outer membrane vesicles (OMV) are critical elements in many host-cell/microbe interactions. Previous studies of the symbiotic association between Euprymna scolopes and Vibrio fischeri had shown that, within 12 h of colonizing crypts deep within the squid’s light organ, the symbionts trigger an irreversible program of tissue development in the host. Here, we report that OMV produced by V. fischeri are powerful contributors to this process. The first detectable host response to the OMV is an increased trafficking of macrophage-like cells called hemocytes into surface epithelial tissues. We showed that exposing the squid to other Vibrio species fails to induce this trafficking; however, addition of a high concentration of their OMV, which can diffuse into the crypts, does. We also provide evidence that tracheal cytotoxin (TCT) release by the symbionts, which can induce hemocyte trafficking, is not part of the OMV cargo, suggesting two distinct mechanisms to induce the same morphogenesis event. By manipulating the timing and localization of OMV signal delivery, we showed that hemocyte trafficking is fully induced only when V. fischeri, the sole species able to reach and grow in the crypts, succeeds in establishing a sustained colonization. Further, our data suggest that the host detection of OMV serves as a symbiotic checkpoint prior to inducing irreversible morphogenesis. PMID:26399913

  16. Vibrio fischeri-derived outer membrane vesicles trigger host development.

    PubMed

    Aschtgen, Marie-Stephanie; Wetzel, Keith; Goldman, William; McFall-Ngai, Margaret; Ruby, Edward

    2016-04-01

    Outer membrane vesicles (OMV) are critical elements in many host-cell/microbe interactions. Previous studies of the symbiotic association between Euprymna scolopes and Vibrio fischeri had shown that within 12 h of colonizing crypts deep within the squid's light organ, the symbionts trigger an irreversible programme of tissue development in the host. Here, we report that OMV produced by V. fischeri are powerful contributors to this process. The first detectable host response to the OMV is an increased trafficking of macrophage-like cells called haemocytes into surface epithelial tissues. We showed that exposing the squid to other Vibrio species fails to induce this trafficking; however, addition of a high concentration of their OMV, which can diffuse into the crypts, does. We also provide evidence that tracheal cytotoxin released by the symbionts, which can induce haemocyte trafficking, is not part of the OMV cargo, suggesting two distinct mechanisms to induce the same morphogenesis event. By manipulating the timing and localization of OMV signal delivery, we showed that haemocyte trafficking is fully induced only when V. fischeri, the sole species able to reach and grow in the crypts, succeeds in establishing a sustained colonization. Further, our data suggest that the host's detection of OMV serves as a symbiotic checkpoint prior to inducing irreversible morphogenesis. PMID:26399913

  17. Extracellular Membrane Vesicles and Immune Regulation in the Brain

    PubMed Central

    Cossetti, Chiara; Smith, Jayden A.; Iraci, Nunzio; Leonardi, Tommaso; Alfaro-Cervello, Clara; Pluchino, Stefano

    2012-01-01

    The brain is characterized by a complex and integrated network of interacting cells in which cell-to-cell communication is critical for proper development and function. Initially considered as an immune privileged site, the brain is now regarded as an immune specialized system. Accumulating evidence reveals the presence of immune components in the brain, as well as extensive bidirectional communication that takes place between the nervous and the immune system both under homeostatic and pathological conditions. In recent years the secretion of extracellular membrane vesicles (EMVs) has been described as a new and evolutionary well-conserved mechanism of cell-to-cell communication, with EMVs influencing the microenvironment through the traffic of bioactive molecules that include proteins and nucleic acids, such as DNA, protein coding, and non-coding RNAs. Increasing evidence suggests that EMVs are a promising candidate to study cross-boundary cell-to-cell communication pathways. Herein we review the role of EMVs secreted by neural cells in modulating the immune response(s) within the brain under physiological and pathological circumstances. PMID:22557978

  18. Liquid General Anesthetics Lower Critical Temperatures in Plasma Membrane Vesicles

    PubMed Central

    Gray, Ellyn; Karslake, Joshua; Machta, Benjamin B.; Veatch, Sarah L.

    2013-01-01

    A large and diverse array of small hydrophobic molecules induce general anesthesia. Their efficacy as anesthetics has been shown to correlate both with their affinity for a hydrophobic environment and with their potency in inhibiting certain ligand-gated ion channels. In this study we explore the effects that n-alcohols and other liquid anesthetics have on the two-dimensional miscibility critical point observed in cell-derived giant plasma membrane vesicles (GPMVs). We show that anesthetics depress the critical temperature (Tc) of these GPMVs without strongly altering the ratio of the two liquid phases found below Tc. The magnitude of this affect is consistent across n-alcohols when their concentration is rescaled by the median anesthetic concentration (AC50) for tadpole anesthesia, but not when plotted against the overall concentration in solution. At AC50 we see a 4°C downward shift in Tc, much larger than is typically seen in the main chain transition at these anesthetic concentrations. GPMV miscibility critical temperatures are also lowered to a similar extent by propofol, phenylethanol, and isopropanol when added at anesthetic concentrations, but not by tetradecanol or 2,6 diterbutylphenol, two structural analogs of general anesthetics that are hydrophobic but have no anesthetic potency. We propose that liquid general anesthetics provide an experimental tool for lowering critical temperatures in plasma membranes of intact cells, which we predict will reduce lipid-mediated heterogeneity in a way that is complimentary to increasing or decreasing cholesterol. Also, several possible implications of our results are discussed in the context of current models of anesthetic action on ligand-gated ion channels. PMID:24359747

  19. DNA-tethered Membranes Formed by Giant Vesicle Rupture

    PubMed Central

    Chung, Minsub; Lowe, Randall; Chan, Yee-Hung M.; Ganesan, Prasad V.; Boxer, Steven G.

    2009-01-01

    We have developed a strategy for preparing tethered lipid bilayer membrane patches on solid surfaces by DNA hybridization. In this way, the tethered membrane patch is held at a controllable distance from the surface by varying the length of the DNA used. Two basic strategies are described. In the first, single-stranded DNA strands are immobilized by click chemistry to a silica surface, whose remaining surface is passivated to prevent direct assembly of a solid supported bilayer. Then giant unilamellar vesicles (GUVs) displaying the antisense strand, using a DNA-lipid conjugate developed in earlier work (Chan, Lengerich et al. 2008), are allowed to tether, spread and rupture to form tethered bilayer patches. In the second, a supported lipid bilayer displaying DNA using the DNA-lipid conjugate is first assembled on the surface. Then GUVs displaying the antisense strand are allowed to tether, spread and rupture to form tethered bilayer patches. The essential difference between these methods is that the tethering hybrid DNA is immobile in the first, while it is mobile in the second. Both strategies are successful; however, with mobile DNA hybrids as tethers, the patches are unstable, while in the first strategy stable patches can be formed. In the case of mobile tethers, if different length DNA hybrids are present, lateral segregation by length occurs and can be visualized by fluorescence interference contrast microscopy making this an interesting model for interactions that occur in cell junctions. In both cases, lipid mobility is high and there is a negligible immobile fraction. Thus, these architectures offer a flexible platform for the assembly of lipid bilayers at a well-defined distance from a solid support. PMID:19560541

  20. Size-dependent, stochastic nature of lipid exchange between nano-vesicles and model membranes.

    PubMed

    Tabaei, Seyed R; Gillissen, Jurriaan J J; Vafaei, Setareh; Groves, Jay T; Cho, Nam-Joon

    2016-07-21

    The interaction of nanoscale lipid vesicles with cell membranes is of fundamental importance for the design and development of vesicular drug delivery systems. Here, we introduce a novel approach to study vesicle-membrane interactions whereby we are able to probe the influence of nanoscale membrane properties on the dynamic adsorption, exchange, and detachment of vesicles. Using total internal reflection fluorescence (TIRF) microscopy, we monitor these processes in real-time upon the electrostatically tuned attachment of individual, sub-100 nm vesicles to a supported lipid bilayer. The observed exponential vesicle detachment rate depends strongly on the vesicle size, but not on the vesicle charge, which suggests that lipid exchange occurs during a single stochastic event, which is consistent with membrane stalk formation. The fluorescence microscopy assay developed in this work may enable measuring of the probability of stalk formation in a controlled manner, which is of fundamental importance in membrane biology, offering a new tool to understand nanoscale phenomena in the context of biological sciences. PMID:27355613

  1. Inward multivesiculation at the basal membrane of adherent giant phospholipid vesicles.

    PubMed

    Moreno-Flores, Susana

    2016-04-01

    Adherent giant vesicles composed of phosphatidylcholine, phosphatidylserine and biotinylated lipids form clusters of inward spherical buds at their basal membrane. The process is spontaneous and occurs when the vesicles undergo a sequence of osmotic swelling and deswelling. The daughter vesicles have a uniform size (diameter ≈ 2-3 μm), engulf small volumes of outer fluid and remain attached to the region of the membrane from which they generate, even after restoring the isotonicity. A pinning-sealing mechanism of long-wavelength modes of membrane fluctuations is proposed, by which the just-deflated vesicles reduce the surplus of membrane area and avoid excessive spreading and compression via biotin anchors. The work discusses the rationale behind the mechanism that furnishes GUVs with basal endovesicles, and its prospective use to simulate cellular events or to create molecular carriers. PMID:26828120

  2. ATP-driven calcium transport in membrane vesicles of Streptococcus sanguis. [Streptococcus sanguis; Streptococcus faecalis; Escherichia coli

    SciTech Connect

    Houng, H.; Lynn, A.R.; Rosen, B.P.

    1986-11-01

    Calcium transport was investigated in membrane vesicles prepared from the oral bacterium Streptococcus sanguis. Procedures were devised for the preparation of membrane vesicles capable of accumulation /sup 45/Ca/sup 2 +/. Uptake was ATP dependent and did not require a proton motive force. Calcium transport in these vesicles was compared with /sup 45/Ca/sup 2 +/ accumulation in membrane vesicles from Streptococcus faecalis and Escherichia coli. The data support the existence of an ATP-driven calcium pump in S. sanguis similar to that in S. faecalis. This pump, which catalyzes uptake into membrane vesicles, would be responsible for extrusion of calcium from intact cells.

  3. Shape bistability of a membrane neck: A toggle switch to control vesicle content release

    PubMed Central

    Frolov, Vadim A.; Lizunov, Vladimir A.; Dunina-Barkovskaya, Antonina Ya.; Samsonov, Andrey V.; Zimmerberg, Joshua

    2003-01-01

    Shape dynamics and permeability of a membrane neck connecting a vesicle and plasma membrane are considered. The neck is modeled by a lipid membrane tubule extended between two parallel axisymmetric rings. Within a range of lengths, defined by system geometry and mechanical properties of the membrane, the tubule has two stable shapes: catenoidal microtubule and cylindrical nanotubule. The permeabilities of these two shapes, measured as ionic conductivity of the tubule interior, differ by up to four orders of magnitude. Near the critical length the transitions between the shapes occur within less than a millisecond. Theoretical estimates show that the shape switching is controlled by a single parameter, the tubule length. Thus the tubule connection can operate as a conductivity microswitch, toggling the release of vesicle content in such cellular processes as “kiss-and-run” exocytosis. In support of this notion, bistable behavior of membrane connections between vesicles and the cell plasma membrane in macrophages is demonstrated. PMID:12857952

  4. Outer membrane vesicles of Lysobacter sp. XL1: biogenesis, functions, and applied prospects.

    PubMed

    Kudryakova, Irina V; Shishkova, Nina A; Vasilyeva, Natalia V

    2016-06-01

    Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have been intensively investigated in recent times. Vesicle formation models have been proposed, some factors affecting the process were established, and important roles vesicles play in vital activities of their producing cells were determined. Studies of pathogenic bacterial vesicles contribute to understanding the causes of acute infection and developing drugs on their basis. Despite intensive research, issues associated with the understanding of vesicle biogenesis, the mechanisms of bacterium-bacterium and pathogen-host interactions with participation of vesicles, still remain unresolved. This review discusses some results obtained in the research into OMVs of Lysobacter sp. XL1 VKM B-1576. This bacterium secretes into the environment a spectrum of bacteriolytic enzymes that hydrolyze peptidoglycan of competing bacteria, thus leading to their lysis. One of these enzymes, lytic endopeptidase L5, has been shown not only to be secreted by means of vesicles but also to be involved in their formation. As part of vesicles, the antimicrobial potential of L5 enzyme has been found to be considerably expanded. Vesicles have been shown to have a therapeutic effect in respect of anthrax infection and staphylococcal sepsis modelled in mice. The scientific basis for constructing liposomal antimicrobial preparations from vesicle phospholipids and recombinant bacteriolytic enzyme L5 has been formed. PMID:27098257

  5. Porin Loss Impacts the Host Inflammatory Response to Outer Membrane Vesicles of Klebsiella pneumoniae.

    PubMed

    Turner, Kelli L; Cahill, Bethaney K; Dilello, Sarah K; Gutel, Dedra; Brunson, Debra N; Albertí, Sebastián; Ellis, Terri N

    2016-03-01

    Antibiotic-resistant strains of Klebsiella pneumoniae often exhibit porin loss. In this study, we investigated how porin loss impacted the composition of secreted outer membrane vesicles as well as their ability to trigger proinflammatory cytokine secretion by macrophages. We hypothesize that porin loss associated with antibiotic resistance will directly impact both the composition of outer membrane vesicles and their interactions with phagocytic cells. Using clonally related clinical isolates of extended-spectrum beta-lactamase (ESBL)-positive Klebsiella pneumoniae with different patterns of porin expression, we demonstrated that altered expression of OmpK35 and OmpK36 results in broad alterations to the protein profile of secreted vesicles. Additionally, the level of OmpA incorporation was elevated in strains lacking a single porin. Porin loss significantly impacted macrophage inflammatory responses to purified vesicles. Outer membrane vesicles lacking both OmpK35 and OmpK36 elicited significantly lower levels of proinflammatory cytokine secretion than vesicles from strains expressing one or both porins. These data demonstrate that antibiotic resistance-associated porin loss has a broad and significant effect on both the composition of outer membrane vesicles and their interactions with phagocytic cells, which may impact bacterial survival and inflammatory reactions in the host.

  6. Porin Loss Impacts the Host Inflammatory Response to Outer Membrane Vesicles of Klebsiella pneumoniae

    PubMed Central

    Turner, Kelli L.; Cahill, Bethaney K.; Dilello, Sarah K.; Gutel, Dedra; Brunson, Debra N.; Albertí, Sebastián

    2015-01-01

    Antibiotic-resistant strains of Klebsiella pneumoniae often exhibit porin loss. In this study, we investigated how porin loss impacted the composition of secreted outer membrane vesicles as well as their ability to trigger proinflammatory cytokine secretion by macrophages. We hypothesize that porin loss associated with antibiotic resistance will directly impact both the composition of outer membrane vesicles and their interactions with phagocytic cells. Using clonally related clinical isolates of extended-spectrum beta-lactamase (ESBL)-positive Klebsiella pneumoniae with different patterns of porin expression, we demonstrated that altered expression of OmpK35 and OmpK36 results in broad alterations to the protein profile of secreted vesicles. Additionally, the level of OmpA incorporation was elevated in strains lacking a single porin. Porin loss significantly impacted macrophage inflammatory responses to purified vesicles. Outer membrane vesicles lacking both OmpK35 and OmpK36 elicited significantly lower levels of proinflammatory cytokine secretion than vesicles from strains expressing one or both porins. These data demonstrate that antibiotic resistance-associated porin loss has a broad and significant effect on both the composition of outer membrane vesicles and their interactions with phagocytic cells, which may impact bacterial survival and inflammatory reactions in the host. PMID:26666932

  7. Delivery of foreign antigens by engineered outer membrane vesicle vaccines.

    PubMed

    Chen, David J; Osterrieder, Nikolaus; Metzger, Stephan M; Buckles, Elizabeth; Doody, Anne M; DeLisa, Matthew P; Putnam, David

    2010-02-16

    As new disease threats arise and existing pathogens grow resistant to conventional interventions, attention increasingly focuses on the development of vaccines to induce protective immune responses. Given their admirable safety records, protein subunit vaccines are attractive for widespread immunization, but their disadvantages include poor immunogenicity and expensive manufacture. We show here that engineered Escherichia coli outer membrane vesicles (OMVs) are an easily purified vaccine-delivery system capable of greatly enhancing the immunogenicity of a low-immunogenicity protein antigen without added adjuvants. Using green-fluorescent protein (GFP) as the model subunit antigen, genetic fusion of GFP with the bacterial hemolysin ClyA resulted in a chimeric protein that elicited strong anti-GFP antibody titers in immunized mice, whereas immunization with GFP alone did not elicit such titers. Harnessing the specific secretion of ClyA to OMVs, the ClyA-GFP fusion was found localized in OMVs, resulting in engineered recombinant OMVs. The anti-GFP humoral response in mice immunized with the engineered OMV formulations was indistinguishable from the response to the purified ClyA-GFP fusion protein alone and equal to purified proteins absorbed to aluminum hydroxide, a standard adjuvant. In a major improvement over current practice, engineered OMVs containing ClyA-GFP were easily isolated by ultracentrifugation, effectively eliminating the need for laborious antigen purification from cell-culture expression systems. With the diverse collection of heterologous proteins that can be functionally localized with OMVs when fused with ClyA, this work signals the possibility of OMVs as a robust and tunable technology platform for a new generation of prophylactic and therapeutic vaccines. PMID:20133740

  8. A practical guide to giant vesicles. Probing the membrane nanoregime via optical microscopy

    NASA Astrophysics Data System (ADS)

    Dimova, Rumiana; Aranda, Said; Bezlyepkina, Natalya; Nikolov, Vesselin; Riske, Karin A.; Lipowsky, Reinhard

    2006-07-01

    Research on giant vesicles is becoming increasingly popular. Giant vesicles provide model biomembrane systems for systematic measurements of mechanical and rheological properties of bilayers as a function of membrane composition and temperature, as well as hydrodynamic interactions. Membrane response to external factors (for example electric fields, ions and amphiphilic molecules) can be directly visualized under the microscope. In this paper we review our current understanding of lipid bilayers as obtained from studies on giant unilamellar vesicles. Because research on giant vesicles increasingly attracts the interest of scientists from various backgrounds, we also try to provide a concise introduction for newcomers in the field. Finally, we summarize some recent developments on curvature effects induced by polymers, domain formation in membranes and shape transitions induced by electric fields.

  9. A practical guide to giant vesicles. Probing the membrane nanoregime via optical microscopy.

    PubMed

    Dimova, Rumiana; Aranda, Said; Bezlyepkina, Natalya; Nikolov, Vesselin; Riske, Karin A; Lipowsky, Reinhard

    2006-07-19

    Research on giant vesicles is becoming increasingly popular. Giant vesicles provide model biomembrane systems for systematic measurements of mechanical and rheological properties of bilayers as a function of membrane composition and temperature, as well as hydrodynamic interactions. Membrane response to external factors (for example electric fields, ions and amphiphilic molecules) can be directly visualized under the microscope. In this paper we review our current understanding of lipid bilayers as obtained from studies on giant unilamellar vesicles. Because research on giant vesicles increasingly attracts the interest of scientists from various backgrounds, we also try to provide a concise introduction for newcomers in the field. Finally, we summarize some recent developments on curvature effects induced by polymers, domain formation in membranes and shape transitions induced by electric fields.

  10. Bacillus anthracis produces membrane-derived vesicles containing biologically active toxins

    PubMed Central

    Rivera, Johanna; Cordero, Radames J. B.; Nakouzi, Antonio S.; Frases, Susana; Nicola, André; Casadevall, Arturo

    2010-01-01

    Extracellular vesicle production is a ubiquitous process in Gram-negative bacteria, but little is known about such process in Gram-positive bacteria. We report the isolation of extracellular vesicles from the supernatants of Bacillus anthracis, a Gram-positive bacillus that is a powerful agent for biological warfare. B. anthracis vesicles formed at the outer layer of the bacterial cell had double-membrane spheres and ranged from 50 to 150 nm in diameter. Immunoelectron microscopy with mAbs to protective antigen, lethal factor, edema toxin, and anthrolysin revealed toxin components and anthrolysin in vesicles, with some vesicles containing more than one toxin component. Toxin-containing vesicles were also visualized inside B. anthracis-infected macrophages. ELISA and immunoblot analysis of vesicle preparations confirmed the presence of B. anthracis toxin components. A mAb to protective antigen protected macrophages against vesicles from an anthrolysin-deficient strain, but not against vesicles from Sterne 34F2 and Sterne δT strains, consistent with the notion that vesicles delivered both toxin and anthrolysin to host cells. Vesicles were immunogenic in BALB/c mice, which produced a robust IgM response to toxin components. Furthermore, vesicle-immunized mice lived significantly longer than controls after B. anthracis challenge. Our results indicate that toxin secretion in B. anthracis is, at least, partially vesicle-associated, thus allowing concentrated delivery of toxin components to target host cells, a mechanism that may increase toxin potency. Our observations may have important implications for the design of vaccines, for passive antibody strategies, and provide a previously unexplored system for studying secretory pathways in Gram-positive bacteria. PMID:20956325

  11. Bacillus anthracis produces membrane-derived vesicles containing biologically active toxins.

    PubMed

    Rivera, Johanna; Cordero, Radames J B; Nakouzi, Antonio S; Frases, Susana; Nicola, André; Casadevall, Arturo

    2010-11-01

    Extracellular vesicle production is a ubiquitous process in Gram-negative bacteria, but little is known about such process in Gram-positive bacteria. We report the isolation of extracellular vesicles from the supernatants of Bacillus anthracis, a Gram-positive bacillus that is a powerful agent for biological warfare. B. anthracis vesicles formed at the outer layer of the bacterial cell had double-membrane spheres and ranged from 50 to 150 nm in diameter. Immunoelectron microscopy with mAbs to protective antigen, lethal factor, edema toxin, and anthrolysin revealed toxin components and anthrolysin in vesicles, with some vesicles containing more than one toxin component. Toxin-containing vesicles were also visualized inside B. anthracis-infected macrophages. ELISA and immunoblot analysis of vesicle preparations confirmed the presence of B. anthracis toxin components. A mAb to protective antigen protected macrophages against vesicles from an anthrolysin-deficient strain, but not against vesicles from Sterne 34F2 and Sterne δT strains, consistent with the notion that vesicles delivered both toxin and anthrolysin to host cells. Vesicles were immunogenic in BALB/c mice, which produced a robust IgM response to toxin components. Furthermore, vesicle-immunized mice lived significantly longer than controls after B. anthracis challenge. Our results indicate that toxin secretion in B. anthracis is, at least, partially vesicle-associated, thus allowing concentrated delivery of toxin components to target host cells, a mechanism that may increase toxin potency. Our observations may have important implications for the design of vaccines, for passive antibody strategies, and provide a previously unexplored system for studying secretory pathways in Gram-positive bacteria.

  12. Atg9 vesicles are an important membrane source during early steps of autophagosome formation

    PubMed Central

    Yamamoto, Hayashi; Kakuta, Soichiro; Watanabe, Tomonobu M.; Kitamura, Akira; Sekito, Takayuki; Kondo-Kakuta, Chika; Ichikawa, Rie; Kinjo, Masataka

    2012-01-01

    During the process of autophagy, cytoplasmic materials are sequestered by double-membrane structures, the autophagosomes, and then transported to a lytic compartment to be degraded. One of the most fundamental questions about autophagy involves the origin of the autophagosomal membranes. In this study, we focus on the intracellular dynamics of Atg9, a multispanning membrane protein essential for autophagosome formation in yeast. We found that the vast majority of Atg9 existed on cytoplasmic mobile vesicles (designated Atg9 vesicles) that were derived from the Golgi apparatus in a process involving Atg23 and Atg27. We also found that only a few Atg9 vesicles were required for a single round of autophagosome formation. During starvation, several Atg9 vesicles assembled individually into the preautophagosomal structure, and eventually, they are incorporated into the autophagosomal outer membrane. Our findings provide conclusive linkage between the cytoplasmic Atg9 vesicles and autophagosomal membranes and offer new insight into the requirement for Atg9 vesicles at the early step of autophagosome formation. PMID:22826123

  13. Characterization of Membrane Protein Interactions in Plasma Membrane Derived Vesicles with Quantitative Imaging FRET

    PubMed Central

    Sarabipour, Sarvenaz; Del Piccolo, Nuala; Hristova, Kalina

    2016-01-01

    CONSPECTUS Here we describe an experimental tool, termed Quantitative Imaging Förster Resonance Energy Transfer (QI-FRET), which enables the quantitative characterization of membrane protein interactions. The QI-FRET methodology allows us to acquire binding curves and calculate association constants for complex membrane proteins in the native plasma membrane environment. The method utilizes FRET detection, and thus requires that the proteins of interest are labeled with florescent proteins, either FRET donors or FRET acceptors. Since plasma membranes of cells have complex topologies precluding the acquisition of two-dimensional binding curves, the FRET measurements are performed in plasma membrane derived vesicles which bud off cells as a result of chemical or osmotic stress. The results overviewed here are acquired in vesicles produced with an osmotic vesiculation buffer developed in our laboratory, which does not utilize harsh chemicals. The concentrations of the donor-labeled and the acceptor-labeled proteins are determined, along with the FRET efficiencies, in each vesicle. The experiments utilize transient transfection, such that a wide variety of concentrations is sampled. Then, data from hundreds of vesicles are combined to yield dimerization curves. Here we discuss recent findings about the dimerization of receptor tyrosine kinases (RTKs), membrane proteins that control cell growth and differentiation via lateral dimerization in the plasma membrane. We focus on the dimerization of fibroblast growth factor receptor 3 (FGFR3), an RTK that plays a critically important role in skeletal development. We study the role of different FGFR3 domains in FGFR3 dimerization in the absence of ligand, and we show that FGFR3 extracellular domains inhibit unliganded dimerization, while contacts between the juxtamembrane domains, which connect the transmembrane domains to the kinase domains, stabilize the unliganded FGFR3 dimers. Since FGFR3 has been documented to harbor

  14. Trypsin digestion for determining orientation of ATPase in Halobacterium saccharovorum membrane vesicles

    NASA Technical Reports Server (NTRS)

    Kristjansson, H.; Hochstein, L. I.

    1986-01-01

    Membranes prepared by low pressure disruption of cells exhibited no ATPase activity in the absence of Triton X-100, although 43% of the total menadione reductase activity was detected. Trypsin digestion reduced menadione reductase activity by 45% whereas ATPase activity was not affected. Disruption of the membrane fraction at higher pressure solubilized about 45% of the ATPase activity. The soluble activity was still enhanced by Triton X-100, suggesting that the detergent, besides disrupting membrane vesicles, also activated the ATPase. The discrepancy in localization of menadione reductase and ATPase activities raised questions regarding the reliability of using a single marker enzyme as an indicator of vesicle orientation.

  15. Formation of polyhedral vesicles and polygonal membrane tubes induced by banana-shaped proteins

    NASA Astrophysics Data System (ADS)

    Noguchi, Hiroshi

    2015-12-01

    The shape transformations of fluid membranes induced by curved protein rods are studied using meshless membrane simulations. The rod assembly at low rod density induces a flat membrane tube and oblate vesicle. It is found that the polyhedral shapes are stabilized at high rod densities. The discrete shape transition between triangular and buckled discoidal tubes is obtained and their curvature energies are analyzed by a simple geometric model. For vesicles, triangular hosohedron and elliptic-disk shapes are formed in equilibrium, whereas tetrahedral and triangular prism shapes are obtained as metastable states.

  16. Towards fully automated Identification of Vesicle-Membrane Fusion Events in TIRF Microscopy

    NASA Astrophysics Data System (ADS)

    Vallotton, Pascal; James, David E.; Hughes, William E.

    2007-11-01

    Total Internal Reflection Fluorescence Microscopy (TIRFM) is imposing itself as the tool of choice for studying biological activity in close proximity to the plasma membrane. For example, the exquisite selectivity of TIRFM allows monitoring the diffusion of GFP-phogrin vesicles and their recruitment to the plasma membrane in pancreatic β-cells. We present a novel computer vision system for automatically identifying the elusive fusion events of GFP-phogrin vesicles with the plasma membrane. Our method is based on robust object tracking and matched filtering. It should accelerate the quantification of TIRFM data and allow the extraction of more biological information from image data to support research in diabetes and obesity.

  17. 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.

  18. Bacterial Membrane Vesicles Mediate the Release of Mycobacterium tuberculosis Lipoglycans and Lipoproteins from Infected Macrophages.

    PubMed

    Athman, Jaffre J; Wang, Ying; McDonald, David J; Boom, W Henry; Harding, Clifford V; Wearsch, Pamela A

    2015-08-01

    Mycobacterium tuberculosis is an intracellular pathogen that infects lung macrophages and releases microbial factors that regulate host defense. M. tuberculosis lipoproteins and lipoglycans block phagosome maturation, inhibit class II MHC Ag presentation, and modulate TLR2-dependent cytokine production, but the mechanisms for their release during infection are poorly defined. Furthermore, these molecules are thought to be incorporated into host membranes and released from infected macrophages within exosomes, 40-150-nm extracellular vesicles that derive from multivesicular endosomes. However, our studies revealed that extracellular vesicles released from infected macrophages include two distinct, largely nonoverlapping populations: one containing host cell markers of exosomes (CD9, CD63) and the other containing M. tuberculosis molecules (lipoglycans, lipoproteins). These vesicle populations are similar in size but have distinct densities, as determined by separation on sucrose gradients. Release of lipoglycans and lipoproteins from infected macrophages was dependent on bacterial viability, implicating active bacterial mechanisms in their secretion. Consistent with recent reports of extracellular vesicle production by bacteria (including M. tuberculosis), we propose that bacterial membrane vesicles are secreted by M. tuberculosis within infected macrophages and subsequently are released into the extracellular environment. Furthermore, extracellular vesicles released from M. tuberculosis-infected cells activate TLR2 and induce cytokine responses by uninfected macrophages. We demonstrate that these activities derive from the bacterial membrane vesicles rather than exosomes. Our findings suggest that bacterial membrane vesicles are the primary means by which M. tuberculosis exports lipoglycans and lipoproteins to impair effector functions of infected macrophages and circulate bacterial components beyond the site of infection to regulate immune responses by uninfected

  19. Bacterial membrane vesicles mediate the release of Mycobacterium tuberculosis lipoglycans and lipoproteins from infected macrophages

    PubMed Central

    Athman, Jaffre J.; Wang, Ying; McDonald, David J.; Boom, W. Henry; Harding, Clifford V.; Wearsch, Pamela A.

    2015-01-01

    Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that infects lung macrophages and releases microbial factors that regulate host defense. Mtb lipoproteins and lipoglycans block phagosome maturation, inhibit MHC-II antigen presentation, and modulate TLR2-dependent cytokine production, but the mechanisms for their release during infection are poorly defined. Furthermore, these molecules are thought to be incorporated into host membranes and released from infected macrophages within exosomes, 40-150 nm extracellular vesicles that derive from multivesicular endosomes. However, our studies revealed that extracellular vesicles released from Mtb-infected macrophages include two distinct, largely non-overlapping populations, one containing host cell markers of exosomes (CD9, CD63) and the other containing Mtb molecules (lipoglycans, lipoproteins). These vesicle populations are similar in size, but have distinct densities as determined by separation on sucrose gradients. Release of Mtb lipoglycans and lipoproteins from infected macrophages was dependent on bacterial viability, implicating active bacterial mechanisms in their genesis. Consistent with recent reports of extracellular vesicle production by bacteria (including Mtb), we propose that bacterial membrane vesicles are secreted by Mtb within infected macrophages and subsequently released into the extracellular environment. Extracellular vesicles released from Mtb-infected cells activate TLR2 and induce cytokine responses by uninfected macrophages. We demonstrate that these activities derive from the bacterial membrane vesicles rather than exosomes. Our findings suggest that bacterial membrane vesicles are the primary means by which Mtb exports lipoglycans and lipoproteins to impair effector functions within infected macrophages and circulate bacterial components beyond the site of infection to regulate immune responses by uninfected cells. PMID:26109643

  20. Solvent drag measurement of transcellular and basolateral membrane NaCl reflection coefficient in kidney proximal tubule.

    PubMed

    Shi, L B; Fushimi, K; Verkman, A S

    1991-08-01

    The NaCl reflection coefficient in proximal tubule has important implications for the mechanisms of near isosmotic volume reabsorption. A new fluorescence method was developed and applied to measure the transepithelial (sigma NaClTE) and basolateral membrane (sigma NaClcl) NaCl reflection coefficients in the isolated proximal straight tubule from rabbit kidney. For sigma NaClTE measurement, tubules were perfused with buffers containing 0 Cl, the Cl-sensitive fluorescent indicator 6-methoxy-N-[3-sulfopropyl] quinolinium and a Cl-insensitive indicator fluorescein sulfonate, and bathed in buffers of differing cryoscopic osmolalities containing NaCl. The transepithelial Cl gradient along the length of the tubule was measured in the steady state by a quantitative ratio imaging technique. A mathematical model based on the Kedem-Katchalsky equations was developed to calculate the axial profile of [Cl] from tubule geometry, lumen flow, water (Pf) and NaCl (PNaCl) permeabilities, and sigma NaClTE. A fit of experimental results to the model gave PNaCl = (2.25 +/- 0.2) x 10(-5) cm/s and sigma NaClTE = 0.98 +/- 0.03 at 23 degrees C. For measurement of sigma NaClbl, tubule cells were loaded with SPQ in the absence of Cl. NaCl solvent drag was measured from the time course of NaCl influx in response to rapid (less than 1 s) Cl addition to the bath solution. With bath-to-cell cryoscopic osmotic gradients of 0, -60, and +30 mosmol, initial Cl influx was 1.23, 1.10, and 1.25 mM/s; a fit to a mathematical model gave sigma NaClbl = 0.97 +/- 0.04. These results indicate absence of NaCl solvent drag in rabbit proximal tubule. The implications of these findings for water and NaCl movement in proximal tubule are evaluated.

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

    SciTech Connect

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

    1990-02-26

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

  2. A continuum model of docking of synaptic vesicle to plasma membrane

    PubMed Central

    Liu, Tianshu; Singh, Pankaj; Jenkins, James T.; Jagota, Anand; Bykhovskaia, Maria; Hui, Chung-Yuen

    2015-01-01

    Neurotransmitter release from neuronal terminals is governed by synaptic vesicle fusion. Vesicles filled with transmitters are docked at the neuronal membrane by means of the SNARE machinery. After a series of events leading up to the fusion pore formation, neurotransmitters are released into the synaptic cleft. In this paper, we study the mechanics of the docking process. A continuum model is used to determine the deformation of a spherical vesicle and a plasma membrane, under the influence of SNARE-machinery forces and electrostatic repulsion. Our analysis provides information on the variation of in-plane stress in the membranes, which is known to affect fusion. Also, a simple model is proposed to study hemifusion. PMID:25551140

  3. Bacterial Social Networks: Structure and composition of Myxococcus xanthus outer membrane vesicle chains

    PubMed Central

    Remis, Jonathan P.; Wei, Doug; Gorur, Amita; Zemla, Marcin; Haraga, Jessica; Allen, Simon; Witkowska, H. Ewa; Costerton, J. William; Berleman, James E.; Auer, Manfred

    2014-01-01

    Summary The social soil bacterium, Myxococcus xanthus, displays a variety of complex and highly coordinated behaviors, including social motility, predatory rippling and fruiting body formation. Here we show that M. xanthus cells produce a network of outer membrane extensions in the form of vesicles and vesicle chains that interconnect cells. We observed peritrichous display of vesicles and vesicle chains and increased abundance in biofilms compared to planktonic cultures. By applying a range of imaging techniques, including 3D Focused Ion Beam Scanning Electron Microscopy (FIB/SEM), we determined these structures to range between 30-60 nm in width and up to 5 μm in length. Purified vesicle chains consist of typical M. xanthus lipids, fucose, mannose, N-acetylglucosamine (GlcNAc) and N-acetylgalactoseamine (GalNAc) carbohydrates and a small set of cargo protein. The protein content includes CglB and Tgl membrane proteins transferred in a contact-dependent manner. Most significantly, the 3D organization of cells within biofilms indicates that cells are connected via an extensive network of membrane extensions that may connect cells at the level of the periplasmic space. Such a network would allow the transfer of membrane proteins and other molecules between cells, and likely provides a mechanism for the coordination of social activities. PMID:23848955

  4. Vesicle Photonics

    SciTech Connect

    Vasdekis, Andreas E.; Scott, E. A.; Roke, Sylvie; Hubbell, J. A.; Psaltis, D.

    2013-04-03

    Thin membranes, under appropriate boundary conditions, can self-assemble into vesicles, nanoscale bubbles that encapsulate and hence protect or transport molecular payloads. In this paper, we review the types and applications of light fields interacting with vesicles. By encapsulating light-emitting molecules (e.g. dyes, fluorescent proteins, or quantum dots), vesicles can act as particles and imaging agents. Vesicle imaging can take place also under second harmonic generation from vesicle membrane, as well as employing mass spectrometry. Light fields can also be employed to transport vesicles using optical tweezers (photon momentum) or directly pertrurbe the stability of vesicles and hence trigger the delivery of the encapsulated payload (photon energy).

  5. Bacterial social networks: structure and composition of Myxococcus xanthus outer membrane vesicle chains.

    PubMed

    Remis, Jonathan P; Wei, Dongguang; Gorur, Amita; Zemla, Marcin; Haraga, Jessica; Allen, Simon; Witkowska, H Ewa; Costerton, J William; Berleman, James E; Auer, Manfred

    2014-02-01

    The social soil bacterium, Myxococcus xanthus, displays a variety of complex and highly coordinated behaviours, including social motility, predatory rippling and fruiting body formation. Here we show that M. xanthus cells produce a network of outer membrane extensions in the form of outer membrane vesicle chains and membrane tubes that interconnect cells. We observed peritrichous display of vesicles and vesicle chains, and increased abundance in biofilms compared with planktonic cultures. By applying a range of imaging techniques, including three-dimensional (3D) focused ion beam scanning electron microscopy, we determined these structures to range between 30 and 60 nm in width and up to 5 μm in length. Purified vesicle chains consist of typical M. xanthus lipids, fucose, mannose, N-acetylglucosamine and N-acetylgalactoseamine carbohydrates and a small set of cargo protein. The protein content includes CglB and Tgl outer membrane proteins known to be transferable between cells in a contact-dependent manner. Most significantly, the 3D organization of cells within biofilms indicates that cells are connected via an extensive network of membrane extensions that may connect cells at the level of the periplasmic space. Such a network would allow the transfer of membrane proteins and other molecules between cells, and therefore could provide a mechanism for the coordination of social activities.

  6. Inhibition of Sendai virus fusion with phospholipid vesicles and human erythrocyte membranes by hydrophobic peptides

    SciTech Connect

    Kelsey, D.R.; Flanagan, T.D.; Young, J.E.; Yeagle, P.L. )

    1991-06-01

    Hydrophobic di- and tripeptides which are capable of inhibiting enveloped virus infection of cells are also capable of inhibiting at least three different types of membrane fusion events. Large unilamellar vesicles (LUV) of N-methyl dioleoylphosphatidylethanolamine (N-methyl DOPE), containing encapsulated 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS) and/or p-xylene bis(pyridinium bromide) (DPX), were formed by extrusion. Vesicle fusion and leakage were then monitored with the ANTS/DPX fluorescence assay. Sendai virus fusion with lipid vesicles and Sendai virus fusion with human erythrocyte membranes were measured by following the relief of fluorescence quenching of virus labeled with octadecylrhodamine B chloride (R18). This study found that the effectiveness of the peptides carbobenzoxy-L-Phe-L-Phe (Z-L-Phe-L-Phe), Z-L-Phe, Z-D-Phe, and Z-Gly-L-Phe-L-Phe in inhibiting N-methyl DOPE LUV fusion or fusion of virus with N-methyl DOPE LUV also paralleled their reported ability to block viral infectivity. Furthermore, Z-D-Phe-L-PheGly and Z-Gly-L-Phe inhibited Sendai virus fusion with human erythrocyte membranes with the same relative potency with which they inhibited vesicle-vesicle and virus-vesicle fusion. The evidence suggests a mechanism by which these peptides exert their inhibition of plaque formation by enveloped viruses. This class of inhibitors apparently acts by inhibiting fusion of the viral envelope with the target cell membrane, thereby preventing viral infection. The physical pathway by which these peptides inhibit membrane fusion was investigated. {sup 31}P nuclear magnetic resonance (NMR) of proposed intermediates in the pathway for membrane fusion in LUV revealed that the potent fusion inhibitor Z-D-Phe-L-PheGly selectively altered the structure (or dynamics) of the hypothesized fusion intermediates and that the poor inhibitor Z-Gly-L-Phe did not.

  7. Chronic and selective inhibition of basolateral membrane Na-K-ATPase uniquely regulates brush border membrane Na absorption in intestinal epithelial cells

    PubMed Central

    Manoharan, Palanikumar; Gayam, Swapna; Arthur, Subha; Palaniappan, Balasubramanian; Singh, Soudamani; Dick, Gregory M.

    2015-01-01

    Na-K-ATPase, an integral membrane protein in mammalian cells, is responsible for maintaining the favorable intracellular Na gradient necessary to promote Na-coupled solute cotransport processes [e.g., Na-glucose cotransport (SGLT1)]. Inhibition of brush border membrane (BBM) SGLT1 is, at least in part, due to the diminished Na-K-ATPase in villus cells from chronically inflamed rabbit intestine. The aim of the present study was to determine the effect of Na-K-ATPase inhibition on the two major BBM Na absorptive pathways, specifically Na-glucose cotransport and Na/H exchange (NHE), in intestinal epithelial (IEC-18) cells. Na-K-ATPase was inhibited using 1 mM ouabain or siRNA for Na-K-ATPase-α1 in IEC-18 cells. SGLT1 activity was determined as 3-O-methyl-d-[3H]glucose uptake. Na-K-ATPase activity was measured as the amount of inorganic phosphate released. Treatment with ouabain resulted in SGLT1 inhibition at 1 h but stimulation at 24 h. To further characterize this unexpected stimulation of SGLT1, siRNA silencing was utilized to inhibit Na-K-ATPase-α1. SGLT1 activity was significantly upregulated by Na-K-ATPase silencing, while NHE3 activity remained unaltered. Kinetics showed that the mechanism of stimulation of SGLT1 activity was secondary to an increase in affinity of the cotransporter for glucose without a change in the number of cotransporters. Molecular studies demonstrated that the mechanism of stimulation was not secondary to altered BBM SGLT1 protein levels. Chronic and direct silencing of basolateral Na-K-ATPase uniquely regulates BBM Na absorptive pathways in intestinal epithelial cells. Specifically, while BBM NHE3 is unaffected, SGLT1 is stimulated secondary to enhanced affinity of the cotransporter. PMID:25652450

  8. Chronic and selective inhibition of basolateral membrane Na-K-ATPase uniquely regulates brush border membrane Na absorption in intestinal epithelial cells.

    PubMed

    Manoharan, Palanikumar; Gayam, Swapna; Arthur, Subha; Palaniappan, Balasubramanian; Singh, Soudamani; Dick, Gregory M; Sundaram, Uma

    2015-04-15

    Na-K-ATPase, an integral membrane protein in mammalian cells, is responsible for maintaining the favorable intracellular Na gradient necessary to promote Na-coupled solute cotransport processes [e.g., Na-glucose cotransport (SGLT1)]. Inhibition of brush border membrane (BBM) SGLT1 is, at least in part, due to the diminished Na-K-ATPase in villus cells from chronically inflamed rabbit intestine. The aim of the present study was to determine the effect of Na-K-ATPase inhibition on the two major BBM Na absorptive pathways, specifically Na-glucose cotransport and Na/H exchange (NHE), in intestinal epithelial (IEC-18) cells. Na-K-ATPase was inhibited using 1 mM ouabain or siRNA for Na-K-ATPase-α1 in IEC-18 cells. SGLT1 activity was determined as 3-O-methyl-D-[(3)H]glucose uptake. Na-K-ATPase activity was measured as the amount of inorganic phosphate released. Treatment with ouabain resulted in SGLT1 inhibition at 1 h but stimulation at 24 h. To further characterize this unexpected stimulation of SGLT1, siRNA silencing was utilized to inhibit Na-K-ATPase-α1. SGLT1 activity was significantly upregulated by Na-K-ATPase silencing, while NHE3 activity remained unaltered. Kinetics showed that the mechanism of stimulation of SGLT1 activity was secondary to an increase in affinity of the cotransporter for glucose without a change in the number of cotransporters. Molecular studies demonstrated that the mechanism of stimulation was not secondary to altered BBM SGLT1 protein levels. Chronic and direct silencing of basolateral Na-K-ATPase uniquely regulates BBM Na absorptive pathways in intestinal epithelial cells. Specifically, while BBM NHE3 is unaffected, SGLT1 is stimulated secondary to enhanced affinity of the cotransporter.

  9. Ursodeoxycholate stabilizes phospholipid-rich membranes and mimics the effect of cholesterol: investigations on large unilamellar vesicles.

    PubMed

    Güldütuna, S; Deisinger, B; Weiss, A; Freisleben, H J; Zimmer, G; Sipos, P; Leuschner, U

    1997-06-12

    Ursodeoxycholate is used to treat primary biliary cirrhosis and is incorporated into hepatocyte plasma membranes. Its steroid nucleus binds to the apolar domain of the membrane, in a similar position to cholesterol. Therefore the question arises whether ursodeoxycholate has a similar effect on membrane structure and stability as cholesterol. Using differential scanning calorimetry the thermotropic behavior of egg phosphatidylcholine and dimyristoylphosphatidylcholine were studied after incubation with cholesterol or ursodeoxycholate. Large unilamellar vesicles were prepared with cholesterol contents of 0-50%. Following incubation of these vesicles with different amounts of ursodeoxycholate, vesicle stability in a gravitational field was investigated by measuring the phospholipid and cholesterol release. Vesicle size was studied by laser light scattering after incubation with cheno- and ursodeoxycholate, and the release of entrapped carboxyfluorescein was measured by means of fluorescence spectroscopy. Increasing cholesterol diminished the enthalpy of the phase transition in the membrane. Ursodeoxycholate decreased the enthalpy of the phase transition at even lower concentrations. Lipid release from vesicles in a high gravitational field diminished with increasing cholesterol content of the vesicles. Ursodeoxycholate had a comparable effect, which increased as the cholesterol content of the vesicles was decreased. Chenodeoxycholate damaged vesicles, whereas ursodeoxycholate did not. Cholesterol and ursodeoxycholate (below its critical micellar concentration) decreased the carboxyfluorescein release from vesicles induced by chenodeoxycholate. Thus like cholesterol, ursodeoxycholate is incorporated into phospholipid model membranes and reduces the change in enthalpy of the gel to liquid-crystalline phase transition. Like cholesterol ursodeoxycholate also maintains membrane stability and prevents membrane damage induced by mechanical and chemical stress.

  10. Single vesicle imaging indicates distinct modes of rapid membrane retrieval during nerve growth

    PubMed Central

    2012-01-01

    Background During nerve growth, cytoplasmic vesicles add new membrane preferentially to the growth cone located at the distal tip of extending axons. Growth cone membrane is also retrieved locally, and asymmetric retrieval facilitates membrane remodeling during growth cone repulsion by a chemorepellent gradient. Moreover, growth inhibitory factors can stimulate bulk membrane retrieval and induce growth cone collapse. Despite these functional insights, the processes mediating local membrane remodeling during axon extension remain poorly defined. Results To investigate the spatial and temporal dynamics of membrane retrieval in actively extending growth cones, we have used a transient labeling and optical recording method that can resolve single vesicle events. Live-cell confocal imaging revealed rapid membrane retrieval by distinct endocytic modes based on spatial distribution in Xenopus spinal neuron growth cones. These modes include endocytic "hot-spots" triggered at the base of filopodia, at the lateral margins of lamellipodia, and along dorsal ridges of the growth cone. Additionally, waves of endocytosis were induced when individual filopodia detached from the substrate and fused with the growth cone dorsal surface or with other filopodia. Vesicle formation at sites of membrane remodeling by self-contact required F-actin polymerization. Moreover, bulk membrane retrieval by macroendocytosis correlated positively with the substrate-dependent rate of axon extension and required the function of Rho-family GTPases. Conclusions This study provides insight into the dynamic membrane remodeling processes essential for nerve growth by identifying several distinct modes of rapid membrane retrieval in the growth cone during axon extension. We found that endocytic membrane retrieval is intensified at specific subdomains and may drive the dynamic membrane ruffling and re-absorption of filopodia and lamellipodia in actively extending growth cones. The findings offer a platform

  11. The lantibiotic nisin induces lipid II aggregation, causing membrane instability and vesicle budding.

    PubMed

    Scherer, Katharina M; Spille, Jan-Hendrik; Sahl, Hans-Georg; Grein, Fabian; Kubitscheck, Ulrich

    2015-03-10

    The antimicrobial peptide nisin exerts its activity by a unique dual mechanism. It permeates the cell membranes of Gram-positive bacteria by binding to the cell wall precursor Lipid II and inhibits cell wall synthesis. Binding of nisin to Lipid II induces the formation of large nisin-Lipid II aggregates in the membrane of bacteria as well as in Lipid II-doped model membranes. Mechanistic details of the aggregation process and its impact on membrane permeation are still unresolved. In our experiments, we found that fluorescently labeled nisin bound very inhomogeneously to bacterial membranes as a consequence of the strong aggregation due to Lipid II binding. A correlation between cell membrane damage and nisin aggregation was observed in vivo. To further investigate the aggregation process of Lipid II and nisin, we assessed its dynamics by single-molecule microscopy of fluorescently labeled Lipid II molecules in giant unilamellar vesicles using light-sheet illumination. We observed a continuous reduction of Lipid II mobility due to a steady growth of nisin-Lipid II aggregates as a function of time and nisin concentration. From the measured diffusion constants of Lipid II, we estimated that the largest aggregates contained tens of thousands of Lipid II molecules. Furthermore, we observed that the formation of large nisin-Lipid II aggregates induced vesicle budding in giant unilamellar vesicles. Thus, we propose a membrane permeation mechanism that is dependent on the continuous growth of nisin-Lipid II aggregation and probably involves curvature effects on the membrane.

  12. The Human Pathogen Streptococcus pyogenes Releases Lipoproteins as Lipoprotein-rich Membrane Vesicles*

    PubMed Central

    Biagini, Massimiliano; Garibaldi, Manuela; Aprea, Susanna; Pezzicoli, Alfredo; Doro, Francesco; Becherelli, Marco; Taddei, Anna Rita; Tani, Chiara; Tavarini, Simona; Mora, Marirosa; Teti, Giuseppe; D'Oro, Ugo; Nuti, Sandra; Soriani, Marco; Margarit, Immaculada; Rappuoli, Rino; Grandi, Guido; Norais, Nathalie

    2015-01-01

    Bacterial lipoproteins are attractive vaccine candidates because they represent a major class of cell surface-exposed proteins in many bacteria and are considered as potential pathogen-associated molecular patterns sensed by Toll-like receptors with built-in adjuvanticity. Although Gram-negative lipoproteins have been extensively characterized, little is known about Gram-positive lipoproteins. We isolated from Streptococcus pyogenes a large amount of lipoproteins organized in vesicles. These vesicles were obtained by weakening the bacterial cell wall with a sublethal concentration of penicillin. Lipid and proteomic analysis of the vesicles revealed that they were enriched in phosphatidylglycerol and almost exclusively composed of lipoproteins. In association with lipoproteins, a few hypothetical proteins, penicillin-binding proteins, and several members of the ExPortal, a membrane microdomain responsible for the maturation of secreted proteins, were identified. The typical lipidic moiety was apparently not necessary for lipoprotein insertion in the vesicle bilayer because they were also recovered from the isogenic diacylglyceryl transferase deletion mutant. The vesicles were not able to activate specific Toll-like receptor 2, indicating that lipoproteins organized in these vesicular structures do not act as pathogen-associated molecular patterns. In light of these findings, we propose to name these new structures Lipoprotein-rich Membrane Vesicles. PMID:26018414

  13. Biliary transport of glutathione S-conjugate by rat liver canalicular membrane vesicles.

    PubMed

    Inoue, M; Akerboom, T P; Sies, H; Kinne, R; Thao, T; Arias, I M

    1984-04-25

    Transport of S-dinitrophenyl glutathione, a model compound of glutathione S-conjugates, was studied in isolated rat liver canalicular membrane vesicles by a rapid filtration technique. The membrane vesicles exhibited time-dependent uptake of [2-3H]glycine-glutathione conjugate into an osmotically sensitive intravesicular space. Inactivation of vesicle-associated gamma-glutamyltransferase by affinity labeling with L-(alpha-S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazole-acetic acid had no effect on the initial rate of transport. Chemical analysis revealed that the intact glutathione conjugate accounted for most vesicle-associated radioactivity, reflecting the low transferase activity in the liver and membrane vesicles. The initial rate of transport followed saturation kinetics with respect to conjugate concentrations; an apparent Km of 1.0 mM and Vmax of 1.7 nmol/mg of protein X 20 s were calculated. These results indicate that transport of the glutathione S-conjugate across the canalicular membranes is a carrier-mediated process. Sodium chloride in the transport medium could be replaced by KCl, LiCl, or choline chloride without any changes in transport activity. The rate of conjugate transport was enhanced by a valinomycin-induced K+ diffusion potential (vesicle-inside-positive). The rate of conjugate uptake was enhanced by replacing KCl in the transport medium with K gluconate, providing a less permeant anion, and was reduced by replacing KCl with KSCN, providing a more permeant anion. These data indicate that conjugate transport is electrogenic and involves the transfer of negative charge. Transport of S-dinitrophenyl glutathione was inhibited by S-benzyl glutathione, oxidized glutathione, or reduced glutathione. This transport system in canalicular membranes may function in biliary secretion of glutathione S-conjugates of xenobiotics whose synthesis in hepatocytes requires glutathione S-transferases.

  14. A novel micropipet method for measuring the bending modulus of vesicle membranes.

    PubMed Central

    Zhelev, D V; Needham, D; Hochmuth, R M

    1994-01-01

    A theoretical model and an experiment are presented for determining the bending modulus of a bilayer vesicle membrane. The vesicle is held with a pipet having a radius between 1 and 2 microns, and the tension in the membrane is changed by changing the suction pressure. Then the vesicle membrane is deformed by aspirating it into a smaller pipet having a radius on the order of 0.5 microns. The relationship between the suction pressures in the two pipets is found to be linear, as predicted by the theoretical model. The curvature of the vesicle membrane at the pipet orifice and the bending modulus are found with the help of the model from the slope and the intercept of the linear experimental relationship between the suction pressures in the two pipets. The bending modulus for the two SOPC membranes studied in these experiments was found to be either 0.6 or 1.15 x 10(-19) J, which is similar to the values measured previously. Images FIGURE 1 PMID:7948685

  15. Selective Field Effects on Intracellular Vacuoles and Vesicle Membranes with Nanosecond Electric Pulses

    PubMed Central

    Tekle, Ephrem; Oubrahim, Hammou; Dzekunov, Sergey M.; Kolb, Juergen F.; Schoenbach, Karl H.; Chock, P. B.

    2005-01-01

    Electric pulses across intact vesicles and cells can lead to transient increase in permeability of their membranes. We studied the integrity of these membranes in response to external electric pulses of high amplitude and submicrosecond duration with a primary aim of achieving selective permeabilization. These effects were examined in two separate model systems comprising of 1), a mixed population of 1,2-di-oleoyl-sn-glycero-3-phosphocholine phospholipid vesicles and in 2), single COS-7 cells, in which large endosomal membrane vacuoles were induced by stimulated endocytosis. It has been shown that large and rapidly varying external electric fields, with pulses shorter than the charging time of the outer-cell membrane, could substantially increase intracellular fields to achieve selective manipulations of intracellular organelles. The underlying principle of this earlier work is further developed and applied to the systems studied here. Under appropriate conditions, we show preferential permeabilization of one vesicle population in a mixed preparation of vesicles of similar size distribution. It is further shown that large endocytosed vacuoles in COS-7 cells can be selectively permeabilized with little effect on the integrity of outer cell membrane. PMID:15821165

  16. Selective field effects on intracellular vacuoles and vesicle membranes with nanosecond electric pulses.

    PubMed

    Tekle, Ephrem; Oubrahim, Hammou; Dzekunov, Sergey M; Kolb, Juergen F; Schoenbach, Karl H; Chock, P B

    2005-07-01

    Electric pulses across intact vesicles and cells can lead to transient increase in permeability of their membranes. We studied the integrity of these membranes in response to external electric pulses of high amplitude and submicrosecond duration with a primary aim of achieving selective permeabilization. These effects were examined in two separate model systems comprising of 1), a mixed population of 1,2-di-oleoyl-sn-glycero-3-phosphocholine phospholipid vesicles and in 2), single COS-7 cells, in which large endosomal membrane vacuoles were induced by stimulated endocytosis. It has been shown that large and rapidly varying external electric fields, with pulses shorter than the charging time of the outer-cell membrane, could substantially increase intracellular fields to achieve selective manipulations of intracellular organelles. The underlying principle of this earlier work is further developed and applied to the systems studied here. Under appropriate conditions, we show preferential permeabilization of one vesicle population in a mixed preparation of vesicles of similar size distribution. It is further shown that large endocytosed vacuoles in COS-7 cells can be selectively permeabilized with little effect on the integrity of outer cell membrane.

  17. Comparative Proteome Analysis of Spontaneous Outer Membrane Vesicles and Purified Outer Membranes of Neisseria meningitidis

    PubMed Central

    Otto, Andreas; Becher, Dörte; Vogel, Ulrich

    2013-01-01

    Outer membrane vesicles (OMVs) of Gram-negative bacteria receive increasing attention because of various biological functions and their use as vaccines. However, the mechanisms of OMV release and selective sorting of proteins into OMVs remain unclear. Comprehensive quantitative proteome comparisons between spontaneous OMVs (SOMVs) and the outer membrane (OM) have not been conducted so far. Here, we established a protocol for metabolic labeling of neisserial proteins with 15N. SOMV and OM proteins labeled with 15N were used as an internal standard for proteomic comparison of the SOMVs and OMs of two different strains. This labeling approach, coupled with high-sensitivity mass spectrometry, allowed us to comprehensively unravel the proteome of the SOMVs and OMs. We quantified the relative distribution of 155 proteins between SOMVs and the OM. Complement regulatory proteins, autotransporters, proteins involved in iron and zinc acquisition, and a two-partner secretion system were enriched in SOMVs. The highly abundant porins PorA and PorB and proteins connecting the OM with peptidoglycan or the inner membrane, such as RmpM, MtrE, and PilQ, were depleted in SOMVs. Furthermore, the three lytic transglycosylases MltA, MltB, and Slt were less abundant in SOMVs. In conclusion, SOMVs are likely to be released from surface areas with a low local abundance of membrane-anchoring proteins and lytic transglycosylases. The enrichment of complement regulatory proteins, autotransporters, and trace metal binding and transport proteins needs to be explored in the context of the pathogenesis of meningococcal disease. PMID:23893116

  18. ER arrival sites for COPI vesicles localize to hotspots of membrane trafficking.

    PubMed

    Schröter, Saskia; Beckmann, Sabrina; Schmitt, Hans Dieter

    2016-09-01

    COPI-coated vesicles mediate retrograde membrane traffic from the cis-Golgi to the endoplasmic reticulum (ER) in all eukaryotic cells. However, it is still unknown whether COPI vesicles fuse everywhere or at specific sites with the ER membrane. Taking advantage of the circumstance that the vesicles still carry their coat when they arrive at the ER, we have visualized active ER arrival sites (ERAS) by monitoring contact between COPI coat components and the ER-resident Dsl tethering complex using bimolecular fluorescence complementation (BiFC). ERAS form punctate structures near Golgi compartments, clearly distinct from ER exit sites. Furthermore, ERAS are highly polarized in an actin and myosin V-dependent manner and are localized near hotspots of plasma membrane expansion. Genetic experiments suggest that the COPI•Dsl BiFC complexes recapitulate the physiological interaction between COPI and the Dsl complex and that COPI vesicles are mistargeted in dsl1 mutants. We conclude that the Dsl complex functions in confining COPI vesicle fusion sites.

  19. Molecular cloning and expression of a cDNA encoding the rabbit ileal villus cell basolateral membrane Na+/H+ exchanger.

    PubMed Central

    Tse, C M; Ma, A I; Yang, V W; Watson, A J; Levine, S; Montrose, M H; Potter, J; Sardet, C; Pouyssegur, J; Donowitz, M

    1991-01-01

    A cDNA clone encoding a rabbit ileal villus cell Na+/H+ exchanger was isolated and its complete nucleotide sequence was determined. The cDNA is 4 kb long and contains 322 bp of 5'-untranslated region, 2451 bp of open reading frame and 1163 bp of 3'-untranslated area, with 70%, 91% and 40% identity to the human sequence, respectively. Amino acid sequence deduced from the longest open reading frame indicated a protein of 816 residues (predicted Mr 90,716) which exhibits 95% amino acid identity to the human Na+/H+ exchanger. The two putative glycosylation sites in the human Na+/H+ exchanger are conserved in this protein, suggesting that it is a glycoprotein. Stable transfection of the cDNA into an Na+/H+ exchanger deficient fibroblast cell line, established Na+/H+ exchange. The Na+/H+ exchanger was stimulated by serum and a phorbol ester but not by 8-Br-cAMP. In Northern blot analysis, the cDNA hybridized to a 4.8 kb message in rabbit ileal villus cells, kidney cortex, kidney medulla, adrenal gland, brain and descending colon and to a 5.2 kb message in cultured human colonic cancer cell lines, HT29-18 and Caco-2. In immunoblotting, a polyclonal antibody raised against a fusion protein of beta-galactosidase and the C-terminal 158 amino acids of the human Na+/H+ exchanger identified a rabbit ileal basolateral membrane protein of 94 kd and only weakly interacted with the ileal brush border membrane. In immunocytochemical studies using ileal villus and crypt epithelial cells, the same antibody identified basolateral and not brush border epitopes. Restriction analysis of genomic DNA with a 462 bp PstI-AccI fragment of the rabbit Na+/H+ exchanger strongly suggests the existence of closely related Na+/H+ exchanger genes. The near identity of the basolateral Na+/H+ exchanger and the human Na+/H+ exchanger plus the ubiquitous expression of this message suggests that the ileal basolateral Na+/H+ exchanger is the 'housekeeping' Na+/H+ exchanger. Images PMID:1712287

  20. Temperature-reversible eruptions of vesicles in model membranes studied by NMR.

    PubMed Central

    Nezil, F A; Bayerl, S; Bloom, M

    1992-01-01

    Deuterium (2H) and phosphorus (31P) nuclear magnetic resonance (NMR) and freeze-fracture electron microscopy were used to study spontaneous vesiculation in model membranes composed of POPC:POPS with or without cholesterol. The NMR spectra indicated the presence of a central isotropic line, the intensity of which is reversibly and linearly dependent upon temperature in the L alpha phase, with no hysteresis when cycling between higher and lower temperatures. Freeze-fracture microscopy showed small, apparently connected vesicles that were only present when the samples were frozen (for freeze-fracture) from an initial temperature of 40-60 degrees C, and absent when the samples are frozen from an initial temperature of 20 degrees C. Analysis of motional narrowing was consistent with the isotropic lines being due to lateral diffusion in (and tumbling of) small vesicles (diameters approximately 50 nm). These results were interpreted in terms of current theories of shape fluctuations in large unilamellar vesicles which predict that small daughter vesicles may spontaneously "erupt" from larger parent vesicles in order to expel the excess area created by thermal expansion of the bilayer surface at constant volume. Assuming that all the increased area due to increasing temperature is associated with the isotropic lines, the NMR results allowed a novel estimate of the coefficient of area expansion alpha A in multilamellar vesicles (MLVs) which is in good agreement with micromechanical measurements upon giant unilamellar vesicles of similar composition. Experiments performed on unilamellar vesicles, which had been placed upon glass beads, confirmed that alpha A determined in this way is unchanged compared with the MLV case. Addition of the highly positively charged (extrinsic) myelin basic protein (MBP) to a POPC:POPS system showed that membrane eruptions of the type described here occur in response to the presence of this protein. Images FIGURE 5 FIGURE 5 FIGURE 5 FIGURE 6

  1. Bioinspired polymer vesicles and membranes for biological and medical applications.

    PubMed

    Palivan, Cornelia G; Goers, Roland; Najer, Adrian; Zhang, Xiaoyan; Car, Anja; Meier, Wolfgang

    2016-01-21

    Biological membranes play an essential role in living organisms by providing stable and functional compartments, preserving cell architecture, whilst supporting signalling and selective transport that are mediated by a variety of proteins embedded in the membrane. However, mimicking cell membranes - to be applied in artificial systems - is very challenging because of the vast complexity of biological structures. In this respect a highly promising strategy to designing multifunctional hybrid materials/systems is to combine biological molecules with polymer membranes or to design membranes with intrinsic stimuli-responsive properties. Here we present supramolecular polymer assemblies resulting from self-assembly of mostly amphiphilic copolymers either as 3D compartments (polymersomes, PICsomes, peptosomes), or as planar membranes (free-standing films, solid-supported membranes, membrane-mimetic brushes). In a bioinspired strategy, such synthetic assemblies decorated with biomolecules by insertion/encapsulation/attachment, serve for development of multifunctional systems. In addition, when the assemblies are stimuli-responsive, their architecture and properties change in the presence of stimuli, and release a cargo or allow "on demand" a specific in situ reaction. Relevant examples are included for an overview of bioinspired polymer compartments with nanometre sizes and membranes as candidates in applications ranging from drug delivery systems, up to artificial organelles, or active surfaces. Both the advantages of using polymer supramolecular assemblies and their present limitations are included to serve as a basis for future improvements. PMID:26563574

  2. Two-compartment behavior during transport of folate compounds in L1210 cell plasma membrane vesicles

    SciTech Connect

    Yang, C.H.; Dembo, M.; Sirotnak, F.M.

    1982-01-01

    The transport of (/sup 3/H) 1,L 5-formyltetrahydrofolate, (/sup 3/H) folic acid, and (/sup 3/H)methotrexate by L1210 cell plasma membrane vesicles exhibited multicompartmental behavior. Two separate vesicular compartments (parallel relationship) of approximately equal volume were revealed during measurements of influx and efflux. Flux in one compartment was rapid, saturable, highly temperature-sensitive, and inhibited by pCMBS. Flux in the other compartment exhibited all of the characteristics of passive diffusion. These results imply that our plasma membrane vesicle preparations consist of a mixture of two functional species. Transport of folate into one of these species occurs by passive diffusion alone, whereas transport into the other kind of vesicle occurs by both passive diffusion and carrier-facilitated transport.

  3. Resonance energy transfer imaging of phospholipid vesicle interaction with a planar phospholipid membrane: undulations and attachment sites in the region of calcium-mediated membrane--membrane adhesion

    PubMed Central

    1996-01-01

    Membrane fusion of a phospholipid vesicle with a planar lipid bilayer is preceded by an initial prefusion stage in which a region of the vesicle membrane adheres to the planar membrane. A resonance energy transfer (RET) imaging microscope, with measured spectral transfer functions and a pair of radiometrically calibrated video cameras, was used to determine both the area of the contact region and the distances between the membranes within this zone. Large vesicles (5-20 microns diam) were labeled with the donor fluorophore coumarin- phosphatidylethanolamine (PE), while the planar membrane was labeled with the acceptor rhodamine-PE. The donor was excited with 390 nm light, and separate images of donor and acceptor emission were formed by the microscope. Distances between the membranes at each location in the image were determined from the RET rate constant (kt) computed from the acceptor:donor emission intensity ratio. In the absence of an osmotic gradient, the vesicles stably adhered to the planar membrane, and the dyes did not migrate between membranes. The region of contact was detected as an area of planar membrane, coincident with the vesicle image, over which rhodamine fluorescence was sensitized by RET. The total area of the contact region depended biphasically on the Ca2+ concentration, but the distance between the bilayers in this zone decreased with increasing [Ca2+]. The changes in area and separation were probably related to divalent cation effects on electrostatic screening and binding to charged membranes. At each [Ca2+], the intermembrane separation varied between 1 and 6 nm within each contact region, indicating membrane undulation prior to adhesion. Intermembrane separation distances < or = 2 nm were localized to discrete sites that formed in an ordered arrangement throughout the contact region. The area of the contact region occupied by these punctate attachment sites was increased at high [Ca2+]. Membrane fusion may be initiated at these sites of

  4. Membrane viscosity determined from shear-driven flow in giant vesicles.

    PubMed

    Honerkamp-Smith, Aurelia R; Woodhouse, Francis G; Kantsler, Vasily; Goldstein, Raymond E

    2013-07-19

    The viscosity of lipid bilayer membranes plays an important role in determining the diffusion constant of embedded proteins and the dynamics of membrane deformations, yet it has historically proven very difficult to measure. Here we introduce a new method based on quantification of the large-scale circulation patterns induced inside vesicles adhered to a solid surface and subjected to simple shear flow in a microfluidic device. Particle image velocimetry based on spinning disk confocal imaging of tracer particles inside and outside of the vesicle and tracking of phase-separated membrane domains are used to reconstruct the full three-dimensional flow pattern induced by the shear. These measurements show excellent agreement with the predictions of a recent theoretical analysis, and allow direct determination of the membrane viscosity.

  5. Microfluidic trapping of giant unilamellar vesicles to study transport through a membrane pore

    PubMed Central

    Robinson, T.; Kuhn, P.; Eyer, K.; Dittrich, P. S.

    2013-01-01

    We present a microfluidic platform able to trap single GUVs in parallel. GUVs are used as model membranes across many fields of biophysics including lipid rafts, membrane fusion, and nanotubes. While their creation is relatively facile, handling and addressing single vesicles remains challenging. The PDMS microchip used herein contains 60 chambers, each with posts able to passively capture single GUVs without compromising their integrity. The design allows for circular valves to be lowered from the channel ceiling to isolate the vesicles from rest of the channel network. GUVs containing calcein were trapped and by rapidly opening the valves, the membrane pore protein α-hemolysin (αHL) was introduced to the membrane. Confocal microscopy revealed the kinetics of the small molecule efflux for different protein concentrations. This microfluidic approach greatly improves the number of experiments possible and can be applied to a wide range of biophysical applications. PMID:24404039

  6. Membrane viscosity determined from shear-driven flow in giant vesicles.

    PubMed

    Honerkamp-Smith, Aurelia R; Woodhouse, Francis G; Kantsler, Vasily; Goldstein, Raymond E

    2013-07-19

    The viscosity of lipid bilayer membranes plays an important role in determining the diffusion constant of embedded proteins and the dynamics of membrane deformations, yet it has historically proven very difficult to measure. Here we introduce a new method based on quantification of the large-scale circulation patterns induced inside vesicles adhered to a solid surface and subjected to simple shear flow in a microfluidic device. Particle image velocimetry based on spinning disk confocal imaging of tracer particles inside and outside of the vesicle and tracking of phase-separated membrane domains are used to reconstruct the full three-dimensional flow pattern induced by the shear. These measurements show excellent agreement with the predictions of a recent theoretical analysis, and allow direct determination of the membrane viscosity. PMID:23909365

  7. Polymeric capsule-cushioned leukocyte cell membrane vesicles as a biomimetic delivery platform

    NASA Astrophysics Data System (ADS)

    Gao, Changyong; Wu, Zhiguang; Lin, Zhihua; Lin, Xiankun; He, Qiang

    2016-02-01

    We report a biomimetic delivery of microsized capsule-cushioned leukocyte membrane vesicles (CLMVs) through the conversion of freshly reassembled leukocyte membrane vesicles (LMVs), including membrane lipids and membrane-bound proteins onto the surface of layer-by-layer assembled polymeric multilayer microcapsules. The leukocyte membrane coating was verified by using electron microscopy, a quartz crystal microbalance, dynamic light scattering, and confocal laser scanning microscopy. The resulting CLMVs have the ability to effectively evade clearance by the immune system and thus prolong the circulation time in mice. Moreover, we also show that the right-side-out leukocyte membrane coating can distinctly improve the accumulation of capsules in tumor sites through the molecular recognition of membrane-bound proteins of CLMVs with those of tumor cells in vitro and in vivo. The natural cell membrane camouflaged polymeric multilayer capsules with the immunosuppressive and tumor-recognition functionalities of natural leukocytes provide a new biomimetic delivery platform for disease therapy.We report a biomimetic delivery of microsized capsule-cushioned leukocyte membrane vesicles (CLMVs) through the conversion of freshly reassembled leukocyte membrane vesicles (LMVs), including membrane lipids and membrane-bound proteins onto the surface of layer-by-layer assembled polymeric multilayer microcapsules. The leukocyte membrane coating was verified by using electron microscopy, a quartz crystal microbalance, dynamic light scattering, and confocal laser scanning microscopy. The resulting CLMVs have the ability to effectively evade clearance by the immune system and thus prolong the circulation time in mice. Moreover, we also show that the right-side-out leukocyte membrane coating can distinctly improve the accumulation of capsules in tumor sites through the molecular recognition of membrane-bound proteins of CLMVs with those of tumor cells in vitro and in vivo. The natural

  8. Vesicle fusion to planar membranes is enhanced by cholesterol and low temperature.

    PubMed

    Lee, David E; Lew, Matthew G; Woodbury, Dixon J

    2013-01-01

    Lipid composition and properties play an important role in many cellular properties such as fusion of vesicles to cell membranes, an essential process for exocytosis. Using a model system composed of artificial vesicles (liposomes) and artificial membranes (planar lipid bilayers), we observed that fusion is significantly affected by the lipid phase of the planar membrane. To determine the effect of lipid phases on fusion rates, we utilized the nystatin/ergosterol fusion assay and stimulated fusion with an osmotic gradient. Phase of the planar membrane was altered by changing cholesterol or temperature while the vesicular lipids were held constant. Liquid disordered (L(d) or L(α)) planar membranes were formed from phosphatidylethanolamine and phosphatidylcholine with unsaturated acyl chains. Addition of cholesterol shifts these membranes to the liquid ordered (L(o)) phase and increases liposome fusion. Planar membranes in the L(α) phase were also made from dipalmitoylphoshatidylcholine (DPPC) above the transition temperature (T(m)) of 41.5 ° C. Decreasing the temperature below T(m) shifts these membranes into the ripple phase (P(β')) and also increases liposome fusion. The cholesterol and temperature data are consistent with the hypothesis that fusion is promoted in membranes that have greater exposure of their lipid tails or in membranes which can form leaflet domains with negative curvature. The data are not consistent with the hypothesis that lipid mismatch drives fusion.

  9. Proteome Profiles of Outer Membrane Vesicles and Extracellular Matrix of Pseudomonas aeruginosa Biofilms.

    PubMed

    Couto, Narciso; Schooling, Sarah R; Dutcher, John R; Barber, Jill

    2015-10-01

    In the present work, two different proteomic platforms, gel-based and gel-free, were used to map the matrix and outer membrane vesicle exoproteomes of Pseudomonas aeruginosa PAO1 biofilms. These two proteomic strategies allowed us a confident identification of 207 and 327 proteins from enriched outer membrane vesicles and whole matrix isolated from biofilms. Because of the physicochemical characteristics of these subproteomes, the two strategies showed complementarity, and thus, the most comprehensive analysis of P. aeruginosa exoproteome to date was achieved. Under our conditions, outer membrane vesicles contribute approximately 20% of the whole matrix proteome, demonstrating that membrane vesicles are an important component of the matrix. The proteomic profiles were analyzed in terms of their biological context, namely, a biofilm. Accordingly relevant metabolic processes involved in cellular adaptation to the biofilm lifestyle as well as those related to P. aeruginosa virulence capabilities were a key feature of the analyses. The diversity of the matrix proteome corroborates the idea of high heterogeneity within the biofilm; cells can display different levels of metabolism and can adapt to local microenvironments making this proteomic analysis challenging. In addition to analyzing our own primary data, we extend the analysis to published data by other groups in order to deepen our understanding of the complexity inherent within biofilm populations. PMID:26303878

  10. ATP-dependent bile-salt transport in canalicular rat liver plasma-membrane vesicles.

    PubMed Central

    Stieger, B; O'Neill, B; Meier, P J

    1992-01-01

    The present study identifies and characterizes a novel ATP-dependent bile-salt transport system in isolated canalicular rat liver plasma-membrane (cLPM) vesicles. ATP (1-5 mM) stimulated taurocholate uptake into cLPM vesicles between 6- and 8-fold above equilibrium uptake values (overshoot) and above values for incubations in the absence of ATP. The ATP-dependent portion of taurocholate uptake was 2-fold higher in the presence of equilibrated KNO3 as compared with potassium gluconate, indicating that the stimulatory effect of ATP was not due to the generation of an intravesicular positive membrane potential. Saturation kinetics revealed a very high affinity (Km approximately 2.1 microM) of the system for taurocholate. The system could only minimally be stimulated by nucleotides other than ATP. Furthermore, it was preferentially inhibited by conjugated univalent bile salts. Further strong inhibitory effects were observed with valinomycin, oligomycin, 4,4'-di-isothiocyano-2,2'-stilbene disulphonate, sulphobromophthalein, leukotriene C4 and N-ethylmaleimide, whereas nigericin, vanadate, GSH, GSSG and daunomycin exerted only weak inhibitory effects or none at all. These results indicate the presence of a high-affinity primary ATP-dependent bile-salt transport system in cLPM vesicles. This transport system might be regulated in vivo by the number of carriers present at the perspective transport site(s), which, in addition to the canalicular membrane, might also include pericanalicular membrane vesicles. PMID:1599411

  11. Hemifusion and fusion of giant vesicles induced by reduction of inter-membrane distance

    NASA Astrophysics Data System (ADS)

    Heuvingh, J.; Pincet, F.; Cribier, S.

    2004-07-01

    Proteins involved in membrane fusion, such as SNARE or influenza virus hemagglutinin, share the common function of pulling together opposing membranes in closer contact. The reduction of inter-membrane distance can be sufficient to induce a lipid transition phase and thus fusion. We have used functionalized lipids bearing DNA bases as head groups incorporated into giant unilamellar vesicles in order to reproduce the reduction of distance between membranes and to trigger fusion in a model system. In our experiments, two vesicles were isolated and brought into adhesion by the mean of micromanipulation; their evolution was monitored by fluorescence microscopy. Actual fusion only occurred in about 5% of the experiments. In most cases, a state of “hemifusion” is observed and quantified. In this state, the outer leaflets of both vesicles' bilayers merged whereas the inner leaflets and the aqueous inner contents remained independent. The kinetics of the lipid probes redistribution is in good agreement with a diffusion model in which lipids freely diffuse at the circumference of the contact zone between the two vesicles. The minimal density of bridging structures, such as stalks, necessary to explain this redistribution kinetics can be estimated.

  12. Direct membrane retrieval into large vesicles after exocytosis in sea urchin eggs

    PubMed Central

    1995-01-01

    At fertilization in sea urchin eggs, elevated cytosolic Ca2+ leads to the exocytosis of 15,000-18,000 1.3-microns-diam cortical secretory granules to form the fertilization envelope. Cortical granule exocytosis more than doubles the surface area of the egg. It is thought that much of the added membrane is retrieved by subsequent endocytosis. We have investigated how this is achieved by activating eggs in the presence of aqueous- and lipid-phase fluorescent dyes. We find rapid endocytosis of membrane into 1.5-microns-diam vesicles starting immediately after cortical granule exocytosis and persisting over the following 15 min. The magnitude of this membrane retrieval can compensate for the changes in the plasma membrane of the egg caused by exocytosis. This membrane retrieval is not stimulated by PMA treatment which activates the endocytosis of clathrin-coated vesicles. When eggs are treated with short wave-length ultraviolet light, cortical granule exocytosis still occurs, but granule cores fail to disperse. After egg activation, large vesicles containing semi-intact cortical granule protein cores are observed. These data together with experiments using sequential pulses of fluid-phase markers support the hypothesis that the bulk of membrane retrieval immediately after cortical granule exocytosis is achieved through direct retrieval into large endocytotic structures. PMID:8522582

  13. Sodium- and adenosine-triphosphate-dependent calcium movements in membrane vesicles prepared from dog erythrocytes.

    PubMed Central

    Ortiz, O E; Sjodin, R A

    1984-01-01

    Inside-out vesicles from the membranes of dog erythrocytes were obtained by the method of Lew & Seymour (1982) for study of Ca movements. In the absence of ATP, 45Ca accumulation by the vesicles was inhibited by external Na and stimulated by internal Na. The presence of either MgCl2, quinidine sulphate, or LaCl3 in the incubation medium inhibited 45Ca accumulation in the absence of ATP. The release of 45Ca from 45Ca-loaded vesicles was specifically promoted by Na+ in the absence as well as in the presence of ATP. The accumulation of 45Ca by vesicles was stimulated by ATP and the effect of ATP was entirely dependent on the presence of Mg. The Mg- and ATP-dependent 45Ca accumulation was stimulated by the presence of either K or Na in the medium, was hyperbolically activated by increasing the Ca2+ concentration in the medium, was stimulated by calmodulin and inhibited by orthovanadate (10(-4) M) or LaCl3 (10(-3) M). The data demonstrate the presence of two mechanisms for controlling Ca movements in inside-out vesicles from dog erythrocyte membranes, a Na-dependent one similar to the Na-Ca exchange described for squid axons and cardiac muscle and a Ca pump utilizing ATP with characteristics similar to those described for human erythrocytes and squid axons. PMID:6090650

  14. Raman spectroscopy of single extracellular vesicles reveals subpopulations with varying membrane content (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Smith, Zachary J.; Lee, Changwon; Rojalin, Tatu; Carney, Randy P.; Hazari, Sidhartha; Knudson, Alisha; Lam, Kit S.; Saari, Heikki; Lazaro Ibañez, Elisa; Viitala, Tapani; Laaksonen, Timo; Yliperttula, Marjo; Wachsmann-Hogiu, Sebastian

    2016-03-01

    Exosomes are small (~100nm) membrane bound vesicles excreted by cells as part of their normal biological processes. These extracellular vesicles are currently an area of intense research, since they were recently found to carry functional mRNA that allows transfer of proteins and other cellular instructions between cells. Exosomes have been implicated in a wide range of diseases, including cancer. Cancer cells are known to have increased exosome production, and may use those exosomes to prepare remote environments for metastasis. Therefore, there is a strong need to develop characterization methods to help understand the structure and function of these vesicles. However, current techniques, such as proteomics and genomics technologies, rely on aggregating a large amount of exosome material and reporting on chemical content that is averaged over many millions of exosomes. Here we report on the use of laser-tweezers Raman spectroscopy (LTRS) to probe individual vesicles, discovering distinct heterogeneity among exosomes both within a cell line, as well as between different cell lines. Through principal components analysis followed by hierarchical clustering, we have identified four "subpopulations" of exosomes shared across seven cell lines. The key chemical differences between these subpopulations, as determined by spectral analysis of the principal component loadings, are primarily related to membrane composition. Specifically, the differences can be ascribed to cholesterol content, cholesterol to phospholipid ratio, and surface protein expression. Thus, we have shown LTRS to be a powerful method to probe the chemical content of single extracellular vesicles.

  15. Fundamental Studies of Assembly and Mechanical Properties of Lipid Bilayer Membranes and Unilamellar Vesicles

    NASA Astrophysics Data System (ADS)

    Wang, Xi

    This dissertation work focuses on: (i) obtaining a phospholipid bilayer membrane (LBM)/conducting electrode system with low defect density and optimized rigidity; (ii) investigating vesicle stability and mechanical properties. LBM is a simplified yet representative cell membrane model. LBMs assembled on conductive surfaces can probe protein-LBM interactions activities electrochemically. Sterically stabilized vesicles could be used as cell models or for drug delivery. The main challenges for LBM assembly on gold are vesicles do not spontaneously rupture to form LBMs on gold and the roughness of the gold substrate has considerable influence on molecular film defect density. In this study, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles were functionalized with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine- N-poly(ethylene glycol)-2000-N-[3-(2-pyridyldithio)propionate] (DSPE-PEG-PDP) to yield stable LBMs on gold without surface modification. A template-stripping method was used to obtain atomically flat and pristine gold surfaces. The critical force to initiate vesicle rupture decreases with increasing DSPE-PEG-PDP concentration, indicating that gold-thiolate bonding between DSPE-PEG-PDP and gold substrates promotes LBM formation. Mechanical properties of LBMs and vesicles were investigated as a function of DSPE-PEG-PDP concentration via Atomic Force Microscopy. The elastic moduli of LBMs were determined with DSPE-PEG-PDP concentration ranging from 0mol% to 24mol% and were found to depend on PEG chain conformation. Incorporating DSPE-PEG-PDP molecules with PEG in mushroom conformation results in a decrease of LBM rigidity, while incorporating PEG in brush conformation leads to LBM stiffening. Contrarily, mechanical properties of functionalized vesicles did not vary significantly by varying DSPE-PEG-PDP concentration. LBM with tunable rigidity by adjusting DSPE-PEG-PDP concentration provides a versatile cell membrane model for studying protein or

  16. Identification of a plastid protein involved in vesicle fusion and/or membrane protein translocation.

    PubMed Central

    Hugueney, P; Bouvier, F; Badillo, A; d'Harlingue, A; Kuntz, M; Camara, B

    1995-01-01

    Structural evidence has accumulated suggesting that fusion and/or translocation factors are involved in plastid membrane biogenesis. To test this hypothesis, we have developed an in vitro system in which the extent of fusion and/or translocation is monitored by the conversion of the xanthophyll epoxide (antheraxanthin) into the red ketocarotenoid (capsanthin). Only chromoplast membrane vesicles from red pepper fruits (Capsicum annuum) contain the required enzyme. Vesicles prepared from the mutant yellow cultivar are devoid of this enzyme and accumulate antheraxanthin. The fusion and/or translocation activity is characterized by complementation due to the synthesis of capsanthin and the parallel decrease of antheraxanthin when the two types of vesicles are incubated together in the presence of plastid stroma. We show that the extent of conversion is dependent upon an ATP-requiring protein that is sensitive to N-ethylmaleimide. Further purification and immunological analysis have revealed that the active factor, designated plastid fusion and/or translocation factor (Pftf), resides in a protein of 72 kDa. cDNA cloning revealed that mature Pftf has significant homology to yeast and animal (NSF) or bacterial (Ftsh) proteins involved in vesicle fusion or membrane protein translocation. Images Fig. 1 Fig. 3 Fig. 4 PMID:7777561

  17. Genome-Wide Assessment of Outer Membrane Vesicle Production in Escherichia coli.

    PubMed

    Kulp, Adam J; Sun, Bo; Ai, Teresa; Manning, Andrew J; Orench-Rivera, Nichole; Schmid, Amy K; Kuehn, Meta J

    2015-01-01

    The production of outer membrane vesicles by Gram-negative bacteria has been well documented; however, the mechanism behind the biogenesis of these vesicles remains unclear. Here a high-throughput experimental method and systems-scale analysis was conducted to determine vesiculation values for the whole genome knockout library of Escherichia coli mutant strains (Keio collection). The resultant dataset quantitatively recapitulates previously observed phenotypes and implicates nearly 150 new genes in the process of vesiculation. Gene functional and biochemical pathway analyses suggest that mutations that truncate outer membrane structures such as lipopolysaccharide and enterobacterial common antigen lead to hypervesiculation, whereas mutants in oxidative stress response pathways result in lower levels. This study expands and refines the current knowledge regarding the cellular pathways required for outer membrane vesiculation in E. coli.

  18. Genome-Wide Assessment of Outer Membrane Vesicle Production in Escherichia coli

    PubMed Central

    Kulp, Adam J.; Sun, Bo; Ai, Teresa; Manning, Andrew J.; Orench-Rivera, Nichole

    2015-01-01

    The production of outer membrane vesicles by Gram-negative bacteria has been well documented; however, the mechanism behind the biogenesis of these vesicles remains unclear. Here a high-throughput experimental method and systems-scale analysis was conducted to determine vesiculation values for the whole genome knockout library of Escherichia coli mutant strains (Keio collection). The resultant dataset quantitatively recapitulates previously observed phenotypes and implicates nearly 150 new genes in the process of vesiculation. Gene functional and biochemical pathway analyses suggest that mutations that truncate outer membrane structures such as lipopolysaccharide and enterobacterial common antigen lead to hypervesiculation, whereas mutants in oxidative stress response pathways result in lower levels. This study expands and refines the current knowledge regarding the cellular pathways required for outer membrane vesiculation in E. coli. PMID:26406465

  19. Assessing the efficacy of vesicle fusion with planar membrane arrays using a mitochondrial porin as reporter

    SciTech Connect

    Pszon-Bartosz, Kamila; Hansen, Jesper S.; Stibius, Karin B.; Groth, Jesper S.; Helix-Nielsen, Claus

    2011-03-04

    Research highlights: {yields} We have established a vesicle fusion efficacy assay based on the major non-specific porin of Fusobacterium nucleatum (FomA). {yields} Maximal fusion obtained was almost 150,000 porin insertions during 20 min. {yields} Incorporation can be either first order or exponential kinetics which has implications for establishing protein delivery to biomimetic membranes. -- Abstract: Reconstitution of functionally active membrane protein into artificially made lipid bilayers is a challenge that must be overcome to create a membrane-based biomimetic sensor and separation device. In this study we address the efficacy of proteoliposome fusion with planar membrane arrays. We establish a protein incorporation efficacy assay using the major non-specific porin of Fusobacterium nucleatum (FomA) as reporter. We use electrical conductance measurements and fluorescence microscopy to characterize proteoliposome fusion with an array of planar membranes. We show that protein reconstitution in biomimetic membrane arrays may be quantified using the developed FomA assay. Specifically, we show that FomA vesicles are inherently fusigenic. Optimal FomA incorporation is obtained with a proteoliposome lipid-to-protein molar ratio (LPR) = 50 more than 10{sup 5} FomA proteins could be incorporated in a bilayer array with a total membrane area of 2 mm{sup 2} within 20 min. This novel assay for quantifying protein delivery into lipid bilayers may be a useful tool in developing biomimetic membrane applications.

  20. Modulation by small hydrophobic molecules of valinomycin-mediated potassium transport across phospholipid vesicle membranes

    SciTech Connect

    Clement, N.R.; Gould, J.M.

    1981-01-01

    The effects of small hydrophobic molecules on valinonycin-mediated K/sup +/ transport in small unilamellar soybean phospholipid vesicles have been studied by using a vesicle-entrapped pH-sensitive hydropholic fluorescence probe to monitor counterion-limited, passive H/sup +/ diffusion into vesicles after an abrupt decrease in external pH. Under conditions where, even in the absence of valinomycin, transmembrane K/sup +/ movement represented the primary and limiting counterion flux, <1 valinomycin molecule/vesicle was sufficient to accelerate the rate of H/sup +/ entry into all of the vesicles. Incorporation of the bulkily substituted molecules butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and p-di-tert-butylbenzene into soybean lipid bilayers had no effect upon K/sup +/ diffusion in the absence of valinomycin. However, the presence of these hydrophobic molecules increased the apparent efficacy for K/sup +/ transport of a given valinomycin concentration by as much as 4-6 fold. The less bulky membrane perturbants tert-butyl alcohol, phenol, and heptane showed very much less dramatic effects. While the rate of valinomycin-mediated K/sup +/ transport (in the presence or absence of BHT) was very sensitive to temperature-induced changes in membrane fluidity, the degree of synergistic interaction between valinomycin and BHT was independent of temperature. Furthermore, BHT, BHA, and p-di-tert-butylbenzene, at levels which alter valinomycin-mediated K/sup +/ transport, did not by themselves induce changes in membrane fluidity. It is postulated that changes in phospholipid head-group packing and/or surface charge density brought about by the presence of bulky perturber molecules leads to changes in partitioning of valinomycin or the valinomycin-K/sup +/ complex between the aqueous and membrane phases.

  1. Transcytotic vesicle fusion with canalicular membranes is modulated by phospholipid species: implications for biliary lipid secretion.

    PubMed

    Hirano, N; Tazuma, S; Kajiyama, G

    1997-07-01

    Phospholipid species modulate bile metastability and the subselection of such species for biliary secretion occurs at the canalicular membrane. In this study, the role of phospholipid head groups and hydrophobic indices in transcytotic vesicle fusion with the canalicular membrane inner leaflet was investigated using rat canalicular membrane vesicles (CMV) and liposomes. The CMV were purified from Sprague-Dawley rat liver, and small unilamellar vesicles (SUV) of phosphatidylserine (PS), phosphatidylcholine (PC) and mixtures of PS/PC (1:1, 2:1 and 4:1) were labelled with 8 mol% of octadecyl rhodamine B chloride (R18). The PC species used in this study were egg yolk PC (EYPC), soybean PC (SBPC), dipalmitoyl PC (DPPC) and dilinoleoyl PC (DLPC). Fusion of SUV with CMV was initiated by the addition of a millimolar concentration of Ca2+ and the degree of fusion was estimated by the increase of R18 fluorescence. Ca(2+)-dependent fusion of SUV consisting of PS, and PS/PC (4:1) with CMV was observed (PS > PS/PC; 4:1), whereas no detectable fusion was evident between CMV and SUV of PC alone or PS/PC (1:1 or 2:1). The rank order of fusibility between CMV and SUV of PS/PC (4:1) containing various PC species was PS/DLPC > PS/SBPC > PS/EYPC > PS/DPPC. The hydrophobic index of PC as determined by high performance liquid chromatography (HPLC) was related closely to liposome fusibility (r = -0.88). These results suggest that transcytotic vesicle fusion with the canalicular membrane inner leaflet is regulated by the phospholipid hydrophobicity of the vesicles.

  2. Versatile cellular uptake mediated by catanionic vesicles: simultaneous spontaneous membrane fusion and endocytosis.

    PubMed

    Mauroy, Chloé; Castagnos, Pauline; Orio, Julie; Blache, Marie-Claire; Rico-Lattes, Isabelle; Teissié, Justin; Rols, Marie-Pierre; Blanzat, Muriel

    2015-01-01

    Lactose-derived catanionic vesicles offer unique opportunities to overcome cellular barriers. These potential nanovectors, very easy to formulate as drug delivery systems, are able to encapsulate drugs of various hydrophilicity. This article highlights versatile interaction mechanisms between these catanionic vesicles, labeled with hydrophilic and amphiphilic fluorescent probes, and a mammalian cell line, Chinese Hamster Ovary. Confocal microscopy and flow cytometry techniques show that these vesicles are internalized by cells through cellular energy dependent processes, as endocytosis, but are simultaneously able to spontaneously fuse with cell plasma membranes and release their hydrophilic content directly inside the cytosol. Such innovative and polyvalent nanovectors, able to deliver their content via different internalization pathways, would positively be a great progress for the coadministration of drugs of complementary efficiency. PMID:25310849

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

    PubMed

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

    2016-07-01

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

  4. Fluorescence assays to monitor membrane fusion: potential application in biliary lipid secretion and vesicle interactions.

    PubMed

    Hoekstra, D

    1990-09-01

    Membrane fusion constitutes an essential, intermediate step in numerous cell biological processes, occurring for example during endocytosis, membrane recycling and exocytosis. Also less desirable events such as the infection of cells by animal viruses are mediated by membrane fusion during which the viral envelope merges with a cellular membrane, causing the expulsion of the viral nucleocapsid into the cytoplasm of the cell as an initial step in virus replication. Much of our current knowledge concerning the mechanism of membrane fusion has been derived from studies using simple artificial membranes, such as liposomes or phospholipid vesicles, as model systems. A most essential feature of these studies has been the development of membrane fusion assays that register in a sensitive and continuous fashion the mixing of membranes or the aqueous volumes initially enclosed by these membranes. Not only do these assays allow one to readily detect and quantify fusion, but they also provide the possibility to relate the kinetics of fusion to the rate by which certain molecular changes in membranes take place. Obviously, this insight is of relevance for understanding the mechanism of membrane fusion. The principles and applications of some representative assays that rely on the use of fluorescence spectroscopy will be discussed. Assays that monitor membrane mixing are commonly based on the detection of changes in resonance energy transfer efficiency or the relief of fluorescence self-quenching of appropriate fluorescent lipid analogs. Contents mixing assays rely on either the formation of a (aqueous-soluble) fluorescent complex or quenching of a fluorophore, encapsulated in one vesicle population, by a suitable quencher, entrapped in a second population.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. The Lantibiotic Nisin Induces Lipid II Aggregation, Causing Membrane Instability and Vesicle Budding

    PubMed Central

    Scherer, Katharina M.; Spille, Jan-Hendrik; Sahl, Hans-Georg; Grein, Fabian; Kubitscheck, Ulrich

    2015-01-01

    The antimicrobial peptide nisin exerts its activity by a unique dual mechanism. It permeates the cell membranes of Gram-positive bacteria by binding to the cell wall precursor Lipid II and inhibits cell wall synthesis. Binding of nisin to Lipid II induces the formation of large nisin-Lipid II aggregates in the membrane of bacteria as well as in Lipid II-doped model membranes. Mechanistic details of the aggregation process and its impact on membrane permeation are still unresolved. In our experiments, we found that fluorescently labeled nisin bound very inhomogeneously to bacterial membranes as a consequence of the strong aggregation due to Lipid II binding. A correlation between cell membrane damage and nisin aggregation was observed in vivo. To further investigate the aggregation process of Lipid II and nisin, we assessed its dynamics by single-molecule microscopy of fluorescently labeled Lipid II molecules in giant unilamellar vesicles using light-sheet illumination. We observed a continuous reduction of Lipid II mobility due to a steady growth of nisin-Lipid II aggregates as a function of time and nisin concentration. From the measured diffusion constants of Lipid II, we estimated that the largest aggregates contained tens of thousands of Lipid II molecules. Furthermore, we observed that the formation of large nisin-Lipid II aggregates induced vesicle budding in giant unilamellar vesicles. Thus, we propose a membrane permeation mechanism that is dependent on the continuous growth of nisin-Lipid II aggregation and probably involves curvature effects on the membrane. PMID:25762323

  6. The lantibiotic nisin induces lipid II aggregation, causing membrane instability and vesicle budding.

    PubMed

    Scherer, Katharina M; Spille, Jan-Hendrik; Sahl, Hans-Georg; Grein, Fabian; Kubitscheck, Ulrich

    2015-03-10

    The antimicrobial peptide nisin exerts its activity by a unique dual mechanism. It permeates the cell membranes of Gram-positive bacteria by binding to the cell wall precursor Lipid II and inhibits cell wall synthesis. Binding of nisin to Lipid II induces the formation of large nisin-Lipid II aggregates in the membrane of bacteria as well as in Lipid II-doped model membranes. Mechanistic details of the aggregation process and its impact on membrane permeation are still unresolved. In our experiments, we found that fluorescently labeled nisin bound very inhomogeneously to bacterial membranes as a consequence of the strong aggregation due to Lipid II binding. A correlation between cell membrane damage and nisin aggregation was observed in vivo. To further investigate the aggregation process of Lipid II and nisin, we assessed its dynamics by single-molecule microscopy of fluorescently labeled Lipid II molecules in giant unilamellar vesicles using light-sheet illumination. We observed a continuous reduction of Lipid II mobility due to a steady growth of nisin-Lipid II aggregates as a function of time and nisin concentration. From the measured diffusion constants of Lipid II, we estimated that the largest aggregates contained tens of thousands of Lipid II molecules. Furthermore, we observed that the formation of large nisin-Lipid II aggregates induced vesicle budding in giant unilamellar vesicles. Thus, we propose a membrane permeation mechanism that is dependent on the continuous growth of nisin-Lipid II aggregation and probably involves curvature effects on the membrane. PMID:25762323

  7. Uptake of auxins into membrane vesicles isolated from pea stems: an in vitro auxin transport system

    SciTech Connect

    Slone, J.H.

    1985-01-01

    The objective of this research was to test the applicability of the chemiosmotic theory of auxin transport to a subcellular system. Membrane vesicles were isolated from the basal portion of the third internode of etiolated pea plants (Pisum sativum L. var. Alaska) by differential centrifugation. Uptake of auxin was determined by adding /sup 14/C-labeled indoleacetic acid (IAA) to vesicles. Nigericin, a monovalent cation ionophore, and the electrogenic protonophore, carbonyl-cyanide m-chlorophenylhydrazone (CCCP), at micromolar concentrations abolished saturable uptake. Bursting vesicles by sonication, osmotic shock and freeze/thawing also eliminated saturable uptake. As the temperature increased from 0 to 30/sup 0/C, saturable uptake decreased markedly. Nonsaturable auxin uptake was less affected by these treatments. The pH gradient-dependent uptake of auxin appeared to be a transmembrane uptake of auxin into the vesicles rather than surface binding. Unlabeled IAA, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-naphthaleneacetic acid (NAA) at low concentrations reduced the saturable accumulation of (/sup 14/C)IAA in vesicles, while phenylacetic acid, benzoic acid, and 1-NAA were effective only at high concentrations. Kinetic analysis revealed two types of sites: a high affinity site with an uptake capacity of 25 to 40 pmoles/g tissue, and a low affinity site with an uptake capacity of 260 to 600 pmole/g tissue, fresh wt. In conclusion, several principal elements of an auxin transport system, as specific by the chemiosmotic theory of polar auxin transport, were present in membrane vesicles isolated from relatively mature pea stem tissue. However, one important aspect of the theory was not demonstrated in this in vitro system - a TIBA/NPA-sensitive auxin efflux. The kinetics and specificity of auxin uptake strongly suggested that this system was physiologically significant.

  8. Separation of the osmotically driven fusion event from vesicle-planar membrane attachment in a model system for exocytosis

    PubMed Central

    1984-01-01

    We demonstrate that there are two experimentally distinguishable steps in the fusion of phospholipid vesicles with planar bilayer membranes. In the first step, the vesicles form a stable, tightly bound pre-fusion state with the planar membrane; divalent cations (Ca++) are required for the formation of this state if the vesicular and/or planar membrane contain negatively charged lipids. In the second step, the actual fusion of vesicular and planar membranes occurs. The driving force for this step is the osmotic swelling of vesicles attached (in the pre- fusion state) to the planar membrane. We suggest that osmotic swelling of vesicles may also be crucial for biological fusion and exocytosis. PMID:6699082

  9. Membrane growth can generate a transmembrane pH gradient in fatty acid vesicles.

    PubMed

    Chen, Irene A; Szostak, Jack W

    2004-05-25

    Electrochemical proton gradients are the basis of energy transduction in modern cells, and may have played important roles in even the earliest cell-like structures. We have investigated the conditions under which pH gradients are maintained across the membranes of fatty acid vesicles, a model of early cell membranes. We show that pH gradients across such membranes decay rapidly in the presence of alkali-metal cations, but can be maintained in the absence of permeable cations. Under such conditions, when fatty acid vesicles grow through the incorporation of additional fatty acid, a transmembrane pH gradient is spontaneously generated. The formation of this pH gradient captures some of the energy released during membrane growth, but also opposes and limits further membrane area increase. The coupling of membrane growth to energy storage could have provided a growth advantage to early cells, once the membrane composition had evolved to allow the maintenance of stable pH gradients.

  10. The structure of the COPII transport-vesicle coat assembled on membranes

    PubMed Central

    Zanetti, Giulia; Prinz, Simone; Daum, Sebastian; Meister, Annette; Schekman, Randy; Bacia, Kirsten; Briggs, John AG

    2013-01-01

    Coat protein complex II (COPII) mediates formation of the membrane vesicles that export newly synthesised proteins from the endoplasmic reticulum. The inner COPII proteins bind to cargo and membrane, linking them to the outer COPII components that form a cage around the vesicle. Regulated flexibility in coat architecture is essential for transport of a variety of differently sized cargoes, but structural data on the assembled coat has not been available. We have used cryo-electron tomography and subtomogram averaging to determine the structure of the complete, membrane-assembled COPII coat. We describe a novel arrangement of the outer coat and find that the inner coat can assemble into regular lattices. The data reveal how coat subunits interact with one another and with the membrane, suggesting how coordinated assembly of inner and outer coats can mediate and regulate packaging of vesicles ranging from small spheres to large tubular carriers. DOI: http://dx.doi.org/10.7554/eLife.00951.001 PMID:24062940

  11. Dynamics of Crowded Vesicles: Local and Global Responses to Membrane Composition

    PubMed Central

    Holdbrook, Daniel A.; Huber, Roland G.; Piggot, Thomas J.; Bond, Peter J.; Khalid, Syma

    2016-01-01

    The bacterial cell envelope is composed of a mixture of different lipids and proteins, making it an inherently complex organelle. The interactions between integral membrane proteins and lipids are crucial for their respective spatial localization within bacterial cells. We have employed microsecond timescale coarse-grained molecular dynamics simulations of vesicles of varying sizes and with a range of protein and lipid compositions, and used novel approaches to measure both local and global system dynamics, the latter based on spherical harmonics analysis. Our results suggest that both hydrophobic mismatch, enhanced by embedded membrane proteins, and curvature based sorting, due to different modes of undulation, may drive assembly in vesicular systems. Interestingly, the modes of undulation of the vesicles were found to be altered by the specific protein and lipid composition of the vesicle. Strikingly, lipid dynamics were shown to be coupled to proteins up to 6 nm from their surface, a substantially larger distance than has previously been observed, resulting in multi-layered annular rings enriched with particular types of phospholipid. Such large protein-lipid complexes may provide a mechanism for long-range communication. Given the complexity of bacterial membranes, our results suggest that subtle changes in lipid composition may have major implications for lipid and protein sorting under a curvature-based membrane-sorting model. PMID:27310814

  12. Chemical modifications to vesicle forming diblock copolymers: Development of smart functional polymersome membranes

    NASA Astrophysics Data System (ADS)

    Katz, Joshua S.

    2011-07-01

    A major limitation to current treatment regimens for diseases is the inability to adequately deliver therapeutics. Many routes to encapsulation of these materials have been explored to improve biodistribution and better protect encapsulants from harsh biological conditions. One vehicle particularly attractive for encapsulation of such materials is the polymersome. While promising for translation to clinical use, there are still limitations in polymer chemistry and resulting polymersome behavior that will slow their adaptation. This thesis addresses several of these limitations. The first major limitation to polymersomes is lack of control over their release rate. Release is generally by simple diffusion, leading to a burst. To address this burst, Aim 1 proposes a route to stabilizing polymersome membranes through their polymerization. PCL-PEG copolymers were terminally acrylated and the acrylates polymerized in the membrane following vesicle assembly. Polymerization enhanced mechanical robustness of the membranes and reduced diffusion of encapsulated contents. To ultimately trigger release, Aim 2 presents a novel route to synthesizing diblock copolymers, enabling insertion of a functional group at the blocks' junction. To facilitate triggering of release, we inserted UV-cleavable 2-nitrophenylalanine. Polymersomes assembled from this polymer collapse upon exposure to light and molecules release. Demonstrating further utility of this synthetic route, fluorescent vesicles were prepared using fluorescent lysine as the joining molecule. These vesicles labeled dendritic cells, providing a novel route to cell labeling and tracking. The second limitation to vesicles promising for biomedical applications (made of PCL-PEG) is their solid membranes. Aim 3 demonstrates partial (or full) replacement of the PCL block with a caprolactone analogue, TOSUO, which is non-crystalline and assembles into soft, deformable vesicles. Increasing TOSUO content in the copolymer leads to

  13. Comparative Transport Activity of Intact Cells, Membrane Vesicles, and Mesosomes of Bacillus licheniformis

    PubMed Central

    MacLeod, Robert A.; Thurman, Paul; Rogers, H. J.

    1973-01-01

    Sodium ion was shown to stimulate strongly the transport of l-glutamic acid into cells of Bacillus licheniformis 6346 His−. Lithium ion had a slight capacity to replace Na+ in this capacity, but K+ was without effect. Three of five amino acids tested. l-glutamic acid, l-aspartic acid, and l-alanine, were concentrated against a gradient in the cells. Intracellular pools of these amino acids were extractable with 5% trichloroacetic acid. Pools of l-histidine and l-lysine could not be detected. No evidence of active transport of lysine into cells could be detected, and histidine was taken up in the absence of chloramphenicol but not in its presence. The uptake of glutamic acid by membrane vesicle preparations was strongly stimulated by reduced nicotinamide adenine dinucleotide (NADH) and to a lesser extent by succinate. The presence of phenazine methosulfate increased uptake in the presence of succinate. Either l- or d-lactate and adenosine triphosphate were without effect. None of these compounds stimulated the uptake of glutamic acid by mesosomes, although some mesosome preparations contained separable membrane which was very active. NADH strongly stimulated the uptake of aspartic acid and alanine by membrane vesicles but had only a slight effect on the uptake of histidine and lysine. No evidence of active transport of any of the amino acids into mesosomes could be detected either in the presence or absence of NADH. NADH stimulation of the uptake of glutamic acid by membrane vesicles was destroyed by exposure to light of 360 nm; this inactivation was reversible by vitamin K2(5) or K2(10). Sodium ion stimulated transport of glutamic acid by membrane vesicles. PMID:4347247

  14. Direct Visualization of the Action of Triton X-100 on Giant Vesicles of Erythrocyte Membrane Lipids

    PubMed Central

    Casadei, Bruna R.; Domingues, Cleyton C.; de Paula, Eneida; Riske, Karin A.

    2014-01-01

    The raft hypothesis proposes that microdomains enriched in sphingolipids, cholesterol, and specific proteins are transiently formed to accomplish important cellular tasks. Equivocally, detergent-resistant membranes were initially assumed to be identical to membrane rafts, because of similarities between their compositions. In fact, the impact of detergents in membrane organization is still controversial. Here, we use phase contrast and fluorescence microscopy to observe giant unilamellar vesicles (GUVs) made of erythrocyte membrane lipids (erythro-GUVs) when exposed to the detergent Triton X-100 (TX-100). We clearly show that TX-100 has a restructuring action on biomembranes. Contact with TX-100 readily induces domain formation on the previously homogeneous membrane of erythro-GUVs at physiological and room temperatures. The shape and dynamics of the formed domains point to liquid-ordered/liquid-disordered (Lo/Ld) phase separation, typically found in raft-like ternary lipid mixtures. The Ld domains are then separated from the original vesicle and completely solubilized by TX-100. The insoluble vesicle left, in the Lo phase, represents around 2/3 of the original vesicle surface at room temperature and decreases to almost 1/2 at physiological temperature. This chain of events could be entirely reproduced with biomimetic GUVs of a simple ternary lipid mixture, 2:1:2 POPC/SM/chol (phosphatidylcholine/sphyngomyelin/cholesterol), showing that this behavior will arise because of fundamental physicochemical properties of simple lipid mixtures. This work provides direct visualization of TX-100-induced domain formation followed by selective (Ld phase) solubilization in a model system with a complex biological lipid composition. PMID:24896120

  15. Ferlins: regulators of vesicle fusion for auditory neurotransmission, receptor trafficking and membrane repair.

    PubMed

    Lek, Angela; Evesson, Frances J; Sutton, R Bryan; North, Kathryn N; Cooper, Sandra T

    2012-02-01

    Ferlins are a family of multiple C2 domain proteins with emerging roles in vesicle fusion and membrane trafficking. Ferlin mutations are associated with muscular dystrophy (dysferlin) and deafness (otoferlin) in humans, and infertility in Caenorhabditis elegans (Fer-1) and Drosophila (misfire), demonstrating their importance for normal cellular functioning. Ferlins show ancient origins in eukaryotic evolution and are detected in all eukaryotic kingdoms, including unicellular eukaryotes and apicomplexian protists, suggesting origins in a common ancestor predating eukaryotic evolutionary branching. The characteristic feature of the ferlin family is their multiple tandem cytosolic C2 domains (five to seven C2 domains), the most of any protein family, and an extremely rare feature amongst eukaryotic proteins. Ferlins also bear a unique nested DysF domain and small conserved 60-70 residue ferlin-specific sequences (Fer domains). Ferlins segregate into two subtypes based on the presence (type I ferlin) or absence (type II ferlin) of the DysF and FerA domains. Ferlins have diverse tissue-specific and developmental expression patterns, with ferlin animal models united by pathologies arising from defects in vesicle fusion. Consistent with their proposed role in vesicle trafficking, ferlin interaction partners include cytoskeletal motors, other vesicle-associated trafficking proteins and transmembrane receptors or channels. Herein we summarize the research history of the ferlins, an intriguing family of structurally conserved proteins with a preserved ancestral function as regulators of vesicle fusion and receptor trafficking.

  16. Model Systems of Precursor Cellular Membranes: Long-Chain Alcohols Stabilize Spontaneously Formed Oleic Acid Vesicles

    PubMed Central

    Rendón, Adela; Carton, David Gil; Sot, Jesús; García-Pacios, Marcos; Montes, Ruth; Valle, Mikel; Arrondo, José-Luis R.; Goñi, Felix M.; Ruiz-Mirazo, Kepa

    2012-01-01

    Oleic acid vesicles have been used as model systems to study the properties of membranes that could be the evolutionary precursors of more complex, stable, and impermeable phospholipid biomembranes. Pure fatty acid vesicles in general show high sensitivity to ionic strength and pH variation, but there is growing evidence that this lack of stability can be counterbalanced through mixtures with other amphiphilic or surfactant compounds. Here, we present a systematic experimental analysis of the oleic acid system and explore the spontaneous formation of vesicles under different conditions, as well as the effects that alcohols and alkanes may have in the process. Our results support the hypothesis that alcohols (in particular 10- to 14-C-atom alcohols) contribute to the stability of oleic acid vesicles under a wider range of experimental conditions. Moreover, studies of mixed oleic-acid-alkane and oleic-acid-alcohol systems using infrared spectroscopy and Langmuir trough measurements indicate that precisely those alcohols that increased vesicle stability also decreased the mobility of oleic acid polar headgroups, as well as the area/molecule of lipid. PMID:22339864

  17. Effect of medium-chain glycerides on the membrane transport of D-glucose and sulfanilic acid in the intestinal brush-border membrane vesicles.

    PubMed

    Sagara, K; Higaki, K; Yamazaki, A; Hashida, M; Sezaki, H

    1990-01-01

    To clarify the influence of medium-chain glycerides (MCG) on a biological membrane, we investigated the membrane transport of D-glucose and sulfanilic acid in the brush-border membrane (BBM) vesicles pretreated with MCG. The size distribution of the BBM vesicles determined by electron microscopic observation was not significantly different between the vesicles incorporated with MCG and those of the control. However, the amount of D-glucose taken up by the vesicles at an equilibrated stage (30 min) was significantly decreased in the MCG-treated ones based on unit content of protein. Based on these results we estimated the membrane transport of D-glucose and sulfanilic acid in consideration of vesiculation or filter-capturing efficiency in MCG-treated vesicles. The rates of Na+ gradient-independent D-glucose transport and sulfanilic acid transport were significantly greater in MCG-treated vesicles than in the control. On the other hand, the magnitude of overshooting effect in Na+ gradient-dependent uptake of D-glucose in MCG-treated vesicles was maintained similar to the control. Comparison of kinetic parameters for active D-glucose transport at different concentrations indicated that Km and Vmax were not significantly different between MCG-treated and the control vesicles. These results indicated that passive diffusion of D-glucose and sulfanilic acid was significantly increased but Na(+)-glucose cotransporter was not significantly changed by the incorporation of MCG in the intestinal BBM vesicles.

  18. Subfractionation, characterization and in-depth proteomic analysis of glomerular membrane vesicles in human urine

    PubMed Central

    Hogan, Marie C.; Johnson, Kenneth L.; Zenka, Roman M.; Charlesworth, M. Cristine; Madden, Benjamin J.; Mahoney, Doug W.; Oberg, Ann L.; Huang, Bing Q.; Nesbitt, Lisa L.; Bakeberg, Jason L.; Bergen, H. Robert; Ward, Christopher J.

    2014-01-01

    Urinary exosome-like vesicles (ELVs) are a heterogenous mixture (diameter 40–200nm) containing vesicles shed from all segments of the nephron including glomerular podocytes. Contamination with Tamm Horsfall protein (THP) oligomers has hampered their isolation and proteomic analysis. Here we improved ELV isolation protocols employing density centrifugation to remove THP and albumin, and isolated a glomerular membranous vesicle (GMV) enriched subfraction from 7 individuals identifying 1830 proteins and in 3 patients with glomerular disease identifying 5657 unique proteins. The GMV fraction was composed of podocin/podocalyxin positive irregularly shaped membranous vesicles and podocin/podocalyxin negative classical exosomes. Ingenuity pathway analysis identified integrin, actin cytoskeleton and RhoGDI signaling in the top three canonical represented signaling pathways and 19 other proteins associated with inherited glomerular diseases. The GMVs are of podocyte origin and the density gradient technique allowed isolation in a reproducible manner. We show many nephrotic syndrome proteins, proteases and complement proteins involved in glomerular disease are in GMVs and some were shed in the disease state (nephrin, TRPC6 and INF2 and PLA2R). We calculated sample sizes required to identify new glomerular disease biomarkers, expand the ELV proteome and provide a reference proteome in a database that may prove useful in the search for biomarkers of glomerular disease. PMID:24196483

  19. Dense core secretory vesicles revealed as a dynamic Ca2+ store in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera

    PubMed Central

    Mitchell, Kathryn J.; Pinton, Paolo; Varadi, Aniko; Tacchetti, Carlo; Ainscow, Edward K.; Pozzan, Tullio; Rizzuto, Rosario; Rutter, Guy A.

    2001-01-01

    The role of dense core secretory vesicles in the control of cytosolic-free Ca2+ concentrations ([Ca2+]c) in neuronal and neuroendocrine cells is enigmatic. By constructing a vesicle-associated membrane protein 2–synaptobrevin.aequorin chimera, we show that in clonal pancreatic islet β-cells: (a) increases in [Ca2+]c cause a prompt increase in intravesicular-free Ca2+ concentration ([Ca2+]SV), which is mediated by a P-type Ca2+-ATPase distinct from the sarco(endo) plasmic reticulum Ca2+-ATPase, but which may be related to the PMR1/ATP2C1 family of Ca2+ pumps; (b) steady state Ca2+ concentrations are 3–5-fold lower in secretory vesicles than in the endoplasmic reticulum (ER) or Golgi apparatus, suggesting the existence of tightly bound and more rapidly exchanging pools of Ca2+; (c) inositol (1,4,5) trisphosphate has no impact on [Ca2+]SV in intact or permeabilized cells; and (d) ryanodine receptor (RyR) activation with caffeine or 4-chloro-3-ethylphenol in intact cells, or cyclic ADPribose in permeabilized cells, causes a dramatic fall in [Ca2+]SV. Thus, secretory vesicles represent a dynamic Ca2+ store in neuroendocrine cells, whose characteristics are in part distinct from the ER/Golgi apparatus. The presence of RyRs on secretory vesicles suggests that local Ca2+-induced Ca2+ release from vesicles docked at the plasma membrane could participate in triggering exocytosis. PMID:11571310

  20. Dense core secretory vesicles revealed as a dynamic Ca(2+) store in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera.

    PubMed

    Mitchell, K J; Pinton, P; Varadi, A; Tacchetti, C; Ainscow, E K; Pozzan, T; Rizzuto, R; Rutter, G A

    2001-10-01

    The role of dense core secretory vesicles in the control of cytosolic-free Ca(2+) concentrations ([Ca(2+)](c)) in neuronal and neuroendocrine cells is enigmatic. By constructing a vesicle-associated membrane protein 2-synaptobrevin.aequorin chimera, we show that in clonal pancreatic islet beta-cells: (a) increases in [Ca(2+)](c) cause a prompt increase in intravesicular-free Ca(2+) concentration ([Ca(2+)]SV), which is mediated by a P-type Ca(2+)-ATPase distinct from the sarco(endo) plasmic reticulum Ca(2+)-ATPase, but which may be related to the PMR1/ATP2C1 family of Ca(2+) pumps; (b) steady state Ca(2+) concentrations are 3-5-fold lower in secretory vesicles than in the endoplasmic reticulum (ER) or Golgi apparatus, suggesting the existence of tightly bound and more rapidly exchanging pools of Ca(2+); (c) inositol (1,4,5) trisphosphate has no impact on [Ca(2+)](SV) in intact or permeabilized cells; and (d) ryanodine receptor (RyR) activation with caffeine or 4-chloro-3-ethylphenol in intact cells, or cyclic ADPribose in permeabilized cells, causes a dramatic fall in [Ca(2+)](SV). Thus, secretory vesicles represent a dynamic Ca(2+) store in neuroendocrine cells, whose characteristics are in part distinct from the ER/Golgi apparatus. The presence of RyRs on secretory vesicles suggests that local Ca(2+)-induced Ca(2+) release from vesicles docked at the plasma membrane could participate in triggering exocytosis.

  1. Urothelial endocytic vesicle recycling and lysosomal degradative pathway regulated by lipid membrane composition.

    PubMed

    Grasso, E J; Calderón, R O

    2013-02-01

    The urothelium, a specialized epithelium that covers the mucosa cell surface of the urinary bladder, undergoes dramatic morphological changes during the micturition cycle that involve a membrane apical traffic. This traffic was first described as a lysosomal pathway, in addition to the known endocytosis/exocytosis membrane recycling. In an attempt to understand the role of membrane lipid composition in those effects, we previously described the lipid-dependent leakage of the endocytosed vesicle content. In this work, we demonstrated clear differences in the traffic of both the fluid probe and the membrane-bound probe in urothelial umbrella cells by using spectrofluorometry and/or confocal and epifluorescence microscopy. Different membrane lipid compositions were established by using three diet formulae enriched in oleic acid, linoleic acid and a commercial formula. Between three and five animals for each dietary treatment were used for each analysis. The decreased endocytosis of both fluid and membrane-bound probes (approximately 32 and 49 % lower, respectively) in oleic acid-derived umbrella cells was concomitant with an increased recycling (approximately 4.0 and 3.7 times, respectively) and diminished sorting to the lysosome (approximately 23 and 37 %, respectively) when compared with the control umbrella cells. The higher intravesicular pH and the impairment of the lysosomal pathway of oleic acid diet-derived vesicles compared to linoleic acid diet-derived vesicles and control diet-derived vesicles correlate with our findings of a lower V-ATPase activity previously reported. We integrated the results obtained in the present and previous work to determine the sorting of endocytosed material (fluid and membrane-bound probes) into the different cell compartments. Finally, the weighted average effect of the individual alterations on the intracellular distribution was evaluated. The results shown in this work add evidences for the modulatory role of the membrane

  2. The Xylella fastidiosa PD1063 protein is secreted in association with outer membrane vesicles.

    PubMed

    Pierce, Brittany K; Voegel, Tanja; Kirkpatrick, Bruce C

    2014-01-01

    Xylella fastidiosa is a gram-negative, xylem-limited plant pathogenic bacterium that causes disease in a variety of economically important agricultural crops including Pierce's disease of grapevines. Xylella fastidiosa biofilms formed in the xylem vessels of plants play a key role in early colonization and pathogenicity by providing a protected niche and enhanced cell survival. Here we investigate the role of Xylella fastidiosa PD1063, the predicted ortholog of Xanthomonas oryzae pv. oryzae PXO_03968, which encodes an outer membrane protein. To assess the function of the Xylella fastidiosa ortholog, we created Xylella fastidiosa mutants deleted for PD1063 and then assessed biofilm formation, cell-cell aggregation and cell growth in vitro. We also assessed disease severity and pathogen titers in grapevines mechanically inoculated with the Xylella fastidiosa PD1063 mutant. We found a significant decrease in cell-cell aggregation among PD1063 mutants but no differences in cell growth, biofilm formation, disease severity or titers in planta. Based on the demonstration that Xanthomonas oryzae pv. oryzae PXO_03968 encodes an outer membrane protein, secreted in association with outer membrane vesicles, we predicted that PD1063 would also be secreted in a similar manner. Using anti-PD1063 antibodies, we found PD1063 in the supernatant and secreted in association with outer membrane vesicles. PD1063 purified from the supernatant, outer membrane fractions and outer membrane vesicles was 19.2 kD, corresponding to the predicted size of the processed protein. Our findings suggest Xylella fastidiosa PD1063 is not essential for development of Pierce's disease in Vitis vinifera grapevines although further research is required to determine the function of the PD1063 outer membrane protein in Xylella fastidiosa. PMID:25426629

  3. H+ V-ATPase-Energized Transporters in Brush Border Membrane Vesicles from Whole Larvae of Aedes Aegypti

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Brush Border Membrane vesicles (BBMVs) from Whole larvae of Aedes aegypti (AeBBMVWs ) contain an H+ V-ATPase (V), a Na+/H+ antiporter, NHA1 (A) and a Na+-coupled, nutrient amino acid transporter, NAT8 (N), VAN for short. All V-ATPase subunits are present in the Ae. aegypti genome and in the vesicles...

  4. Interfacial effects on droplet electrohydrodynamics: particle vortices, patchy membranes, and vesicle drums

    NASA Astrophysics Data System (ADS)

    Vlahovska, Petia

    2014-11-01

    The analytical work by Acrivos group on drop dynamics in linear flows and rheology of dilute emulsions (papers with Frankel and Barthes-Biesel) have provided solid basis for more than 40 years of research on drops and capsules. These classical papers have inspired my research on drops with ``complex'' interfaces - surfactant-laden and particle-covered drops, and vesicles (drops encapsulated with lipid bilayer membranes). I will present some of our recent experimental observations on these systems in uniform DC and AC electric fields, where the coupling of the electric-field-induced flow and complex mechanics of the interface drives peculiar (and yet to be explained) behaviors: drum-like and asymmetric dumbbell shapes of vesicles; domains formation and motion in multicomponent membranes; particle assembly in dynamic vortices; drop kayaking. Possible implications of our findings to the design of patchy particles and electrorheology of emulsions will be discussed. Supported by NSF-CBET 1132614.

  5. Lipid binding and membrane penetration of polymyxin B derivatives studied in a biomimetic vesicle system.

    PubMed Central

    Katz, Marina; Tsubery, Haim; Kolusheva, Sofiya; Shames, Alex; Fridkin, Mati; Jelinek, Raz

    2003-01-01

    Understanding membrane interactions and cell-wall permeation of Gram-negative bacteria is of great importance, owing to increasing bacterial resistance to existing drugs and therapeutic treatments. Here we use biomimetic lipid vesicles to analyse membrane association and penetration by synthetic derivatives of polymyxin B (PMB), a potent naturally occurring antibacterial cyclic peptide. The PMB analogues studied were PMB nonapeptide (PMBN), in which the hydrophobic alkyl residue was cleaved, PMBN diastereomer containing D-instead of L-amino acids within the cyclic ring (dPMBN) and PMBN where the hydrophobic alkyl chain was replaced with an Ala6 repeat (Ala6-PMBN). Peptide binding measurements, colorimetric transitions induced within the vesicles, fluorescence quenching experiments and ESR spectroscopy were applied to investigate the structural parameters underlying the different membrane-permeation profiles and biological activities of the analogues. The experiments point to the role of negatively charged lipids in membrane binding and confirm the prominence of lipopolisaccharide (LPS) in promoting membrane association and penetration by the peptides. Examination of the lipid interactions of the PMB derivatives shows that the cyclic moiety of PMB is not only implicated in lipid attachment and LPS binding, but also affects penetration into the inner bilayer core. The addition of the Ala6 peptide moiety, however, does not significantly promote peptide insertion into the hydrophobic lipid environment. The data also indicate that the extent of penetration into the lipid bilayer is not related to the overall affinity of the peptides to the membrane. PMID:12848621

  6. Preparation of biocompatible zwitterionic block copolymer vesicles by direct dissolution in water and subsequent silicification within their membranes.

    PubMed

    Du, Jianzhong; Armes, Steven P

    2009-08-18

    The facile preparation of block copolymer vesicles in pure water and their subsequent stabilization by sol-gel chemistry within the vesicle membrane is described. An amphiphilic biocompatible zwitterionic diblock copolymer, poly(epsilon-caprolactone)-block-poly[2-(methacryloyloxy)ethyl phosphorylcholine], PCL-b-PMPC, was synthesized by (i) ring-opening polymerization of epsilon-caprolactone, (ii) end-group modification by esterification, and (iii) atom transfer radical polymerization (ATRP) of 2-(methacryloyloxy)ethyl phosphorylcholine (MPC). Unusually, block copolymer vesicles were formed instantly upon adding dried copolymer powder into hot water without using organic cosolvents, pH adjustment, or even stirring. This protocol is much more convenient than previously reported methods such as solvent-switching and film rehydration. The PCL vesicle membrane is moderately hydrophobic and fully biodegradable. The highly biocompatible PMPC chains are expressed on both the exterior and interior surface of the membrane. These vesicles can be stabilized by aqueous sol-gel chemistry within the hydrophobic PCL vesicle membrane by using tetramethyl orthosilicate (TMOS) as the silica precursor in the absence of any external catalyst. The water-immiscible TMOS precursor is initially solubilized within the hydrophobic membrane prior to its in situ transformation into silica. The vesicles were characterized by 1H NMR spectroscopy, atomic force microscopy, transmission electron microscopy, and dynamic light scattering.

  7. VESICULAR TRANSPORT. A structure of the COPI coat and the role of coat proteins in membrane vesicle assembly.

    PubMed

    Dodonova, S O; Diestelkoetter-Bachert, P; von Appen, A; Hagen, W J H; Beck, R; Beck, M; Wieland, F; Briggs, J A G

    2015-07-10

    Transport of material within cells is mediated by trafficking vesicles that bud from one cellular compartment and fuse with another. Formation of a trafficking vesicle is driven by membrane coats that localize cargo and polymerize into cages to bend the membrane. Although extensive structural information is available for components of these coats, the heterogeneity of trafficking vesicles has prevented an understanding of how complete membrane coats assemble on the membrane. We combined cryo-electron tomography, subtomogram averaging, and cross-linking mass spectrometry to derive a complete model of the assembled coat protein complex I (COPI) coat involved in traffic between the Golgi and the endoplasmic reticulum. The highly interconnected COPI coat structure contradicted the current "adaptor-and-cage" understanding of coated vesicle formation.

  8. Characterization of outer membrane vesicles released by the psychrotolerant bacterium Pseudoalteromonas antarctica NF3

    PubMed Central

    Nevot, Maria; Deroncelé, Víctor; Messner, Paul; Guinea, Jesús; Mercadé, Elena

    2015-01-01

    Summary Pseudoalteromonas antarctica NF3 is an Antarctic psychrotolerant Gram-negative bacterium that accumulates large amounts of an extracellular polymeric substance (EPS) with high protein content. Transmission electron microscopy analysis after high-pressure freezing and freeze substitution (HPF-FS) shows that the EPS is composed of a capsular polymer and large numbers of outer membrane vesicles (OMVs). These vesicles are bilayered structures and predominantly spherical in shape, with an average diameter of 25–70 nm, which is similar to what has been observed in OMVs from other Gram-negative bacteria. Analyses of lipopolysaccharide (LPS), phospholipids and protein profiles of OMVs are consistent with the bacterial outer membrane origin of these vesicles. In an initial attempt to elucidate the functions of OMVs proteins, we conducted a proteomic analysis on 1D SDS-PAGE bands. Those proteins putatively identified match with outer membrane proteins and proteins related to nutrient processing and transport in Gram-negative bacteria. This approach suggests that OMVs present in the EPS from P. antarctica NF3, might function to deliver proteins to the external media, and therefore play an important role in the survival of the bacterium in the extreme Antarctic environment. PMID:16913913

  9. Deciphering How Pore Formation Causes Strain-Induced Membrane Lysis of Lipid Vesicles.

    PubMed

    Jackman, Joshua A; Goh, Haw Zan; Zhdanov, Vladimir P; Knoll, Wolfgang; Cho, Nam-Joon

    2016-02-01

    Pore formation by membrane-active antimicrobial peptides is a classic strategy of pathogen inactivation through disruption of membrane biochemical gradients. It remains unknown why some membrane-active peptides also inhibit enveloped viruses, which do not depend on biochemical gradients. Here, we employ a label-free biosensing approach based on simultaneous quartz crystal microbalance-dissipation and ellipsometry measurements in order to investigate how a pore-forming, virucidal peptide destabilizes lipid vesicles in a surface-based experimental configuration. A key advantage of the approach is that it enables direct kinetic measurement of the surface-bound peptide-to-lipid (P:L) ratio. Comprehensive experiments involving different bulk peptide concentrations and biologically relevant membrane compositions support a unified model that membrane lysis occurs at or above a critical P:L ratio, which is at least several-fold greater than the value corresponding to the onset of pore formation. That is consistent with peptide-induced pores causing additional membrane strain that leads to lysis of highly curved membranes. Collectively, the work presents a new model that describes how peptide-induced pores may destabilize lipid membranes through a membrane strain-related lytic process, and this knowledge has important implications for the design and application of membrane-active peptides.

  10. The endoplasmic reticulum and casein-containing vesicles contribute to milk fat globule membrane.

    PubMed

    Honvo-Houéto, Edith; Henry, Céline; Chat, Sophie; Layani, Sarah; Truchet, Sandrine

    2016-10-01

    During lactation, mammary epithelial cells secrete huge amounts of milk from their apical side. The current view is that caseins are secreted by exocytosis, whereas milk fat globules are released by budding, enwrapped by the plasma membrane. Owing to the number and large size of milk fat globules, the membrane surface needed for their release might exceed that of the apical plasma membrane. A large-scale proteomics analysis of both cytoplasmic lipid droplets and secreted milk fat globule membranes was used to decipher the cellular origins of the milk fat globule membrane. Surprisingly, differential analysis of protein profiles of these two organelles strongly suggest that, in addition to the plasma membrane, the endoplasmic reticulum and the secretory vesicles contribute to the milk fat globule membrane. Analysis of membrane-associated and raft microdomain proteins reinforces this possibility and also points to a role for lipid rafts in milk product secretion. Our results provide evidence for a significant contribution of the endoplasmic reticulum to the milk fat globule membrane and a role for SNAREs in membrane dynamics during milk secretion. These novel aspects point to a more complex model for milk secretion than currently envisioned.

  11. Uptake of barbituric acid derivatives in small intestinal brush border membrane vesicles from retinyl palmitate-treated rats.

    PubMed

    Tanii, H; Horie, T

    2000-08-01

    Brush border membrane was prepared from the small intestinal (jejunum) cells along the crypt-villus axis. The fluorescence spectra of 1,8-anilinonaphthalene sulfonic acid and the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene were measured in the brush border membrane vesicle suspension. The hydrophobicity of brush border membrane was found to be in the order villus tip >mid villus >lower villus. The fluidity of brush border membrane was in the order villus tip membrane vesicles was well correlated with their partition coefficients (isopentyl acetate/water). No significant difference was observed between the uptake of hexobarbital by brush border membrane vesicles from the villus tip and lower villus. When retinyl palmitate was administered to rats, the fluidity of brush border membrane was found to be higher in the retinyl palmitate-treated rats than in the control rats. However, no significant difference in the uptake of hexobarbital by brush border membrane vesicles was observed between the retinyl palmitate-administered rats and the control rats. Thus, the retinyl palmitate treatment seems unlikely to affect the passively transported ligands like barbituric acid derivatives in brush border membrane vesicles. PMID:10989945

  12. Surfactant-Increased Glyphosate Uptake into Plasma Membrane Vesicles Isolated from Common Lambsquarters Leaves.

    PubMed Central

    Riechers, D. E.; Wax, L. M.; Liebl, R. A.; Bush, D. R.

    1994-01-01

    Plasma membrane vesicles were isolated from mature leaves of lambsquarters (Chenopodium album L.) to investigate whether this membrane is a barrier to glyphosate uptake and whether surfactants possess differential abilities to enhance glyphosate permeability. Amino acids representing several structural classes showed [delta]pH-dependent transport, indicating that the proteins necessary for active, proton-coupled amino acid transport were present and functional. Glyphosate uptake was very low compared to the acidic amino acid glutamate, indicating that glyphosate is not utilizing an endogenous amino acid carrier to enter the leaf cells and that the plasma membrane appears to be a significant barrier to cellular uptake. In addition, glyphosate flux was much lower than that measured for either bentazon or atrazine, both lipid-permeable herbicides that diffuse through the bilayer. Glyphosate uptake was stimulated by 0.01% (v:v) MON 0818, the cationic surfactant used in the commercial formulation of this herbicide for foliar application. This concentration of surfactant did not disrupt the integrity of the plasma membrane vesicles, as evidenced by the stability of imposed pH gradients and active amino acid transport. Nonionic surfactants that disrupt the cuticle but that do not promote glyphosate toxicity in the field also increased glyphosate transport into the membrane vesicles. Thus, no correlation was observed between whole plant toxicity and surfactant-aided uptake. Current data suggest that surfactant efficacy may be the result of charged surfactants' ability to diffuse away from the cuticle into the subtending apoplastic space, where they act directly on the plasma membrane to increase glyphosate uptake. PMID:12232297

  13. The mechanism of uptake of retinol by plasma-membrane vesicles.

    PubMed Central

    Sivaprasadarao, A; Findlay, J B

    1988-01-01

    The mechanism of retinol uptake by human placental brush-border membrane vesicles was investigated using initial-velocity studies of [3H]retinol uptake from the [3H]retinol-RBP (retinol-binding protein) complex. The process was rapid and time- and temperature-dependent. The uptake was specifically reversed by the addition of native or apo-RBP, but not by serum albumin. By contrast, uptake of free [3H]retinol was temperature-independent, partially reversible and showed no requirement for a specific protein for reversibility. Treatment of membrane vesicles with p-chloromercuribenzenesulphonate (PCMBS), which inhibited 125I-RBP binding, also inhibited the uptake of retinol from RBP, but the uptake of free retinol was unaffected. Addition of PCMBS after the attainment of steady-state uptake equilibrium abolished the binding of RBP, but did not affect the retinol already taken up from RBP. The results suggest that binding of RBP to its specific receptor is obligatory for the subsequent delivery of retinol to the membrane. Since the studies were carried out on isolated membrane vesicles, endocytosis of RBP is most unlikely to be involved in the placental transport of retinol. A double-reciprocal plot of initial velocity versus [3H]retinol-RBP concentration gave an apparent Km of 116 +/- 13 nM. Transthyretin decreased the rate of uptake of [3H]retinol from RBP without substantially altering the steady-state uptake levels, suggesting that membranes take up retinol from uncomplexed RBP. High-pressure gel-filtration chromatography showed that [3H]retinol is largely transferred to a membrane component with an apparent molecular mass of 125 kDa. PMID:2849421

  14. Vesicle formation in the membrane of onion cells (Allium cepa) during rapid osmotic dehydration

    PubMed Central

    Assani, Akym; Moundanga, Sylvie; Beney, Laurent; Gervais, Patrick

    2009-01-01

    Background and Aims Optimization of osmotic dehydration in different plant cells has been investigated through the variation of parameters such as the nature of the sugar used, the concentration of osmotic solutions and the processing time. In micro-organisms such as the yeast, Saccharomyces cerevisiae, the exposure of a cell to a slow increase in osmotic pressure preserves cell viability after rehydration, while sudden dehydration involves a lower rate of cell viability, which could be due to membrane vesiculation. The aim of this work is to study cytoplasmic vesicle formation in onion epidermal cells (Allium cepa) as a function of the kinetics of osmotic pressure variation in the external medium. Methods Onion epidermal cells were submitted either to an osmotic shock or to a progressive osmotic shift from an osmotic pressure of 2 to 24 MPa to induce plasmolysis. After 30 min in the treatment solution, deplasmolysis was carried out. Cells were observed by microscopy during the whole cycle of dehydration–rehydration. Key Results The application of an osmotic shock to onion cells, from an initial osmotic pressure of 2 MPa to a final one of 24 MPa for <1 s, led to the formation of numerous exocytotic and osmocytic vesicles visualized through light and confocal microscopy. In contrast, after application of a progressive osmotic shift, from an initial osmotic pressure of 2 MPa to a final one of 24 MPa for 30 min, no vesicles were observed. Additionally, the absence of Hechtian strand connections led to the bursting of vesicles in the case of the osmotic shock. Conclusions It is concluded that the kinetics of osmotic dehydration strongly influence vesicle formation in onion cells, and that Hechtian strand connections between protoplasts and exocytotic vesicles are a prerequisite for successful deplasmolysis. These results suggest that a decrease in the area-to-volume ratio of a cell could cause cell death following an osmotic shock. PMID:19833611

  15. Epoxide-Mediated Differential Packaging of Cif and Other Virulence Factors into Outer Membrane Vesicles

    PubMed Central

    Ballok, Alicia E.; Filkins, Laura M.; Bomberger, Jennifer M.; Stanton, Bruce A.

    2014-01-01

    Pseudomonas aeruginosa produces outer membrane vesicles (OMVs) that contain a number of secreted bacterial proteins, including phospholipases, alkaline phosphatase, and the CFTR inhibitory factor (Cif). Previously, Cif, an epoxide hydrolase, was shown to be regulated at the transcriptional level by epoxides, which serve as ligands of the repressor, CifR. Here, we tested whether epoxides have an effect on Cif levels in OMVs. We showed that growth of P. aeruginosa in the presence of specific epoxides but not a hydrolysis product increased Cif packaging into OMVs in a CifR-independent fashion. The outer membrane protein, OprF, was also increased under these conditions, but alkaline phosphatase activity was not significantly altered. Additionally, we demonstrated that OMV shape and density were affected by epoxide treatment, with two distinct vesicle fractions present when cells were treated with epibromohydrin (EBH), a model epoxide. Vesicles isolated from the two density fractions exhibited different protein profiles in Western blotting and silver staining. We have shown that a variety of clinically or host-relevant treatments, including antibiotics, also alter the proteins packaged in OMVs. Proteomic analysis of purified OMVs followed by an analysis of transposon mutant OMVs yielded mutants with altered vesicle packaging. Finally, epithelial cell cytotoxicity was reduced in the vesicles formed in the presence of EBH, suggesting that this epoxide alters the function of the OMVs. Our data support a model whereby clinically or host-relevant signals mediate differential packaging of virulence factors in OMVs, which results in functional consequences for host-pathogen interactions. PMID:25112474

  16. Membrane vesicles of Clostridium perfringens Type A strains induce innate and adaptive immunity

    PubMed Central

    Jiang, Yanlong; Kong, Qingke; Roland, Kenneth L.; Curtiss, Roy

    2014-01-01

    Vesicle shedding from bacteria is a universal process in most Gram-negative bacteria and a few Gram-positive bacteria. In this report, we isolate extracellular membrane vesicles (MVs) from the supernatants of Gram-positive pathogen Clostridium perfringens (C. perfringens). We demonstrated vesicle production in a variety of virulent and nonvirulent type A strains. MVs did not contain alpha-toxin and NetB toxin demonstrated by negative reaction to specific antibody and absence of specific proteins identified by LC-MS/MS. C. perfringens MVs contained DNA components such as 16S ribosomal RNA gene (16S rRNA), alpha-toxin gene (plc) and the perfringolysin O gene (pfoA) demonstrated by PCR. We also identified a total of 431 proteins in vesicles by 1-D gel separation and LC-MS/MS analysis. In vitro studies demonstrated that vesicles could be internalized into murine macrophage RAW264.7 cells without direct cytotoxicity effects, causing release of inflammation cytokines including granulocyte colony stimulating factor (G-CSF), tumor necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1), which could also be detected in mice injected with MVs through intraperitoneal (i.p.) route. Mice immunized with C. perfringens MVs produced high titer IgG, especially IgG1, antibodies against C. perfringens membrane proteins. However, this kind of antibody could not provide protection in mice following challenge, though it could slightly postpone the time of death. Our results indicate that release of MVs from C. perfringens could provide a previously unknown mechanism to induce release of inflammatory cytokines, especially TNF-α, these findings may contribute to a better understanding of the pathogenesis of C. perfringens infection. PMID:24631214

  17. Rat hepatocytes exhibit basolateral Na+/HCO/sub 3/- cotransport

    SciTech Connect

    Renner, E.L.; Lake, J.R.; Scharschmidt, B.F.; Zimmerli, B.; Meier, P.J.

    1989-04-01

    Primary cultures and plasma membrane vesicles were used to characterize Na+ and HCO3- transport by rat hepatocytes. Na+ uptake into hepatocytes was stimulated approximately 10-fold by 25 mM extracellular HCO3-.HCO3--stimulated Na+ uptake was saturable, abolished by 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene (SITS), and unaffected by amiloride or Cl- removal. Neither propionate nor acetate reproduced this effect of HCO3-. 22Na efflux from preloaded hepatocytes was similarly increased approximately 10-fold by an in greater than out HCO3- concentration gradient. 22Na efflux was also increased by valinomycin and an in greater than out K+ concentration gradient in the presence but not absence of HCO3-. Intracellular pH (pHi) measured with the pH-sensitive fluorochrome 2',7'-bis-(2-carboxyethyl)-5-(and 6-)carboxyfluorescein (BCECF) decreased at a rate of 0.227 (+/- 0.074 SEM) pH units/min when extracellular HCO3- concentration was lowered from 25 to 5 mM at constant PCO2. This intracellular acidification rate was decreased 50-60% in the absence of Na+ or presence of SITS, and was unaffected by amiloride or Cl- removal. Membrane hyperpolarization produced by valinomycin and an in greater than out K+ concentration gradient caused pHi to fall; the rate of fall was decreased 50-70% by Na+ removal or SITS, but not amiloride. An inside positive K+ diffusion potential and a simultaneous out greater than in HCO3- gradient produced a transient 4,4'-diisothiocyano-2,2' disulfonic acid stilbene (DIDS) sensitive, amiloride-insensitive 22Na accumulation in basolateral but not canalicular membrane vesicles. Rat hepatocytes thus exhibit electrogenic basolateral Na+/HCO3- cotransport.

  18. Heterogeneity of auxin-accumulating membrane vesicles from Cucurbita and Zea: a possible reflection of cell polarity.

    PubMed

    Lützelschwab, M; Asard, H; Ingold, U; Hertel, R

    1989-03-01

    When microsomes from hypocotyls of Cucurbita pepo L. or coleoptiles of Zea mays L. were centrifuged on dextran-sucrose gradients a heterogeneity of auxin-accumulating vesicles was observed. Vesicles from the top part of the gradient showed saturable, specific accumulation of indole-3-acetic acid with only a small stimulation by phytotropins, and with very few binding sites for 1-N-naphthylphthalamic acid. In the vesicles from the lower part of the gradient, net accumulation of indole-3-acetic acid could be strongly increased by addition of phytotropins; binding of 1-N-naphthylphthalamic acid was high in this region. After two-phase partitioning, both kinds of vesicles were found in the upper-phase membrane fraction considered to be purified plasma membrane. The hypothesis is discussed that vesicles can be separated from the apical and basal parts of the cell's plasmalemma.

  19. Liposome-based engineering of cells to package hydrophobic compounds in membrane vesicles for tumor penetration.

    PubMed

    Lee, Junsung; Kim, Jiyoung; Jeong, Moonkyoung; Lee, Hyoungjin; Goh, Unbyeol; Kim, Hyaeyeong; Kim, Byungji; Park, Ji-Ho

    2015-05-13

    Natural membrane vesicles (MVs) derived from various types of cells play an essential role in transporting biological materials between cells. Here, we show that exogenous compounds are packaged in the MVs by engineering the parental cells via liposomes, and the MVs mediate autonomous intercellular migration of the compounds through multiple cancer cell layers. Hydrophobic compounds delivered selectively to the plasma membrane of cancer cells using synthetic membrane fusogenic liposomes were efficiently incorporated into the membrane of MVs secreted from the cells and then transferred to neighboring cells via the MVs. This liposome-mediated MV engineering strategy allowed hydrophobic photosensitizers to significantly penetrate both spheroids and in vivo tumors, thereby enhancing the therapeutic efficacy. These results suggest that innate biological transport systems can be in situ engineered via synthetic liposomes to guide the penetration of chemotherapeutics across challenging tissue barriers in solid tumors.

  20. Mechanism of glucose and maltose transport in plasma-membrane vesicles from the yeast Candida utilis.

    PubMed Central

    van den Broek, P J; van Gompel, A E; Luttik, M A; Pronk, J T; van Leeuwen, C C

    1997-01-01

    Transport of glucose and maltose was studied in plasma-membrane vesicles from Candida utilis. The yeast was grown on a mixture of glucose and maltose in aerobic carbon-limited continuous cultures which enabled transport to be studied for both sugars with the same vesicles. Vesicles were prepared by fusion of isolated plasma membranes with proteoliposomes containing bovine heart cytochrome c oxidase as a proton-motive-force-generating system. Addition of reduced cytochrome c generated a proton-motive force, consisting of a membrane potential, negative inside, and a pH gradient, alkaline inside. Energization led to accumulation of glucose and maltose in these vesicles, reaching accumulation ratios of about 40-50. Accumulation also occurred in the presence of valinomycin or nigericin, but was prevented by a combination of the two ionophores or by uncoupler, showing that glucose and maltose transport are dependent on the proton-motive force. Comparison of sugar accumulation with quantitative data on the proton-motive force indicated a 1:1 H+/sugar stoichiometry for both transport systems. Efflux of accumulated glucose was observed on dissipation of the proton-motive force. Exchange and counterflow experiments confirmed the reversible character of the H+-glucose symporter. In contrast, uncoupler or a mixture of valinomycin plus nigericin induced only a slow efflux of accumulated maltose. Moreover under counterflow conditions, the expected transient accumulation was small. Thus the H+-maltose symporter has some characteristics of a carrier that is not readily reversible. It is concluded that in C. utilis the transport systems for glucose and maltose are both driven by the proton-motive force, but the mechanisms are different. PMID:9020885

  1. A membrane vesicle-based assay to enable prediction of human biliary excretion.

    PubMed

    Colombo, Federico; Poirier, Hugo; Rioux, Nathalie; Montecillo, Maria Arias; Duan, Jianmin; Ribadeneira, Maria D

    2013-10-01

    1. Prediction of biliary excretion is a challenge due to the lack of in vitro assays. Our laboratory previously demonstrated a highly significant correlation between in vitro IC50 values against mrp2 using rat canalicular liver plasma membrane vesicles and in vivo biliary excretion (Colombo et al., 2012). This study explores the possibility of predicting in vivo biliary excretion in human using membrane vesicles prepared from MDCKII cells transfected with human ABCC2. 2. In vitro MRP2 activity was determined by measuring the ATP-dependent uptake of 5(6)-carboxy-2',7'-dichlorofluorescein (CDCF) in inside-out membrane vesicles isolated from MDCK-ABCC2 cells. CDCF uptake was time- and concentration-dependent (Km of 4.0 ± 1.2 µM and a Vmax of 7.8 ± 0.9 pmol/mg/min) and inhibited by benzbromarone and MK-571 with IC50 values of 1.2 and 7.6 µM, respectively. 3. A significant linear correlation (r(2 )= 0.790) between the in vitro IC50 values from the described MRP2 assay and in vivo biliary excretion in humans was observed using 11 well-documented drugs covering low to high biliary excretions. 4. This study demonstrates, for the first time, that inhibition of CDCF uptake in MDCKII-ABCC2 vesicles not only provides a screening assay to assess MRP2 drug-drug interaction potential, but is also predictive of human MRP2-mediated biliary excretion.

  2. Energy generation coupled to azoreduction by membranous vesicles from Shewanella decolorationis S12.

    PubMed

    Hong, Yi-Guo; Guo, Jun; Sun, Guo-Ping

    2009-01-01

    Previous studies have demonstrated that Shewanella decolorationis S12 can grow on the azo compound amaranth as the sole electron acceptor. Thus, to explore the mechanism of energy generation in this metabolism, membranous vesicles (MVs) were prepared and the mechanism of energy generation investigated. The membrane, which was fragmentized during preparation, automatically formed vesicles ranging from 37.5-112.5 nm in diameter under electron micrograph observation. Energy was conserved when coupling the azoreduction by the MVs of an azo compound or Fe(III) as the sole electron acceptor with H2, formate, or lactate as the electron donor. The amaranth reduction by the vesicles was found to be inhibited by specific respiratory inhibitors, including Cu(2+) ions, dicumarol, stigmatellin, and metyrapone, indicating that the azoreduction was indeed a respiration reaction. This finding was further confirmed by the fact that the ATP synthesis was repressed by the ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD). Therefore, this study offers solid evidence of a mechanism of microbial dissimilatory azoreduction on a subcell level.

  3. Proteomic and functional characterization of the outer membrane vesicles from the gastric pathogen Helicobacter pylori.

    PubMed

    Mullaney, Erica; Brown, Paul A; Smith, Sinead M; Botting, Catherine H; Yamaoka, Yoshio Y; Terres, Ana M; Kelleher, Dermot P; Windle, Henry J

    2009-07-01

    The gastric pathogen Helicobacter pylori causes a spectrum of gastro-duodenal diseases, which may be mediated in part by the outer membrane vesicles (OMVs) constitutively shed by the pathogen. We aimed to determine the proteome of H. pylori OMV to help evaluate the mechanisms whereby these structures confer their known immuno-modulatory and cytotoxic activities to host cells, as such disease-associated activities are also conferred by the bacterium from which the vesicles are derived. We also evaluated the effect of the OMV on gastric/colonic epithelial cells, duodenal explants and neutrophils. A proteomic analysis of the OMV proteins separated by SDS-PAGE from two strains of H. pylori (J99 and NCTC 11637) was undertaken and 162 OMV-associated proteins were identified in J99 and 91 in NCTC 11637 by LC-MS/MS. The vesicles are rich in membrane proteins, porins, adhesins and several molecules known to modulate chemokine secretion, cell proliferation and other host cellular processes. Further, the OMVs are also vehicles for the carriage of the cytotoxin-associated gene A cytotoxin in addition to the previously documented toxin, vacuolating cytotoxin. Taken together, it is evident from the proteome of H. pylori OMV that these structures are equipped with the molecules required to interact with host cells in a manner not dissimilar from the intact pathogen.

  4. Uptake mechanism of trientine by rat intestinal brush-border membrane vesicles.

    PubMed

    Tanabe, R; Kobayashi, M; Sugawara, M; Iseki, K; Miyazaki, K

    1996-05-01

    The uptake characteristics of trientine by rat intestinal brush-border membrane vesicles were studied. The uptake characteristics of trientine were similar to those of the physiological polyamines with respect to the excessive accumulation in vesicles, the pH dependency, the temperature dependency and the ineffectiveness of K+ diffusion potential (inside negative). The initial uptake of trientine was saturable with a K(m) value of 1.13 mM, which was larger than that of spermine and spermidine. Furthermore, the uptake rate of trientine was dose-dependently inhibited by spermine and spermidine. Spermine competitively inhibited the uptake of trientine with a Ki value of 18.6 microM, and it was close to the K(m) value for spermine (30.4 microM). These data suggested that the uptake of trientine was similar to that of spermine and spermidine in rat small intestinal brush-border membrane vesicles, and these polyamines seem to inhibit the absorption of trientine from the gastrointestinal tract.

  5. Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle

    PubMed Central

    Şener, Melih K.; Olsen, John D.; Hunter, C. Neil; Schulten, Klaus

    2007-01-01

    The photosynthetic unit (PSU) of purple photosynthetic bacteria consists of a network of bacteriochlorophyll–protein complexes that absorb solar energy for eventual conversion to ATP. Because of its remarkable simplicity, the PSU can serve as a prototype for studies of cellular organelles. In the purple bacterium Rhodobacter sphaeroides the PSU forms spherical invaginations of the inner membrane, ≈70 nm in diameter, composed mostly of light-harvesting complexes, LH1 and LH2, and reaction centers (RCs). Atomic force microscopy studies of the intracytoplasmic membrane have revealed the overall spatial organization of the PSU. In the present study these atomic force microscopy data were used to construct three-dimensional models of an entire membrane vesicle at the atomic level by using the known structure of the LH2 complex and a structural model of the dimeric RC–LH1 complex. Two models depict vesicles consisting of 9 or 18 dimeric RC–LH1 complexes and 144 or 101 LH2 complexes, representing a total of 3,879 or 4,464 bacteriochlorophylls, respectively. The in silico reconstructions permit a detailed description of light absorption and electronic excitation migration, including computation of a 50-ps excitation lifetime and a 95% quantum efficiency for one of the model membranes, and demonstration of excitation sharing within the closely packed RC–LH1 dimer arrays. PMID:17895378

  6. Variations on the "dilution" method for reconstituting cytochrome c oxidase into membrane vesicles.

    PubMed

    Ramírez, J; Calahorra, M; Peña, A

    1987-05-15

    A method for the rapid incorporation of cytochrome c oxidase into membranes has been developed. This method essentially consists of obtaining a preparation of the enzyme in which it is isolated and then dissolving it in a medium containing 0.5% of the detergent Tween 20, which gives a final concentration of 0.0125% after reconstitution. These studies revealed an optimal ratio of 1 microgram of enzyme to 5 mg of phospholipids. A similar optimal ratio was found when the amount of protein was varied. The optimum temperature was found to be 30 degrees C. Without a peak value being reached, it was found that the best reconstitution was obtained at pH 7.0-8.0. When measurements were performed either with a fluorescent cyanine (DiSC3) or by the uptake of tetraphenylphosphonium, it was found that the enzyme, with cytochrome c added to the outside, was capable of generating a membrane potential that was negative inside. Using the same procedure, the enzyme could also be reconstituted into vesicles of yeast plasma membrane. The procedure, then, seems adequate for incorporating cytochrome c oxidase into different kinds of membrane vesicles.

  7. Two Rab proteins, vesicle-associated membrane protein 2 (VAMP-2) and secretory carrier membrane proteins (SCAMPs), are present on immunoisolated parietal cell tubulovesicles.

    PubMed Central

    Calhoun, B C; Goldenring, J R

    1997-01-01

    The tubulovesicles of gastric parietal cells sequester H+/K+-ATPase molecules within resting parietal cells. Stimulation of parietal cell secretion elicits delivery of intracellular H+/K+-ATPase to the apically oriented secretory canaliculus. Previous investigations have suggested that this process requires the regulated fusion of intracellular tubulovesicles with the canalicular target membrane. We have sought to investigate the presence of critical putative regulators of vesicle fusion on immunoisolated gastric parietal cell tubulovesicles. Highly purified tubulovesicles were prepared by gradient fractionation and immunoisolation on magnetic beads coated with monoclonal antibodies against the alpha subunit of H+/K+-ATPase. Western blot analysis revealed the presence of Rab11, Rab25, vesicle-associated membrane protein 2 (VAMP-2) and secretory carrier membrane proteins (SCAMPs) on immunoisolated vesicles. The same cohort of proteins was recovered on vesicles immunoisolated with monoclonal antibodies against SCAMPs and VAMP-2. In contrast, whereas immunoreactivities for syntaxin 1A/1B and synaptosome-associated protein (SNAP-25) were present in gradient-isolated vesicles, none of the immunoreactivity was associated with immunoisolated vesicles. The observation of VAMP-2 and two Rab proteins on immunoisolated H+/K+-ATPase-containing tubulovesicles supports the role for tubulovesicles in a regulated vesicle fusion process. In addition, the presence of SCAMPs along with Rab11 and Rab25 implicates the tubulovesicles as a critical apical recycling vesicle population. PMID:9230141

  8. Characterization of procoagulant extracellular vesicles and platelet membrane disintegration in DMSO-cryopreserved platelets

    PubMed Central

    Tegegn, Tseday Z.; De Paoli, Silvia H.; Orecna, Martina; Elhelu, Oumsalama K.; Woodle, Samuel A.; Tarandovskiy, Ivan D.; Ovanesov, Mikhail V.; Simak, Jan

    2016-01-01

    Background Freezing is promising for extended platelet (PLT) storage for transfusion. 6% DMSO cryopreserved PLTs (CPPs) are currently in clinical development. CPPs contain significant amount of platelet membrane vesicles (PMVs). PLT-membrane changes and PMV release in CPP are poorly understood, and haemostatic effects of CPP PMVs are not fully elucidated. This study aims to investigate PLT-membrane alterations in CPPs and provide comprehensive characterization of CPP PMVs, and their contribution to procoagulant activity (PCA) of CPPs. Methods CPPs and corresponding liquid-stored PLTs (LSPs) were characterized by flow cytometry (FC), fluorescence polarization (FP), nanoparticle tracking analysis (NTA), electron microscopy (SEM, TEM), atomic force microscopy (AFM) and thrombin-generation (TG) test. Results SEM and TEM revealed disintegration and vesiculation of the PLT-plasma membrane and loss of intracellular organization in 60% PLTs in CPPs. FP demonstrated that 6% DMSO alone and with freezing–thawing caused marked increase in PLT-membrane fluidity. The FC counts of annexin V-binding PMVs and CD41a+ PMVs were 68- and 56-folds higher, respectively, in CPPs than in LSPs. The AFM and NTA size distribution of PMVs in CPPs indicated a peak diameter of 100 nm, corresponding to exosome-size vesicles. TG-based PCA of CPPs was 2- and 9-folds higher per PLT and per volume, respectively, compared to LSPs. Differential centrifugation showed that CPP supernatant contributed 26% to CPP TG-PCA, mostly by the exosome-size PMVs and their TG-PCA was phosphatidylserine dependent. Conclusions Major portion of CPPs does not show activation phenotype but exhibits grape-like membrane disintegration with significant increase of membrane fluidity induced by 6% DMSO alone and further aggravated by freezing–thawing process. DMSO cryopreservation of PLTs is associated with the release of PMVs and marked increase of TG-PCA, as compared to LSPs. Exosome-size PMVs have significant

  9. The mechanism of biliary secretion of reduced glutathione. Analysis of transport process in isolated rat-liver canalicular membrane vesicles.

    PubMed

    Inoue, M; Kinne, R; Tran, T; Arias, I M

    1983-08-15

    Transport of reduced glutathione (GSH) was studied in isolated rat liver canalicular membrane vesicles by a rapid filtration technique. The membrane vesicles exhibit uptake of [2-3H]glycine--labeled GSH into an osmotically reactive intravesicular space. Although the canalicular membrane vesicles possess gamma-glutamyltransferase and aminopeptidase M, enzymes that hydrolyze glutathione into component amino acids, inactivation of the vesicle-associated transferase by affinity labeling with L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) had no effect on the initial rate of GSH transport. Chemical analysis revealed that intact GSH accounted for most of vesicle-associated radioactivity. The initial rate of transport followed saturation kinetics with respect to GSH concentration; an apparent Km of 0.33 mM and V of 1.47 nmol/mg protein in 20 s were calculated. These results indicate that transport of GSH across the canalicular membranes is a carrier-mediated process. Replacement of NaCl in the transport medium by KCl, LiCl or choline chloride had no effect on the transport activity of the vesicles. The rate of GSH uptake by the vesicles was enhanced by valinomycin-induced K+-diffusion potential (vesicle inside-positive) and was inhibited by probenecid, indicating that GSH transport across the canalicular membranes is electrogenic and involves the transfer of negative charge. The transport of GSH was inhibited by oxidized glutathione or S-benzyl-glutathione. This transport system in canalicular plasma membranes may function in biliary secretion of GSH and its derivatives which are synthesized in hepatocytes by oxidative processes or glutathione S-transferase.

  10. Sec35p, a Novel Peripheral Membrane Protein, Is Required for ER to Golgi Vesicle Docking

    PubMed Central

    VanRheenen, Susan M.; Cao, Xiaochun; Lupashin, Vladimir V.; Barlowe, Charles; Gerard Waters, M.

    1998-01-01

    SEC35 was identified in a novel screen for temperature-sensitive mutants in the secretory pathway of the yeast Saccharomyces cerevisiae (Wuestehube et al., 1996. Genetics. 142:393–406). At the restrictive temperature, the sec35-1 strain exhibits a transport block between the ER and the Golgi apparatus and accumulates numerous vesicles. SEC35 encodes a novel cytosolic protein of 32 kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec35-1 is efficiently suppressed by YPT1, which encodes the rab-like GTPase required early in the secretory pathway, or by SLY1-20, which encodes a dominant form of the ER to Golgi target -SNARE–associated protein Sly1p. Weaker suppression is evident upon overexpression of genes encoding the vesicle-SNAREs SEC22, BET1, or YKT6. The cold-sensitive lethality that results from deleting SEC35 is suppressed by YPT1 or SLY1-20. These genetic relationships suggest that Sec35p acts upstream of, or in conjunction with, Ypt1p and Sly1p as was previously found for Uso1p. Using a cell-free assay that measures distinct steps in vesicle transport from the ER to the Golgi, we find Sec35p is required for a vesicle docking stage catalyzed by Uso1p. These genetic and biochemical results suggest Sec35p acts with Uso1p to dock ER-derived vesicles to the Golgi complex. PMID:9606204

  11. Fusion of phospholipid vesicles arrested by quick-freezing. The question of lipidic particles as intermediates in membrane fusion.

    PubMed

    Bearer, E L; Düzgünes, N; Friend, D S; Papahadjopoulos, D

    1982-12-01

    We have examined the early events in Ca2+-induced fusion of large (0.2 microns diameter) unilamellar cardiolipin/phosphatidylcholine and phosphatidylserine/phosphatidylethanolamine vesicles by quick-freezing freeze-fracture electron microscopy, eliminating the necessity of using glycerol as a cryoprotectant. Freeze-fracture replicas of vesicle suspensions frozen after 1-2 s of stimulation revealed that the majority of vesicles had already undergone membrane fusion, as evidenced by dumbbell-shaped structures and large vesicles. In the absence of glycerol, lipidic particles or the hexagonal HII phase, which have been proposed to be intermediate structures in membrane fusion, were not observed at the sites of fusion. Lipidic particles were evident in less than 5% of the cardiolipin/phosphatidylcholine vesicles after long-term incubation with Ca2+, and the addition of glycerol produced more vesicles displaying the particles. We have also shown that rapid fusion occurred within seconds of Ca2+ addition by the time-course of fluorescence emission produced by the intermixing of aqueous contents of two separate vesicle populations. These studies therefore have produced no evidence that lipidic particles are necessary intermediates for membrane fusion. On the contrary, they indicate that lipidic particles are structures obtained at equilibrium long after fusion has occurred and they become particularly prevalent in the presence of glycerol.

  12. Molecular characterization of outer membrane vesicles released from Acinetobacter radioresistens and their potential roles in pathogenesis.

    PubMed

    Fulsundar, Shweta; Kulkarni, Heramb M; Jagannadham, Medicharla V; Nair, Rashmi; Keerthi, Sravani; Sant, Pooja; Pardesi, Karishma; Bellare, Jayesh; Chopade, Balu Ananda

    2015-01-01

    Acinetobacter radioresistens is an important member of genus Acinetobacter from a clinical point of view. In the present study, we report that a clinical isolate of A. radioresistens releases outer membrane vesicles (OMVs) under in vitro growth conditions. OMVs were released in distinctive size ranges with diameters from 10 to 150 nm as measured by the dynamic light scattering (DLS) technique. Additionally, proteins associated with or present into OMVs were identified using LC-ESI-MS/MS. A total of 71 proteins derived from cytosolic, cell membrane, periplasmic space, outer membrane (OM), extracellular and undetermined locations were found in OMVs. The initial characterization of the OMV proteome revealed a correlation of some proteins to biofilm, quorum sensing, oxidative stress tolerance, and cytotoxicity functions. Thus, the OMVs of A. radioresistens are suggested to play a role in biofilm augmentation and virulence possibly by inducing apoptosis.

  13. Evidence that membrane transduction of oligoarginine does not require vesicle formation

    SciTech Connect

    Zaro, Jennica L.; Shen Weichiang . E-mail: weishen@usc.edu

    2005-07-01

    The involvement of vesicular formation processes in the membrane transduction and nuclear transport of oligoarginine is currently a subject of controversy. In this report, a novel quantitative method which allows for the selective measurement of membrane transduction excluding concurrent endocytosis was used to determine the effects of temperature, endosomal acidification, endosomolysis, and several known inhibitors of endocytic pathways on the internalization of oligoarginine. The results show that, unlike endocytosis, transduction of oligoarginine was not affected by incubation at 16 deg. C as compared to the 37 deg. C control, and was only partially inhibited at 4 deg. C incubation. Additionally, membrane transduction was not inhibited to the same extent as endocytosis following treatment with ammonium chloride, hypertonic medium, amiloride, or filipin. The endosomolytic activity of oligoarginine was investigated by examining the leakage of FITC-dextran into the cytosolic compartment, which was not higher in the presence of oligoarginine. Furthermore, ammonium chloride showed no effect on the nuclear transport of oligoarginine. The data presented in this report indicate that membrane transduction is likely to occur at the plasma membrane without the formation of membrane vesicles, and the nuclear localization involves membrane transduction, rather than endocytosis of oligoarginine.

  14. Electrostatically Driven Assembly of Charged Amphiphiles Forming Crystallized Membranes, Vesicles and Nanofiber Arrays

    NASA Astrophysics Data System (ADS)

    Leung, Cheuk Yui Curtis

    Charged amphiphilic molecules can self-assemble into a large variety of objects including membranes, vesicles and fibers. These micro to nano-scale structures have been drawing increasing attention due to their broad applications, especially in biotechnology and biomedicine. In this dissertation, three self-assembled systems were investigated: +3/-1 self-assembled catanionic membranes, +2/-1 self-assembled catanionic membranes and +1 self-assembled nanofibers. Transmission electron microscopy (TEM) combined with synchrotron small and wide angle x-ray scattering (SAXS and WAXS) were used to characterize the coassembled structures from the mesoscopic to nanometer scale. We designed a system of +3 and -1 ionic amphiphiles that coassemble into crystalline ionic bilayer vesicles with large variety of geometries that resemble polyhedral cellular crystalline shells and archaea wall envelopes. The degree of ionization of the amphiphiles and their intermolecular electrostatic interactions can be controlled by varying pH. The molecular packing of these membranes showed a hexagonal to rectangular-C to hexagonal phase transition with increasing pH, resulting in significant changes to the membrane morphology. A similar mixture of +2 and -1 ionic amphiphiles was also investigated. In addition to varying pH, which controls the headgroup attractions, we also adjust the tail length of the amphiphiles to control the van der Waals interactions between the tails. A 2D phase diagram was developed to show how pH and tail length can be used to control the intermolecular packing within the membranes. Another system of self-assembled nanofiber network formed by positively charged amphiphiles was also studied. These highly charged fibers repel each other and are packed in hexagonal lattice with lattice constant at least eight times of the fiber diameter. The d-spacing and the crystal structure can be controlled by varying the solution concentration and temperature.

  15. Effects of food restriction and insulin treatment on (Ca2+ + Mg2+)-ATPase response to insulin in kidney basolateral membranes of noninsulin-dependent diabetic rats.

    PubMed

    Levy, J; Grunberger, G; Karl, I; Gavin, J R

    1990-01-01

    Insulin increases (Ca2+ + Mg2+)-ATPase activity in cell membranes of normal rats but fails to do so in membranes of non-insulin-dependent diabetic (NIDD) rats. The loss of regulatory effect of the hormone on the enzyme might contribute to the insulin resistance observed in the NIDD animals. To further test this hypothesis, the effects of insulin treatment and acute food restriction on the ability of insulin to regulate the ATPase activity in kidney basolateral membranes (BLM) of NIDD rats were studied. Although insulin levels in NIDD and control rats were similar, plasma glucose was higher in the NIDD rats (18.3 +/- 1.5 v 19.3 +/- 1.7 microU/mL and 236 +/- 32 v 145 +/- 3 mg/dL, respectively). Insulin treatment (2 U/100 g), which increased plasma insulin in the NIDD rats (47.8 +/- 11.5 microU/mL; P less than .05), did not decrease their glucose (221 +/- 25 mg/dL). Higher insulin dose (4 U/100 g) decreased glucose level in the NIDD rats (73 +/- 3 mg/dL; P less than .001) but increased their plasma insulin 10-fold (202.5 +/- 52.5 microU/mL). Acute food restriction decreased glucose levels in the NIDD rats to levels seen in controls (135 +/- 3 mg/dL), while their insulin decreased by half (8.5 +/- 1.0 microU/mL; P less than .05). Basal (Ca2+ + Mg2+)-ATPase activity in BLM of all diabetic rats was higher than in controls (P less than .05). None of the treatments reversed this defect.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2136760

  16. 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

  17. Characterization of L-carnitine transport by rat kidney brush-border-membrane vesicles.

    PubMed Central

    Stieger, B; O'Neill, B; Krähenbühl, S

    1995-01-01

    In the presence of a 100 mM Na+ gradient, transport of L-carnitine into rat renal brush-border-membrane vesicles was linear over 30 s and showed an overshoot at 5 min. The uptake of L-carnitine was clearly less active in the presence of other cations such as Li+, K+, Cs+ or choline. In the presence of a Na+ gradient, L-carnitine uptake after 20 s was much higher for chloride as an anion than for SCN-, NO3-, gluconate or SO4(2-). In comparison with conditions with inside positive or no membrane potential, transport was higher in vesicles with an inside negative membrane potential, suggesting an electrogenic mechanism. The kinetic characterization of the Na(+)-dependent portion of L-carnitine transport revealed two transport systems with Km values of 17.4 +/- 3.9 microM and 15.0 +/- 6.0 mM, respectively. The transport could be inhibited in a concentration-dependent fashion by structural analogues such as butyrobetaine, L-acetylcarnitine, trimethyl-lysine and D-carnitine, but not by L-arginine or glycinebetaine. PMID:7626031

  18. Biochemical requirements for the targeting and fusion of ER-derived transport vesicles with purified yeast Golgi membranes

    PubMed Central

    1996-01-01

    In order for secretion to progress, ER-derived transport vesicles must target to, and fuse with the cis-Golgi compartment. These processes have been reconstituted using highly enriched membrane fractions and partially purified soluble components. The functionally active yeast Golgi membranes that have been purified are highly enriched in the cis- Golgi marker enzymes alpha 1,6 mannosyltransferase and GDPase. Fusion of transport vesicles with these membranes requires both GTP and ATP hydrolysis, and depends on cytosolic and peripheral membrane proteins. At least two protein fractions from yeast cytosol are required for the reconstitution of ER-derived vesicle fusion. Soluble fractions prepared from temperature-sensitive mutants revealed requirements for the Ypt1p, Sec19p, Sly1p, Sec7p, and Uso1 proteins. A model for the sequential involvement of these components in the targeting and fusion reaction is proposed. PMID:8636207

  19. Chloride permeability of rat brain membrane vesicles correlates with thiamine triphosphate content.

    PubMed

    Bettendorff, L; Hennuy, B; De Clerck, A; Wins, P

    1994-07-25

    Incubation of rat brain homogenates with thiamine or thiamine diphosphate (TDP) leads to a synthesis of thiamine triphosphate (TTP). In membrane vesicles subsequently prepared from the homogenates, increased TTP content correlates with increased 36Cl- uptake. A hyperbolic relationship was obtained with a K0.5 of 0.27 nmol TTP/mg protein. In crude mitochondrial fractions from the brains of animals previously treated with thiamine or sulbutiamine, a positive correlation between 36Cl- uptake and TTP content was found. These results, together with other results previously obtained with the patch-clamp technique, suggest that TTP is an activator of chloride channels having a large unit conductance. PMID:7953714

  20. Membrane Vesicles Nucleate Mineralo-organic Nanoparticles and Induce Carbonate Apatite Precipitation in Human Body Fluids*

    PubMed Central

    Wu, Cheng-Yeu; Martel, Jan; Cheng, Wei-Yun; He, Chao-Chih; Ojcius, David M.; Young, John D.

    2013-01-01

    Recent studies indicate that membrane vesicles (MVs) secreted by various cells are associated with human diseases, including arthritis, atherosclerosis, cancer, and chronic kidney disease. The possibility that MVs may induce the formation of mineralo-organic nanoparticles (NPs) and ectopic calcification has not been investigated so far. Here, we isolated MVs ranging in size between 20 and 400 nm from human serum and FBS using ultracentrifugation and sucrose gradient centrifugation. The MV preparations consisted of phospholipid-bound vesicles containing the serum proteins albumin, fetuin-A, and apolipoprotein A1; the mineralization-associated enzyme alkaline phosphatase; and the exosome proteins TNFR1 and CD63. Notably, we observed that MVs induced mineral precipitation following inoculation and incubation in cell culture medium. The mineral precipitates consisted of round, mineralo-organic NPs containing carbonate hydroxyapatite, similar to previous descriptions of the so-called nanobacteria. Annexin V-immunogold staining revealed that the calcium-binding lipid phosphatidylserine (PS) was exposed on the external surface of serum MVs. Treatment of MVs with an anti-PS antibody significantly decreased their mineral seeding activity, suggesting that PS may provide nucleating sites for calcium phosphate deposition on the vesicles. These results indicate that MVs may represent nucleating agents that induce the formation of mineral NPs in body fluids. Given that mineralo-organic NPs represent precursors of calcification in vivo, our results suggest that MVs may initiate ectopic calcification in the human body. PMID:23990473

  1. A comprehensive characterization of membrane vesicles released by autophagic human endothelial cells.

    PubMed

    Pallet, Nicolas; Sirois, Isabelle; Bell, Christina; Hanafi, Laïla-Aïcha; Hamelin, Katia; Dieudé, Mélanie; Rondeau, Christiane; Thibault, Pierre; Desjardins, Michel; Hebert, Marie-Josée

    2013-04-01

    The stress status of the apoptotic cell can promote phenotypic changes that have important consequences on the immunogenicity of the dying cell. Autophagy is one of the biological processes activated in response to a stressful condition. It is an important mediator of intercellular communications, both by regulating the unconventional secretion of molecules, including interleukin 1β, and by regulating the extracellular release of ATP from early stage apoptotic cells. Additionally, autophagic components can be released in a caspase-dependent manner by serum-starved human endothelial cells that have engaged apoptotic and autophagic processes. The nature and the components of the extracellular vesicles released by dying autophagic cells are not known. In this study, we have identified extracellular membrane vesicles that are released by human endothelial cells undergoing apoptosis and autophagy, and characterized their biochemical, ultrastructural, morphological properties as well as their proteome. These extracellular vesicles differ from classical apoptotic bodies because they do not contain nucleus components and are released independently of Rho-associated, coiled-coil containing protein kinase 1 activation. Instead, they are enriched with autophagosomes and mitochondria and convey various danger signals, including ATP, suggesting that they could be involved in the modulation of innate immunity. PMID:23436686

  2. Salmonella enterica delivers its genotoxin through outer membrane vesicles secreted from infected cells.

    PubMed

    Guidi, Riccardo; Levi, Laura; Rouf, Syed Fazle; Puiac, Speranta; Rhen, Mikael; Frisan, Teresa

    2013-12-01

    Cytolethal-distending toxins (CDTs) belong to a family of DNA damage inducing exotoxins that are produced by several Gram-negative bacteria. Salmonella enterica serovar Typhi expresses its CDT (named as Typhoid toxin) only in the Salmonella-containing vacuole (SCV) of infected cells, which requires its export for cell intoxication. The mechanisms of secretion, release in the extracellular space and uptake by bystander cells are poorly understood. We have addressed these issues using a recombinant S. Typhimurium strain, MC71-CDT, where the genes encoding for the PltA, PltB and CdtB subunits of the Typhoid toxin are expressed under control of the endogenous promoters. MC71-CDT grown under conditions that mimic the SCV secreted the holotoxin in outer membrane vesicles (OMVs). Epithelial cells infected with MC71-CDT also secreted OMVs-like vesicles. The release of these extracellular vesicles required an intact SCV and relied on anterograde transport towards the cellular cortex on microtubule and actin tracks. Paracrine internalization of Typhoid toxin-loaded OMVs by bystander cells was dependent on dynamin-1, indicating active endocytosis. The subsequent induction of DNA damage required retrograde transport of the toxin through the Golgi complex. These data provide new insights on the mode of secretion of exotoxins by cells infected with intracellular bacteria.

  3. Membrane vesicles nucleate mineralo-organic nanoparticles and induce carbonate apatite precipitation in human body fluids.

    PubMed

    Wu, Cheng-Yeu; Martel, Jan; Cheng, Wei-Yun; He, Chao-Chih; Ojcius, David M; Young, John D

    2013-10-18

    Recent studies indicate that membrane vesicles (MVs) secreted by various cells are associated with human diseases, including arthritis, atherosclerosis, cancer, and chronic kidney disease. The possibility that MVs may induce the formation of mineralo-organic nanoparticles (NPs) and ectopic calcification has not been investigated so far. Here, we isolated MVs ranging in size between 20 and 400 nm from human serum and FBS using ultracentrifugation and sucrose gradient centrifugation. The MV preparations consisted of phospholipid-bound vesicles containing the serum proteins albumin, fetuin-A, and apolipoprotein A1; the mineralization-associated enzyme alkaline phosphatase; and the exosome proteins TNFR1 and CD63. Notably, we observed that MVs induced mineral precipitation following inoculation and incubation in cell culture medium. The mineral precipitates consisted of round, mineralo-organic NPs containing carbonate hydroxyapatite, similar to previous descriptions of the so-called nanobacteria. Annexin V-immunogold staining revealed that the calcium-binding lipid phosphatidylserine (PS) was exposed on the external surface of serum MVs. Treatment of MVs with an anti-PS antibody significantly decreased their mineral seeding activity, suggesting that PS may provide nucleating sites for calcium phosphate deposition on the vesicles. These results indicate that MVs may represent nucleating agents that induce the formation of mineral NPs in body fluids. Given that mineralo-organic NPs represent precursors of calcification in vivo, our results suggest that MVs may initiate ectopic calcification in the human body.

  4. Membrane vesicles nucleate mineralo-organic nanoparticles and induce carbonate apatite precipitation in human body fluids.

    PubMed

    Wu, Cheng-Yeu; Martel, Jan; Cheng, Wei-Yun; He, Chao-Chih; Ojcius, David M; Young, John D

    2013-10-18

    Recent studies indicate that membrane vesicles (MVs) secreted by various cells are associated with human diseases, including arthritis, atherosclerosis, cancer, and chronic kidney disease. The possibility that MVs may induce the formation of mineralo-organic nanoparticles (NPs) and ectopic calcification has not been investigated so far. Here, we isolated MVs ranging in size between 20 and 400 nm from human serum and FBS using ultracentrifugation and sucrose gradient centrifugation. The MV preparations consisted of phospholipid-bound vesicles containing the serum proteins albumin, fetuin-A, and apolipoprotein A1; the mineralization-associated enzyme alkaline phosphatase; and the exosome proteins TNFR1 and CD63. Notably, we observed that MVs induced mineral precipitation following inoculation and incubation in cell culture medium. The mineral precipitates consisted of round, mineralo-organic NPs containing carbonate hydroxyapatite, similar to previous descriptions of the so-called nanobacteria. Annexin V-immunogold staining revealed that the calcium-binding lipid phosphatidylserine (PS) was exposed on the external surface of serum MVs. Treatment of MVs with an anti-PS antibody significantly decreased their mineral seeding activity, suggesting that PS may provide nucleating sites for calcium phosphate deposition on the vesicles. These results indicate that MVs may represent nucleating agents that induce the formation of mineral NPs in body fluids. Given that mineralo-organic NPs represent precursors of calcification in vivo, our results suggest that MVs may initiate ectopic calcification in the human body. PMID:23990473

  5. Use of membrane vesicles as a simplified system for studying auxin transport of auxin: Progress report

    SciTech Connect

    Goldsmith, M.H.M.

    1986-01-01

    Indoleacetic acid (IAA), the auxin regulating growth, is transported polarly in plants. IAA stimulates a rapid increase in the rate of electrogenic proton secretion by the plasma membrane. This not only increases the magnitude of the pH and electrical gradients providing the driving force for polar auxin transport and uptake of sugars, amino acids and inorganic ions, but, by acidifying the cell wall, also leads to growth. We find that auxin uptake by membrane vesicles isolated from actively growing plant tissues exhibits some of the same properties as by cells: the accumulation depends on the pH gradient, is saturable and specific for auxin, and enhanced by herbicides that inhibit polar auxin transport. We are using accumulation of a radioactive weak acid to quantify the pH gradient and distribution of fluorescent cyanine dyes to monitor the membrane potential. The magnitude of IAA accumulation exceeds that predicted from the pH gradient, and in the absence of a pH gradient, a membrane potential fails to support any auxin accumulation, leading to the conclusion that the transmembrane potential is not a significant driving force for auxin accumulation in this system. Since increasing the external ionic strength decreases saturable auxin accumulation, we are investigating how modifying the surface potential of the vesicles affects the interaction of the amphipathic IAA molecules with the membranes and whether protein modifying reagents affect the saturability and stimulation by NPA. These studies should provide information on the location and function of the auxin binding site and may enable us to identify the solubilized protein. 5 refs.

  6. In vitro study of interaction of synaptic vesicles with lipid membranes

    NASA Astrophysics Data System (ADS)

    Ghosh, S. K.; Castorph, S.; Konovalov, O.; Jahn, R.; Holt, M.; Salditt, T.

    2010-10-01

    The fusion of synaptic vesicles (SVs) with the plasma membrane in neurons is a crucial step in the release of neurotransmitters, which are responsible for carrying signals between nerve cells. While many of the molecular players involved in this fusion process have been identified, a precise molecular description of their roles in the process is still lacking. A case in point is the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2). Although PIP2 is known to be essential for vesicle fusion, its precise role in the process remains unclear. We have re-investigated the role of this lipid in membrane structure and function using the complementary experimental techniques of x-ray reflectivity, both on lipid monolayers at an air-water interface and bilayers on a solid support, and grazing incidence x-ray diffraction on lipid monolayers. These techniques provide unprecedented access to structural information at the molecular level, and detail the profound structural changes that occur in a membrane following PIP2 incorporation. Further, we also confirm and extend previous findings that the association of SVs with membranes is enhanced by PIP2 incorporation, and reveal the structural changes that underpin this phenomenon. Further, the association is further intensified by a physiologically relevant amount of Ca2+ ions in the subphase of the monolayer, as revealed by the increase in interfacial pressure seen with the lipid monolayer system. Finally, a theoretical calculation concerning the products arising from the fusion of these SVs with proteoliposomes is presented, with which we aim to illustrate the potential future uses of this system.

  7. Reconstitution of membrane proteins into giant unilamellar vesicles via peptide-induced fusion.

    PubMed Central

    Kahya, N; Pécheur, E I; de Boeij, W P; Wiersma, D A; Hoekstra, D

    2001-01-01

    In this work, we present a protocol to reconstitute membrane proteins into giant unilamellar vesicles (GUV) via peptide-induced fusion. In principle, GUV provide a well-defined lipid matrix, resembling a close-to-native state for biophysical studies, including optical microspectroscopy, of transmembrane proteins at the molecular level. Furthermore, reconstitution in this manner would also eliminate potential artifacts arising from secondary interactions of proteins, when reconstituted in planar membranes supported on solid surfaces. However, assembly procedures of GUV preclude direct reconstitution. Here, for the first time, a method is described that allows the controlled incorporation of membrane proteins into GUV. We demonstrate that large unilamellar vesicles (LUV, diameter 0.1 microm), to which the small fusogenic peptide WAE has been covalently attached, readily fuse with GUV, as revealed by monitoring lipid and contents mixing by fluorescence microscopy. To monitor contents mixing, a new fluorescence-based enzymatic assay was devised. Fusion does not introduce changes in the membrane morphology, as shown by fluorescence correlation spectroscopy. Analysis of fluorescence confocal imaging intensity revealed that approximately 6 to 10 LUV fused per microm(2) of GUV surface. As a model protein, bacteriorhodopsin (BR) was reconstituted into GUV, using LUV into which BR was incorporated via detergent dialysis. BR did not affect GUV-LUV fusion and the protein was stably inserted into the GUV and functionally active. Fluorescence correlation spectroscopy experiments show that BR inserted into GUV undergoes unrestricted Brownian motion with a diffusion coefficient of 1.2 microm(2)/s. The current procedure offers new opportunities to address issues related to membrane-protein structure and dynamics in a close-to-native state. PMID:11509360

  8. Immobilization of lipid vesicles on polymer support via an amphiphilic peptidic anchor: application to a membrane enzyme.

    PubMed

    Percot, A; Zhu, X X; Lafleur, M

    2000-01-01

    To immobilize lipid vesicles on a polymer support, we have used a peptidic anchor with the following sequence: Ala-Ala-Leu-Leu-Leu-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-A la-Ala-Ala-Ala-Ala-Ala-Ala-Trp-Lys-Lys-Lys-Lys-Lys-Lys. This amphiphilic peptide was previously designed in our group to interact spontaneously and strongly with vesicles without perturbing their permeability. At the end of the solid-phase peptide synthesis, the peptide was left on the polymer beads and this novel polymer-peptide system was used for vesicle immobilization. It was shown that this polymer-peptide system could immobilize as much as 200 micromol of lipids per gram of dry resin. The amount of immobilized vesicles was decreased by a reduction of the proportion of the negatively charged lipids in the vesicles, indicating the importance of electrostatic interactions in the immobilization of the vesicles. The integrity of the vesicles was mostly preserved after the immobilization. This new polymer-peptide system was used easily and successfully to immobilize a membrane-bound enzyme, gamma-glutamyl transpeptidase. The activity of the membrane-bound enzyme was studied by monitoring the release of p-nitroaniline. The activity of the enzyme was still retained, even after being re-used eight times, indicating the strong immobilization of the enzyme in its active form. The polymer-peptide support could be regenerated by washing with ethanol and reused.

  9. Characterization of membrane protein interactions in plasma membrane derived vesicles with quantitative imaging Förster resonance energy transfer.

    PubMed

    Sarabipour, Sarvenaz; Del Piccolo, Nuala; Hristova, Kalina

    2015-08-18

    Here we describe an experimental tool, termed quantitative imaging Förster resonance energy transfer (QI-FRET), that enables the quantitative characterization of membrane protein interactions. The QI-FRET methodology allows us to acquire binding curves and calculate association constants for complex membrane proteins in the native plasma membrane environment. The method utilizes FRET detection, and thus requires that the proteins of interest are labeled with florescent proteins, either FRET donors or FRET acceptors. Since plasma membranes of cells have complex topologies precluding the acquisition of two-dimensional binding curves, the FRET measurements are performed in plasma membrane derived vesicles that bud off cells as a result of chemical or osmotic stress. The results overviewed here are acquired in vesicles produced with an osmotic vesiculation buffer developed in our laboratory, which does not utilize harsh chemicals. The concentrations of the donor-labeled and the acceptor-labeled proteins are determined, along with the FRET efficiencies, in each vesicle. The experiments utilize transient transfection, such that a wide variety of concentrations is sampled. Then, data from hundreds of vesicles are combined to yield dimerization curves. Here we discuss recent findings about the dimerization of receptor tyrosine kinases (RTKs), membrane proteins that control cell growth and differentiation via lateral dimerization in the plasma membrane. We focus on the dimerization of fibroblast growth factor receptor 3 (FGFR3), a RTK that plays a critically important role in skeletal development. We study the role of different FGFR3 domains in FGFR3 dimerization in the absence of ligand, and we show that FGFR3 extracellular domains inhibit unliganded dimerization, while contacts between the juxtamembrane domains, which connect the transmembrane domains to the kinase domains, stabilize the unliganded FGFR3 dimers. Since FGFR3 has been documented to harbor many pathogenic

  10. Maltose/proton co-transport in Saccharomyces cerevisiae. Comparative study with cells and plasma membrane vesicles.

    PubMed Central

    Van Leeuwen, C C; Weusthuis, R A; Postma, E; Van den Broek, P J; Van Dijken, J P

    1992-01-01

    Maltose/proton co-transport was studied in intact cells and in plasma membrane vesicles of the yeast Saccharomyces cerevisiae. In order to determine uphill transport in vesicles, plasma membranes were fused with proteoliposomes containing cytochrome c oxidase as a proton-motive force-generating system. Maltose accumulation, dependent on the electrical and pH gradients, was observed. The initial uptake velocity and accumulation ratio in vesicles proved to be dependent on the external pH. Moreover, kinetic analysis of maltose transport showed that Vmax. values greatly decreased with increasing pH, whereas the Km remained virtually constant. These observations were in good agreement with results obtained with intact cells, and suggest that proton binding to the carrier proceeds with an apparent pK of 5.7. The observation with intact cells that maltose is co-transported with protons in a one-to-one stoichiometry was ascertained in the vesicle system by measuring the balance between proton-motive force and the chemical maltose gradient. These results show that maltose transport in vesicles prepared by fusion of plasma membranes with cytochrome c oxidase proteoliposomes behaves in a similar way as in intact cells. It is therefore concluded that this vesicle model system offers a wide range of new possibilities for the study of maltose/proton co-transport in more detail. PMID:1318030

  11. bSUM: A bead-supported unilamellar membrane system facilitating unidirectional insertion of membrane proteins into giant vesicles

    PubMed Central

    Zheng, Hui; Lee, Sungsoo; Llaguno, Marc C.

    2016-01-01

    Fused or giant vesicles, planar lipid bilayers, a droplet membrane system, and planar-supported membranes have been developed to incorporate membrane proteins for the electrical and biophysical analysis of such proteins or the bilayer properties. However, it remains difficult to incorporate membrane proteins, including ion channels, into reconstituted membrane systems that allow easy control of operational dimensions, incorporation orientation of the membrane proteins, and lipid composition of membranes. Here, using a newly developed chemical engineering procedure, we report on a bead-supported unilamellar membrane (bSUM) system that allows good control over membrane dimension, protein orientation, and lipid composition. Our new system uses specific ligands to facilitate the unidirectional incorporation of membrane proteins into lipid bilayers. Cryo–electron microscopic imaging demonstrates the unilamellar nature of the bSUMs. Electrical recordings from voltage-gated ion channels in bSUMs of varying diameters demonstrate the versatility of the new system. Using KvAP as a model system, we show that compared with other in vitro membrane systems, the bSUMs have the following advantages: (a) a major fraction of channels are orientated in a controlled way; (b) the channels mediate the formation of the lipid bilayer; (c) there is one and only one bilayer membrane on each bead; (d) the lipid composition can be controlled and the bSUM size is also under experimental control over a range of 0.2–20 µm; (e) the channel activity can be recorded by patch clamp using a planar electrode; and (f) the voltage-clamp speed (0.2–0.5 ms) of the bSUM on a planar electrode is fast, making it suitable to study ion channels with fast gating kinetics. Our observations suggest that the chemically engineered bSUMs afford a novel platform for studying lipid–protein interactions in membranes of varying lipid composition and may be useful for other applications, such as targeted

  12. Vesicles Bearing Toxoplasma Apicoplast Membrane Proteins Persist Following Loss of the Relict Plastid or Golgi Body Disruption

    PubMed Central

    Bouchut, Anne; Geiger, Jennifer A.; DeRocher, Amy E.; Parsons, Marilyn

    2014-01-01

    Toxoplasma gondii and malaria parasites contain a unique and essential relict plastid called the apicoplast. Most apicoplast proteins are encoded in the nucleus and are transported to the organelle via the endoplasmic reticulum (ER). Three trafficking routes have been proposed for apicoplast membrane proteins: (i) vesicular trafficking from the ER to the Golgi and then to the apicoplast, (ii) contiguity between the ER membrane and the apicoplast allowing direct flow of proteins, and (iii) vesicular transport directly from the ER to the apicoplast. Previously, we identified a set of membrane proteins of the T. gondii apicoplast which were also detected in large vesicles near the organelle. Data presented here show that the large vesicles bearing apicoplast membrane proteins are not the major carriers of luminal proteins. The vesicles continue to appear in parasites which have lost their plastid due to mis-segregation, indicating that the vesicles are not derived from the apicoplast. To test for a role of the Golgi body in vesicle formation, parasites were treated with brefeldin A or transiently transfected with a dominant-negative mutant of Sar1, a GTPase required for ER to Golgi trafficking. The immunofluorescence patterns showed little change. These findings were confirmed using stable transfectants, which expressed the toxic dominant-negative sar1 following Cre-loxP mediated promoter juxtaposition. Our data support the hypothesis that the large vesicles do not mediate the trafficking of luminal proteins to the apicoplast. The results further show that the large vesicles bearing apicoplast membrane proteins continue to be observed in the absence of Golgi and plastid function. These data raise the possibility that the apicoplast proteome is generated by two novel ER to plastid trafficking pathways, plus the small set of proteins encoded by the apicoplast genome. PMID:25369183

  13. Explosive cell lysis as a mechanism for the biogenesis of bacterial membrane vesicles and biofilms.

    PubMed

    Turnbull, Lynne; Toyofuku, Masanori; Hynen, Amelia L; Kurosawa, Masaharu; Pessi, Gabriella; Petty, Nicola K; Osvath, Sarah R; Cárcamo-Oyarce, Gerardo; Gloag, Erin S; Shimoni, Raz; Omasits, Ulrich; Ito, Satoshi; Yap, Xinhui; Monahan, Leigh G; Cavaliere, Rosalia; Ahrens, Christian H; Charles, Ian G; Nomura, Nobuhiko; Eberl, Leo; Whitchurch, Cynthia B

    2016-01-01

    Many bacteria produce extracellular and surface-associated components such as membrane vesicles (MVs), extracellular DNA and moonlighting cytosolic proteins for which the biogenesis and export pathways are not fully understood. Here we show that the explosive cell lysis of a sub-population of cells accounts for the liberation of cytosolic content in Pseudomonas aeruginosa biofilms. Super-resolution microscopy reveals that explosive cell lysis also produces shattered membrane fragments that rapidly form MVs. A prophage endolysin encoded within the R- and F-pyocin gene cluster is essential for explosive cell lysis. Endolysin-deficient mutants are defective in MV production and biofilm development, consistent with a crucial role in the biogenesis of MVs and liberation of extracellular DNA and other biofilm matrix components. Our findings reveal that explosive cell lysis, mediated through the activity of a cryptic prophage endolysin, acts as a mechanism for the production of bacterial MVs. PMID:27075392

  14. Explosive cell lysis as a mechanism for the biogenesis of bacterial membrane vesicles and biofilms.

    PubMed

    Turnbull, Lynne; Toyofuku, Masanori; Hynen, Amelia L; Kurosawa, Masaharu; Pessi, Gabriella; Petty, Nicola K; Osvath, Sarah R; Cárcamo-Oyarce, Gerardo; Gloag, Erin S; Shimoni, Raz; Omasits, Ulrich; Ito, Satoshi; Yap, Xinhui; Monahan, Leigh G; Cavaliere, Rosalia; Ahrens, Christian H; Charles, Ian G; Nomura, Nobuhiko; Eberl, Leo; Whitchurch, Cynthia B

    2016-04-14

    Many bacteria produce extracellular and surface-associated components such as membrane vesicles (MVs), extracellular DNA and moonlighting cytosolic proteins for which the biogenesis and export pathways are not fully understood. Here we show that the explosive cell lysis of a sub-population of cells accounts for the liberation of cytosolic content in Pseudomonas aeruginosa biofilms. Super-resolution microscopy reveals that explosive cell lysis also produces shattered membrane fragments that rapidly form MVs. A prophage endolysin encoded within the R- and F-pyocin gene cluster is essential for explosive cell lysis. Endolysin-deficient mutants are defective in MV production and biofilm development, consistent with a crucial role in the biogenesis of MVs and liberation of extracellular DNA and other biofilm matrix components. Our findings reveal that explosive cell lysis, mediated through the activity of a cryptic prophage endolysin, acts as a mechanism for the production of bacterial MVs.

  15. Protein kinase A dependent membrane protein phosphorylation and chloride conductance in endosomal vesicles from kidney cortex

    SciTech Connect

    Reenstra, W.W.; Bae, H.R.; Verkman, A.S. Univ. of California, San Francisco ); Sabolic, I. Harvard Medical School, Charlestown, MA )

    1992-01-14

    Regulation of Cl conductance by protein kinase A action, cell-free measurements of Cl transport and membrane protein phosphorylation were carried out in apical endocytic vesicles from rabbit kidney proximal tubule. Cl transport was measured by a stopped-flow quenching assay in endosomes labeled in vivo with the fluorescent Cl indicator 6-methoxy-N-(3-sulfopropyl)quinolinium. Phosphorylation was studied in a purified endosomal preparation by SDS-PAGE and autoradiography of membrane proteins labeled by ({gamma}-{sup 32}P)ATP. These results suggest that, in a cell-free system, protein kinase A increases Cl conductance in endosomes from kidney proximal tubule by a phosphorylation mechanism. The labeled protein has a size similar to that of the 64-kDa putative kidney Cl channel reported by Landry et al. but is much smaller than the {approximately}170-kDa cystic fibrosis transmembrane conductance regulatory protein.

  16. Positional assembly of enzymes on bacterial outer membrane vesicles for cascade reactions.

    PubMed

    Park, Miso; Sun, Qing; Liu, Fang; DeLisa, Matthew P; Chen, Wilfred

    2014-01-01

    The systematic organization of enzymes is a key feature for the efficient operation of cascade reactions in nature. Here, we demonstrate a facile method to create nanoscale enzyme cascades by using engineered bacterial outer membrane vesicles (OMVs) that are spheroid nanoparticles (roughly 50 nm in diameter) produced by Gram-negative bacteria during all phases of growth. By taking advantage of the fact that OMVs naturally contain proteins found in the outer cell membrane, we displayed a trivalent protein scaffold containing three divergent cohesin domains for the position-specific presentation of a three-enzyme cascade on OMVs through a truncated ice nucleation protein anchoring motif (INP). The positional assembly of three enzymes for cellulose hydrolysis was demonstrated. The enzyme-decorated OMVs provided synergistic cellulose hydrolysis resulting in 23-fold enhancement in glucose production than free enzymes. PMID:24820175

  17. Explosive cell lysis as a mechanism for the biogenesis of bacterial membrane vesicles and biofilms

    PubMed Central

    Turnbull, Lynne; Toyofuku, Masanori; Hynen, Amelia L.; Kurosawa, Masaharu; Pessi, Gabriella; Petty, Nicola K.; Osvath, Sarah R.; Cárcamo-Oyarce, Gerardo; Gloag, Erin S.; Shimoni, Raz; Omasits, Ulrich; Ito, Satoshi; Yap, Xinhui; Monahan, Leigh G.; Cavaliere, Rosalia; Ahrens, Christian H.; Charles, Ian G.; Nomura, Nobuhiko; Eberl, Leo; Whitchurch, Cynthia B.

    2016-01-01

    Many bacteria produce extracellular and surface-associated components such as membrane vesicles (MVs), extracellular DNA and moonlighting cytosolic proteins for which the biogenesis and export pathways are not fully understood. Here we show that the explosive cell lysis of a sub-population of cells accounts for the liberation of cytosolic content in Pseudomonas aeruginosa biofilms. Super-resolution microscopy reveals that explosive cell lysis also produces shattered membrane fragments that rapidly form MVs. A prophage endolysin encoded within the R- and F-pyocin gene cluster is essential for explosive cell lysis. Endolysin-deficient mutants are defective in MV production and biofilm development, consistent with a crucial role in the biogenesis of MVs and liberation of extracellular DNA and other biofilm matrix components. Our findings reveal that explosive cell lysis, mediated through the activity of a cryptic prophage endolysin, acts as a mechanism for the production of bacterial MVs. PMID:27075392

  18. Interaction of multiwalled carbon nanotubes with supported lipid bilayers and vesicles as model biological membranes.

    PubMed

    Yi, Peng; Chen, Kai Loon

    2013-06-01

    The influence of solution chemistry on the kinetics and reversibility of the deposition of multiwalled carbon nanotubes (MWNTs) on model biological membranes was investigated using a quartz crystal microbalance with dissipation monitoring (QCM-D). Supported lipid bilayers (SLBs) comprised of zwitterionic 1,2-dioleoyl-sn-glyero-3-phosphocholine (DOPC), as well as DOPC vesicles, were used as model cell membranes. Under neutral pH conditions, the deposition kinetics of MWNTs on SLBs increased with increasing electrolyte (NaCl and CaCl2) concentrations. In the presence of NaCl, favorable deposition was not achieved even at a concentration of 1 M, which is attributed to the presence of strong repulsive hydration forces due to the highly hydrophilic headgroups of SLBs. Conversely, favorable deposition was observed at CaCl2 concentrations above 0.5 mM when the charge of SLBs was reversed from negative to positive through the binding of Ca(2+) cations to the exposed phosphate headgroups. Favorable nanotube deposition was also observed at pH 2, at which the DOPC SLBs exhibited positive surface charge, since the isoelectric point of DOPC is ca. 4. When MWNTs on SLBs were rinsed with low ionic strength solutions at pH 7.3, only ca. 20% of deposited nanotubes were released, indicating that nanotube deposition was mostly irreversible. The deposition of MWNTs on DOPC vesicles under favorable deposition conditions did not result in any detectable leakage of solution from the vesicles, indicating that MWNTs did not severely disrupt the DOPC bilayers upon attachment.

  19. Decoration of outer membrane vesicles with multiple antigens by using an autotransporter approach.

    PubMed

    Daleke-Schermerhorn, Maria H; Felix, Tristan; Soprova, Zora; Ten Hagen-Jongman, Corinne M; Vikström, David; Majlessi, Laleh; Beskers, Joep; Follmann, Frank; de Punder, Karin; van der Wel, Nicole N; Baumgarten, Thomas; Pham, Thang V; Piersma, Sander R; Jiménez, Connie R; van Ulsen, Peter; de Gier, Jan-Willem; Leclerc, Claude; Jong, Wouter S P; Luirink, Joen

    2014-09-01

    Outer membrane vesicles (OMVs) are spherical nanoparticles that naturally shed from Gram-negative bacteria. They are rich in immunostimulatory proteins and lipopolysaccharide but do not replicate, which increases their safety profile and renders them attractive vaccine vectors. By packaging foreign polypeptides in OMVs, specific immune responses can be raised toward heterologous antigens in the context of an intrinsic adjuvant. Antigens exposed at the vesicle surface have been suggested to elicit protection superior to that from antigens concealed inside OMVs, but hitherto robust methods for targeting heterologous proteins to the OMV surface have been lacking. We have exploited our previously developed hemoglobin protease (Hbp) autotransporter platform for display of heterologous polypeptides at the OMV surface. One, two, or three of the Mycobacterium tuberculosis antigens ESAT6, Ag85B, and Rv2660c were targeted to the surface of Escherichia coli OMVs upon fusion to Hbp. Furthermore, a hypervesiculating ΔtolR ΔtolA derivative of attenuated Salmonella enterica serovar Typhimurium SL3261 was generated, enabling efficient release and purification of OMVs decorated with multiple heterologous antigens, exemplified by the M. tuberculosis antigens and epitopes from Chlamydia trachomatis major outer membrane protein (MOMP). Also, we showed that delivery of Salmonella OMVs displaying Ag85B to antigen-presenting cells in vitro results in processing and presentation of an epitope that is functionally recognized by Ag85B-specific T cell hybridomas. In conclusion, the Hbp platform mediates efficient display of (multiple) heterologous antigens, individually or combined within one molecule, at the surface of OMVs. Detection of antigen-specific immune responses upon vesicle-mediated delivery demonstrated the potential of our system for vaccine development. PMID:25038093

  20. Sulfate uptake by crustacean hepatopancreatic brush border membrane vesicles. [Homarus americanus

    SciTech Connect

    Gerencser, G.A.; Cattey, M.A; Ahearn, G.A. Univ. of Hawaii, Honolulu )

    1990-02-26

    Purified brush border membrane vesicles (BBMV) were prepared from Atlantic lobster (Homarus americanus) hepatopancreas using differential centrifugation and Mg{sup +2} precipitation techniques. Uptake of 0.1 mM {sup 35}SO{sub 4}{sup {minus}2} was stimulated by pre-loading vesicles with Cl{sup {minus}} leading to a transient accumulation of isotope more than twice that at equilibrium. Pre-loading with HCO{sub 3}{sup {minus}} or gluconate had no effect on sulfate uptake. No stimulation of {sup 35}SO{sub 4}{sup {minus}2} was observed in the presence of inwardly directed Na{sup +} or tetramethylammonium{sup +} gradients. Uptake of the divalent anion was strongly stimulated by inwardly directed proton gradients (pH{sub o} < pH{sub i}) and markedly inhibited by outwardly directed proton gradients (pH{sub o} > pH{sub i}). {sup 35}SO{sub 4}{sup {minus}2}/Cl{sup {minus}} exchange was enhanced by imposing a transmembrane inside positive K{sup +} diffusion potential and inhibited by a membrane potential of the opposite polarity (K{sup +}/valinomycin). Results suggest the presence of a proton-dependent, electrogenic anion antiport mechanism in BBMV isolated from the crustacean hepatopancreas.

  1. Vesicles between plasma membrane and cell wall prior to visible senescence of Iris and Dendrobium flowers.

    PubMed

    Kamdee, Channatika; Kirasak, Kanjana; Ketsa, Saichol; van Doorn, Wouter G

    2015-09-01

    Cut Iris flowers (Iris x hollandica, cv. Blue Magic) show visible senescence about two days after full opening. Epidermal cells of the outer tepals collapse due to programmed cell death (PCD). Transmission electron microscopy (TEM) showed irregular swelling of the cell walls, starting prior to cell collapse. Compared to cells in flowers that had just opened, wall thickness increased up to tenfold prior to cell death. Fibrils were visible in the swollen walls. After cell death very little of the cell wall remained. Prior to and during visible wall swelling, vesicles (paramural bodies) were observed between the plasma membrane and the cell walls. The vesicles were also found in groups and were accompanied by amorphous substance. They usually showed a single membrane, and had a variety of diameters and electron densities. Cut Dendrobium hybrid cv. Lucky Duan flowers exhibited visible senescence about 14 days after full flower opening. Paramural bodies were also found in Dendrobium tepal epidermis and mesophyll cells, related to wall swelling and degradation. Although alternative explanations are well possible, it is hypothesized that paramural bodies carry enzymes involved in cell wall breakdown. The literature has not yet reported such bodies in association with senescence/PCD.

  2. Coordinated recruitment of Spir actin nucleators and myosin V motors to Rab11 vesicle membranes

    PubMed Central

    Pylypenko, Olena; Welz, Tobias; Tittel, Janine; Kollmar, Martin; Chardon, Florian; Malherbe, Gilles; Weiss, Sabine; Michel, Carina Ida Luise; Samol-Wolf, Annette; Grasskamp, Andreas Till; Hume, Alistair; Goud, Bruno; Baron, Bruno; England, Patrick; Titus, Margaret A; Schwille, Petra; Weidemann, Thomas

    2016-01-01

    There is growing evidence for a coupling of actin assembly and myosin motor activity in cells. However, mechanisms for recruitment of actin nucleators and motors on specific membrane compartments remain unclear. Here we report how Spir actin nucleators and myosin V motors coordinate their specific membrane recruitment. The myosin V globular tail domain (MyoV-GTD) interacts directly with an evolutionarily conserved Spir sequence motif. We determined crystal structures of MyoVa-GTD bound either to the Spir-2 motif or to Rab11 and show that a Spir-2:MyoVa:Rab11 complex can form. The ternary complex architecture explains how Rab11 vesicles support coordinated F-actin nucleation and myosin force generation for vesicle transport and tethering. New insights are also provided into how myosin activation can be coupled with the generation of actin tracks. Since MyoV binds several Rab GTPases, synchronized nucleator and motor targeting could provide a common mechanism to control force generation and motility in different cellular processes. DOI: http://dx.doi.org/10.7554/eLife.17523.001 PMID:27623148

  3. Coordinated recruitment of Spir actin nucleators and myosin V motors to Rab11 vesicle membranes.

    PubMed

    Pylypenko, Olena; Welz, Tobias; Tittel, Janine; Kollmar, Martin; Chardon, Florian; Malherbe, Gilles; Weiss, Sabine; Michel, Carina Ida Luise; Samol-Wolf, Annette; Grasskamp, Andreas Till; Hume, Alistair; Goud, Bruno; Baron, Bruno; England, Patrick; Titus, Margaret A; Schwille, Petra; Weidemann, Thomas; Houdusse, Anne; Kerkhoff, Eugen

    2016-01-01

    There is growing evidence for a coupling of actin assembly and myosin motor activity in cells. However, mechanisms for recruitment of actin nucleators and motors on specific membrane compartments remain unclear. Here we report how Spir actin nucleators and myosin V motors coordinate their specific membrane recruitment. The myosin V globular tail domain (MyoV-GTD) interacts directly with an evolutionarily conserved Spir sequence motif. We determined crystal structures of MyoVa-GTD bound either to the Spir-2 motif or to Rab11 and show that a Spir-2:MyoVa:Rab11 complex can form. The ternary complex architecture explains how Rab11 vesicles support coordinated F-actin nucleation and myosin force generation for vesicle transport and tethering. New insights are also provided into how myosin activation can be coupled with the generation of actin tracks. Since MyoV binds several Rab GTPases, synchronized nucleator and motor targeting could provide a common mechanism to control force generation and motility in different cellular processes. PMID:27623148

  4. Outer Membrane Vesicle Biosynthesis in Salmonella: Is There More to Gram-Negative Bacteria?

    PubMed Central

    2016-01-01

    ABSTRACT Recent research has focused on the biological role of outer membrane vesicles (OMVs), which are derived from the outer membranes (OMs) of Gram-negative bacteria, and their potential exploitation as therapeutics. OMVs have been characterized in many ways and functions. Until recently, research focused on hypothetical and empirical models that addressed the molecular mechanisms of OMV biogenesis, such as vesicles bulging from the OM in various ways. The recently reported study by Elhenawy et al. (mBio 7:e00940-16, 2016, http://dx.doi.org/10.1128/mBio.00940-16) provided further insights into OMV biogenesis of Salmonella enterica serovar Typhimurium. That study showed that deacylation of lipopolysaccharides (LPS) influences the level of OMV production and, furthermore, determines a sorting of high versus low acylated LPS in OMs and OMVs, respectively. Interestingly, deacylation may inversely correlate with other LPS modifications, suggesting some synergy toward optimized host resistance via best OM compositions for S. Typhimurium. PMID:27531914

  5. Calcium transport in sealed vesicles from red beet (Beta vulgaris L. ) storage tissue. II. Characterization of /sup 45/Ca/sup 2 +/ uptake into plasma membrane vesicles

    SciTech Connect

    Giannini, J.L.; Ruiz-Cristin, J.; Briskin, D.P.

    1987-12-01

    Calcium uptake was examined in sealed plasma membrane vesicles isolated from red beet (Beta vulgaris L.) storage tissue using /sup 45/Ca/sup 2 +/. Uptake of /sup 45/Ca/sup 2 +/ by the vesicles was ATP-dependent and radiotracer accumulated by the vesicles could be released by the addition of the calcium ionophore A23187. The uptake was stimulated by gramicidin D but slightly inhibited by carbonylcyanide m-chlorophenylhydrazone. Although the latter result might suggest some degree of indirect coupling of /sup 45/Ca/sup 2 +/ uptake to ATP utilization via ..delta mu..H/sup +/, no evidence for a secondary H/sup +//Ca/sup 2 +/ antiport in this vesicle system could be found. Following the imposition of an acid-interior pH gradient, proton efflux from the vesicle was not enhanced by the addition of Ca/sup 2 +/ and an imposed pH gradient could not drive /sup 45/Ca/sup 2 +/ uptake. Optimal uptake of /sup 45/Ca/sup 2 +/ occurred broadly between pH 7.0 and 7.5 and the transport was inhibited by orthovanadate, N,N'-dicyclohexylcarbodiimide, and diethylstilbestrol but insensitive to nitrate and azide. The dependence of /sup 45/Ca/sup 2 +/ uptake on both calcium and Mg:ATP concentration demonstrated saturation kinetics with K/sub m/ values of 6 micromolar and 0.37 millimolar, respectively. While ATP was the preferred substrate for driving /sup 45/Ca/sup 2 +/ uptake, GTP could drive transport at about 50% of the level observed for ATP. The results of this study demonstrate the presence of a unique primary calcium transport system associated with the plasma membrane which could drive calcium efflux from the plant cell.

  6. On-chip light sheet illumination enables diagnostic size and concentration measurements of membrane vesicles in biofluids

    NASA Astrophysics Data System (ADS)

    Deschout, Hendrik; Raemdonck, Koen; Stremersch, Stephan; Maoddi, Pietro; Mernier, Guillaume; Renaud, Philippe; Jiguet, Sébastien; Hendrix, An; Bracke, Marc; van den Broecke, Rudy; Röding, Magnus; Rudemo, Mats; Demeester, Jo; de Smedt, Stefaan C.; Strubbe, Filip; Neyts, Kristiaan; Braeckmans, Kevin

    2014-01-01

    Cell-derived membrane vesicles that are released in biofluids, like blood or saliva, are emerging as potential non-invasive biomarkers for diseases, such as cancer. Techniques capable of measuring the size and concentration of membrane vesicles directly in biofluids are urgently needed. Fluorescence single particle tracking microscopy has the potential of doing exactly that by labelling the membrane vesicles with a fluorescent label and analysing their Brownian motion in the biofluid. However, an unbound dye in the biofluid can cause high background intensity that strongly biases the fluorescence single particle tracking size and concentration measurements. While such background intensity can be avoided with light sheet illumination, current set-ups require specialty sample holders that are not compatible with high-throughput diagnostics. Here, a microfluidic chip with integrated light sheet illumination is reported, and accurate fluorescence single particle tracking size and concentration measurements of membrane vesicles in cell culture medium and in interstitial fluid collected from primary human breast tumours are demonstrated.Cell-derived membrane vesicles that are released in biofluids, like blood or saliva, are emerging as potential non-invasive biomarkers for diseases, such as cancer. Techniques capable of measuring the size and concentration of membrane vesicles directly in biofluids are urgently needed. Fluorescence single particle tracking microscopy has the potential of doing exactly that by labelling the membrane vesicles with a fluorescent label and analysing their Brownian motion in the biofluid. However, an unbound dye in the biofluid can cause high background intensity that strongly biases the fluorescence single particle tracking size and concentration measurements. While such background intensity can be avoided with light sheet illumination, current set-ups require specialty sample holders that are not compatible with high-throughput diagnostics

  7. Multiscale modeling of mechanosensing channels on vesicles and cell membranes in 3D constricted flows and shear flows

    NASA Astrophysics Data System (ADS)

    Peng, Zhangli; Pak, On Shun; Young, Yuan-Nan; Liu, Allen; Stone, Howard

    2015-11-01

    We investigate the gating of mechanosensing channels (Mscls) on vesicles and cell membranes under different flow conditions using a multiscale approach. At the cell level (microns), the membrane tension is calculated using a 3D two-component whole-cell membrane model based on dissipative particle dynamics (DPD), including the cortex cytoskeleton and its interactions with the lipid bilayer. At the Mscl level (nanometers), we predict the relation between channel gating and the membrane tension obtained from a cell-level model using a semi-analytical model based on the bilayer hydrophobic mismatch energy. We systematically study the gating of Mscls of vesicles and cell membranes in constricted channel flows and shear flows, and explore the dependence of the gating on flow rate, cell shape and size. The results provide guidance for future experiments in inducing Mscl opening for various purposes such as drug delivery.

  8. Proteome analysis of outer membrane vesicles from a clinical Acinetobacter baumannii isolate.

    PubMed

    Kwon, Sang-Oh; Gho, Yong Song; Lee, Je Chul; Kim, Seung Il

    2009-08-01

    The secretion of outer membrane vesicles (OMVs) is one of the major mechanisms by which Gram-negative bacteria deliver effector molecules to host cells. Acinetobacter baumannii is an important opportunistic pathogen in hospital-acquired infections, but the secretion system for effector molecules to induce host cell damage has not been characterized. In the present study, we investigated the secretion of OMVs from a clinical A. baumannii isolate and analyzed the comprehensive proteome of A. baumannii-derived OMVs. Acinetobacter baumannii secreted OMVs into the extracellular milieu during in vitro growth. Using 1-DE and LC-MS/MS protein identification and assignment analysis, 132 different proteins associated with OMVs were identified. These proteins were derived from outer membranes (n=26), periplasmic space (n=6), inner membranes (n=8), cytoplasm (n=43), and unknown localization or multiple localization sites (n=49) according to the cell location prediction programs. Among the proteins associated with OMVs, a potent cytotoxic molecule, outer membrane protein A, was highly enriched and several putative virulence-associated proteins were also identified. These results suggest that OMVs from A. baumannii are an important vehicle designed to deliver effector molecules to host cells.

  9. Membrane Vesicles of Group B Streptococcus Disrupt Feto-Maternal Barrier Leading to Preterm Birth

    PubMed Central

    Sthanam, Lakshmi Kavitha; Srivastava, Rohit; Basu, Bhakti; Dutta, Suryendu; Sen, Shamik; Modi, Deepak

    2016-01-01

    Infection of the genitourinary tract with Group B Streptococcus (GBS), an opportunistic gram positive pathogen, is associated with premature rupture of amniotic membrane and preterm birth. In this work, we demonstrate that GBS produces membrane vesicles (MVs) in a serotype independent manner. These MVs are loaded with virulence factors including extracellular matrix degrading proteases and pore forming toxins. Mice chorio-decidual membranes challenged with MVs ex vivo resulted in extensive collagen degradation leading to loss of stiffness and mechanical weakening. MVs when instilled vaginally are capable of anterograde transport in mouse reproductive tract. Intra-amniotic injections of GBS MVs in mice led to upregulation of pro-inflammatory cytokines and inflammation mimicking features of chorio-amnionitis; it also led to apoptosis in the chorio-decidual tissue. Instillation of MVs in the amniotic sac also resulted in intrauterine fetal death and preterm delivery. Our findings suggest that GBS MVs can independently orchestrate events at the feto-maternal interface causing chorio-amnionitis and membrane damage leading to preterm birth or fetal death. PMID:27583406

  10. Membrane Vesicles of Group B Streptococcus Disrupt Feto-Maternal Barrier Leading to Preterm Birth.

    PubMed

    Surve, Manalee Vishnu; Anil, Anjali; Kamath, Kshama Ganesh; Bhutda, Smita; Sthanam, Lakshmi Kavitha; Pradhan, Arpan; Srivastava, Rohit; Basu, Bhakti; Dutta, Suryendu; Sen, Shamik; Modi, Deepak; Banerjee, Anirban

    2016-09-01

    Infection of the genitourinary tract with Group B Streptococcus (GBS), an opportunistic gram positive pathogen, is associated with premature rupture of amniotic membrane and preterm birth. In this work, we demonstrate that GBS produces membrane vesicles (MVs) in a serotype independent manner. These MVs are loaded with virulence factors including extracellular matrix degrading proteases and pore forming toxins. Mice chorio-decidual membranes challenged with MVs ex vivo resulted in extensive collagen degradation leading to loss of stiffness and mechanical weakening. MVs when instilled vaginally are capable of anterograde transport in mouse reproductive tract. Intra-amniotic injections of GBS MVs in mice led to upregulation of pro-inflammatory cytokines and inflammation mimicking features of chorio-amnionitis; it also led to apoptosis in the chorio-decidual tissue. Instillation of MVs in the amniotic sac also resulted in intrauterine fetal death and preterm delivery. Our findings suggest that GBS MVs can independently orchestrate events at the feto-maternal interface causing chorio-amnionitis and membrane damage leading to preterm birth or fetal death. PMID:27583406

  11. Modeling the effects of cyclodextrin on intracellular membrane vesicles from Cos-7 cells prepared by sonication and carbonate treatment

    PubMed Central

    Kilbride, Peter; Woodward, Holly J.; Tan, Kuan Boone; Thanh, Nguyễn T.K.; Chu, K.M. Emily; Minogue, Shane

    2015-01-01

    Cholesterol has important functions in the organization of membrane structure and this may be mediated via the formation of cholesterol-rich, liquid-ordered membrane microdomains often referred to as lipid rafts. Methyl-beta-cyclodextrin (cyclodextrin) is commonly used in cell biology studies to extract cholesterol and therefore disrupt lipid rafts. However, in this study we reassessed this experimental strategy and investigated the effects of cyclodextrin on the physical properties of sonicated and carbonate-treated intracellular membrane vesicles isolated from Cos-7 fibroblasts. We treated these membranes, which mainly originate from the trans-Golgi network and endosomes, with cyclodextrin and measured the effects on their equilibrium buoyant density, protein content, represented by the palmitoylated protein phosphatidylinositol 4-kinase type IIα, and cholesterol. Despite the reduction in mass stemming from cholesterol removal, the vesicles became denser, indicating a possible large volumetric decrease, and this was confirmed by measurements of hydrodynamic vesicle size. Subsequent mathematical analyses demonstrated that only half of this change in membrane size was attributable to cholesterol loss. Hence, the non-selective desorption properties of cyclodextrin are also involved in membrane size and density changes. These findings may have implications for preceding studies that interpreted cyclodextrin-induced changes to membrane biochemistry in the context of lipid raft disruption without taking into account our finding that cyclodextrin treatment also reduces membrane size. PMID:26528413

  12. Membrane vesicles: A simplified system for studying auxin transport. Final technical report

    SciTech Connect

    Goldsmith, M.H.M.

    1989-12-31

    Indoleacetic acid (IAA), the auxin responsible for regulation of growth, is transported polarly in plants. Several different models have been suggested to account for IAA transport by cells and its accumulation by membrane vesicles. One model sees diffusion of IAA driven by a pH gradient. The anion of a lipophilic weak acid like IAA or butyrate accumulates in an alkaline compartment in accord with the size of the pH gradient The accumulation of IAA may be diminished by the permeability of its lipophilic anion. This anion leak may be blocked by NPA. With anion efflux blocked, a gradient of two pH units would support an IAA accumulation of less than 50-fold at equilibrium (2) Another model sees diffusion of IAA in parallel with a saturable symport (IAA{sup {minus}} + nH{sup +}), driven by both the pH gradient and membrane voltage. Such a symport should be highly accumulative, however, with a lipophilic weak acid such as IAA, net diffusive efflux of IAAH whenever IAAHI{sub i} > IAAH{sub o} would constitute a leak. (3) A third model sees a pH change driven IAA uptake and saturable symport enhanced by internal binding sites. Following pH gradient-driven accumulation of IAA, the anion may bind to an intravesicular site, permitting further uptake of IAA. NPA, by blocking anion efflux, enhances this binding. We have reported that membrane vesicles isolated from actively growing plant tissues are a good system for studying the mechanisms involved in the transport and accumulation of auxin.

  13. Discovery of Salmonella virulence factors translocated via outer membrane vesicles to murine macrophages.

    PubMed

    Yoon, Hyunjin; Ansong, Charles; Adkins, Joshua N; Heffron, Fred

    2011-06-01

    Salmonella enterica serovar Typhimurium, an intracellular pathogen and leading cause of food-borne illness, encodes a plethora of virulence effectors. Salmonella virulence factors are translocated into host cells and manipulate host cellular activities, providing a more hospitable environment for bacterial proliferation. In this study, we report a new set of virulence factors that is translocated into the host cytoplasm via bacterial outer membrane vesicles (OMV). PagK (or PagK1), PagJ, and STM2585A (or PagK2) are small proteins composed of ∼70 amino acids and have high sequence homology to each other (>85% identity). Salmonella lacking all three homologues was attenuated for virulence in a mouse infection model, suggesting at least partial functional redundancy among the homologues. While each homologue was translocated into the macrophage cytoplasm, their translocation was independent of all three Salmonella gene-encoded type III secretion systems (T3SSs)-Salmonella pathogenicity island 1 (SPI-1) T3SS, SPI-2 T3SS, and the flagellar system. Selected methods, including direct microscopy, demonstrated that the PagK-homologous proteins were secreted through OMV, which were enriched with lipopolysaccharide (LPS) and outer membrane proteins. Vesicles produced by intracellular bacteria also contained lysosome-associated membrane protein 1 (LAMP1), suggesting the possibility of OMV convergence with host cellular components during intracellular trafficking. This study identified novel Salmonella virulence factors secreted via OMV and demonstrated that OMV can function as a vehicle to transfer virulence determinants to the cytoplasm of the infected host cell.

  14. Uptake of (/sup 3/H)serotonin into plasma membrane vesicles from mouse cerebral cortex

    SciTech Connect

    O'Reilly, C.A.; Reith, M.E.A.

    1988-05-05

    Preparations of plasma membrane vesicles were used as a tool to study the properties of the serotonin transporter in the central nervous system. The vesicles were obtained after hypotonic shock of synaptosomes purified from mouse cerebral cortex. Uptake of (/sup 3/H)serotonin had a Na/sup +/-dependent and Na/sup +/-independent component. The Na/sup +/-dependent uptake was inhibited by classical blockers of serotonin uptake and had a K/sub m/ of 63-180 nM, and a V/sub max/ of 0.1-0.3 pmol mg/sup -1/ s/sup -1/ at 77 mM Na/sup +/. The uptake required the presence of external Na/sup +/ and internal K/sup +/. Replacement of Cl/sup -/ by other anions (NO/sub 2//sup -/, S/sub 2/O/sub 3//sup 2 -/) reduced uptake appreciably. Gramicidin prevented uptake. Although valinomycin increased uptake somewhat, the membrane potential per se could not drive uptake because no uptake was observed when a membrane potential was generated by the SCN/sup -/ ion in the absence of internal K/sup +/ and with equal (Na/sup +/) inside and outside. The increase of uptake as a function of (Na/sup +/) indicated a K/sub m/ for Na/sup +/ of 118 mM and a Hill number of 2.0, suggesting a requirement of two sodium ions for serotonin transport. The present results are accommodated very well by the model developed for porcine platelet serotonin transport except for the number of sodium ions that are required for transport.

  15. Analysis of Arf1 GTPase-dependent membrane binding and remodeling using the exomer secretory vesicle cargo adaptor

    PubMed Central

    Paczkowski, Jon E.; Fromme, J. Christopher

    2016-01-01

    Summary Protein-protein and protein-membrane interactions play a critical role in shaping biological membranes through direct physical contact with the membrane surface. This is particularly evident in many steps of membrane trafficking, in which proteins deform the membrane and induce fission to form transport carriers. The small GTPase Arf1 and related proteins have the ability to remodel membranes by insertion of an amphipathic helix into the membrane. Arf1 and the exomer cargo adaptor coordinate cargo sorting into subset of secretory vesicle carriers in the model organism Saccharomyces cerevisiae. Here, we detail the assays we used to explore the cooperative action of Arf1 and exomer to bind and remodel membranes. We expect these methods are broadly applicable to other small GTPase/effector systems where investigation of membrane binding and remodeling is of interest. PMID:27632000

  16. Analysis of Arf1 GTPase-Dependent Membrane Binding and Remodeling Using the Exomer Secretory Vesicle Cargo Adaptor.

    PubMed

    Paczkowski, Jon E; Fromme, J Christopher

    2016-01-01

    Protein-protein and protein-membrane interactions play a critical role in shaping biological membranes through direct physical contact with the membrane surface. This is particularly evident in many steps of membrane trafficking, in which proteins deform the membrane and induce fission to form transport carriers. The small GTPase Arf1 and related proteins have the ability to remodel membranes by insertion of an amphipathic helix into the membrane. Arf1 and the exomer cargo adaptor coordinate cargo sorting into subset of secretory vesicle carriers in the model organism Saccharomyces cerevisiae. Here, we detail the assays we used to explore the cooperative action of Arf1 and exomer to bind and remodel membranes. We expect these methods are broadly applicable to other small GTPase/effector systems where investigation of membrane binding and remodeling is of interest. PMID:27632000

  17. Multidrug Resistance-Associated Protein 2 (MRP2) Mediated Transport of Oxaliplatin-Derived Platinum in Membrane Vesicles.

    PubMed

    Myint, Khine; Li, Yan; Paxton, James; McKeage, Mark

    2015-01-01

    The platinum-based anticancer drug oxaliplatin is important clinically in cancer treatment. However, the role of multidrug resistance-associated protein 2 (MRP2) in controlling oxaliplatin membrane transport, in vivo handling, toxicity and therapeutic responses is unclear. In the current study, preparations of MRP2-expressing and control membrane vesicles, containing inside-out orientated vesicles, were used to directly characterise the membrane transport of oxaliplatin-derived platinum measured by inductively coupled plasma mass spectrometry. Oxaliplatin inhibited the ATP-dependent accumulation of the model MRP2 fluorescent probe, 5(6)-carboxy-2,'7'-dichlorofluorescein, in MRP2-expressing membrane vesicles. MRP2-expressing membrane vesicles accumulated up to 19-fold more platinum during their incubation with oxaliplatin and ATP as compared to control membrane vesicles and in the absence of ATP. The rate of ATP-dependent MRP2-mediated active transport of oxaliplatin-derived platinum increased non-linearly with increasing oxaliplatin exposure concentration, approaching a plateau value (Vmax) of 2680 pmol Pt/mg protein/10 minutes (95%CI, 2010 to 3360 pmol Pt/mg protein/10 minutes), with the half-maximal platinum accumulation rate (Km) at an oxaliplatin exposure concentration of 301 μM (95% CI, 163 to 438 μM), in accordance with Michaelis-Menten kinetics (r2 = 0.954). MRP2 inhibitors (myricetin and MK571) reduced the ATP-dependent accumulation of oxaliplatin-derived platinum in MRP2-expressing membrane vesicles in a concentration-dependent manner. To identify whether oxaliplatin, or perhaps a degradation product, was the likely substrate for this active transport, HPLC studies were undertaken showing that oxaliplatin degraded slowly in membrane vesicle incubation buffer containing chloride ions and glutathione, with approximately 95% remaining intact after a 10 minute incubation time and a degradation half-life of 2.24 hours (95%CI, 2.08 to 2.43 hours). In

  18. Multidrug Resistance-Associated Protein 2 (MRP2) Mediated Transport of Oxaliplatin-Derived Platinum in Membrane Vesicles

    PubMed Central

    Myint, Khine; Li, Yan; Paxton, James; McKeage, Mark

    2015-01-01

    The platinum-based anticancer drug oxaliplatin is important clinically in cancer treatment. However, the role of multidrug resistance-associated protein 2 (MRP2) in controlling oxaliplatin membrane transport, in vivo handling, toxicity and therapeutic responses is unclear. In the current study, preparations of MRP2-expressing and control membrane vesicles, containing inside-out orientated vesicles, were used to directly characterise the membrane transport of oxaliplatin-derived platinum measured by inductively coupled plasma mass spectrometry. Oxaliplatin inhibited the ATP-dependent accumulation of the model MRP2 fluorescent probe, 5(6)-carboxy-2,'7'-dichlorofluorescein, in MRP2-expressing membrane vesicles. MRP2-expressing membrane vesicles accumulated up to 19-fold more platinum during their incubation with oxaliplatin and ATP as compared to control membrane vesicles and in the absence of ATP. The rate of ATP-dependent MRP2-mediated active transport of oxaliplatin-derived platinum increased non-linearly with increasing oxaliplatin exposure concentration, approaching a plateau value (Vmax) of 2680 pmol Pt/mg protein/10 minutes (95%CI, 2010 to 3360 pmol Pt/mg protein/10 minutes), with the half-maximal platinum accumulation rate (Km) at an oxaliplatin exposure concentration of 301 μM (95% CI, 163 to 438 μM), in accordance with Michaelis-Menten kinetics (r2 = 0.954). MRP2 inhibitors (myricetin and MK571) reduced the ATP-dependent accumulation of oxaliplatin-derived platinum in MRP2-expressing membrane vesicles in a concentration-dependent manner. To identify whether oxaliplatin, or perhaps a degradation product, was the likely substrate for this active transport, HPLC studies were undertaken showing that oxaliplatin degraded slowly in membrane vesicle incubation buffer containing chloride ions and glutathione, with approximately 95% remaining intact after a 10 minute incubation time and a degradation half-life of 2.24 hours (95%CI, 2.08 to 2.43 hours). In

  19. The Neurospora crassa exocyst complex tethers Spitzenkörper vesicles to the apical plasma membrane during polarized growth

    PubMed Central

    Riquelme, Meritxell; Bredeweg, Erin L.; Callejas-Negrete, Olga; Roberson, Robert W.; Ludwig, Sarah; Beltrán-Aguilar, Alejandro; Seiler, Stephan; Novick, Peter; Freitag, Michael

    2014-01-01

    Fungal hyphae are among the most highly polarized cells. Hyphal polarized growth is supported by tip-directed transport of secretory vesicles, which accumulate temporarily in a stratified manner in an apical vesicle cluster, the Spitzenkörper. The exocyst complex is required for tethering of secretory vesicles to the apical plasma membrane. We determined that the presence of an octameric exocyst complex is required for the formation of a functional Spitzenkörper and maintenance of regular hyphal growth in Neurospora crassa. Two distinct localization patterns of exocyst subunits at the hyphal tip suggest the dynamic formation of two assemblies. The EXO-70/EXO-84 subunits are found at the peripheral part of the Spitzenkörper, which partially coincides with the outer macrovesicular layer, whereas exocyst components SEC-5, -6, -8, and -15 form a delimited crescent at the apical plasma membrane. Localization of SEC-6 and EXO-70 to the plasma membrane and the Spitzenkörper, respectively, depends on actin and microtubule cytoskeletons. The apical region of exocyst-mediated vesicle fusion, elucidated by the plasma membrane–associated exocyst subunits, indicates the presence of an exocytotic gradient with a tip-high maximum that dissipates gradually toward the subapex, confirming the earlier predictions of the vesicle supply center model for hyphal morphogenesis. PMID:24523289

  20. Proline transport by brush-border membrane vesicles of lobster antennal glands

    SciTech Connect

    Behnke, R.D.; Wong, R.K.; Huse, S.M.; Reshkin, S.J.; Ahearn, G.A. )

    1990-02-01

    Purified brush-border membrane vesicles (BBMV) of lobster antennal gland labyrinth and bladder were separately formed by a magnesium precipitation technique. L-(3H)proline uptake was stimulated by a transmembrane NaCl gradient (outside (o) greater than inside (i)) to a greater extent in BBMV from labyrinth than those from the bladder. Detailed study of the labyrinth proline-transport processes revealed a specific dependence on NaCl, with negligible stimulatory effects by NaSCN, Na-gluconate, or KCl. A transmembrane proton gradient (o greater than i) was without stimulatory effect on proline transport. A transmembrane potential difference alone, in the presence of equilibrated NaCl and L-(3H)proline, led to net influx of the labeled amino acid, suggesting that the uptake process was electrogenic and capable of bringing about the net transfer of positive charge to the vesicle interior. Although a transmembrane Na gradient alone, in the presence of equilibrated Cl and L-(3H)proline, was able to bring about the net influx of the amino acid, a transmembrane Cl gradient alone under Na- and L-(3H)proline-equilibrated conditions was not, suggesting that only the Na gradient could energize the carrier process through cotransport, while the anion served an essential activating role. Proline influx by these vesicles occurred by the combination of at least one saturable Michaelis-Menten carrier system (apparent Kt = 0.37 mM; apparent JM = 1.19 nmol.mg protein-1.10 s-1) and apparent diffusion (P = 0.33 nmol.mg protein-1.10 s-1.mM-1). Static head analysis of the transport process suggested a cotransport stoichiometry of 2 Na:1 proline with essential activation by Cl ion.

  1. Fusion of Legionella pneumophila outer membrane vesicles with eukaryotic membrane systems is a mechanism to deliver pathogen factors to host cell membranes.

    PubMed

    Jäger, Jens; Keese, Susanne; Roessle, Manfred; Steinert, Michael; Schromm, Andra B

    2015-05-01

    The formation and release of outer membrane vesicles (OMVs) is a phenomenon observed in many bacteria, including Legionella pneumophila. During infection, this human pathogen primarily invades alveolar macrophages and replicates within a unique membrane-bound compartment termed Legionella-containing vacuole. In the current study, we analysed the membrane architecture of L. pneumophila OMVs by small-angle X-ray scattering and biophysically characterized OMV membranes. We investigated the interaction of L. pneumophila OMVs with model membranes by Förster resonance energy transfer and Fourier transform infrared spectroscopy. These experiments demonstrated the incorporation of OMV membrane material into liposomes composed of different eukaryotic phospholipids, revealing an endogenous property of OMVs to fuse with eukaryotic membranes. Cellular co-incubation experiments showed a dose- and time-dependent binding of fluorophore-labelled OMVs to macrophages. Trypan blue quenching experiments disclosed a rapid internalization of OMVs into macrophages at 37 and 4 °C. Purified OMVs induced tumour necrosis factor-α production in human macrophages at concentrations starting at 300 ng ml(-1). Experiments on HEK293-TLR2 and TLR4/MD-2 cell lines demonstrated a dominance of TLR2-dependent signalling pathways. In summary, we demonstrate binding, internalization and biological activity of L. pneumophila OMVs on human macrophages. Our data support OMV membrane fusion as a mechanism for the remote delivery of virulence factors to host cells.

  2. Proteomic analysis of outer membrane vesicles from the probiotic strain Escherichia coli Nissle 1917.

    PubMed

    Aguilera, Laura; Toloza, Lorena; Giménez, Rosa; Odena, Antonia; Oliveira, Eliandre; Aguilar, Juan; Badia, Josefa; Baldomà, Laura

    2014-02-01

    Escherichia coli Nissle 1917 (EcN) is a probiotic used for the treatment of intestinal disorders. EcN improves gastrointestinal homeostasis and microbiota balance; however, little is known about how this probiotic delivers effector molecules to the host. Outer membrane vesicles (OMVs) are constitutively produced by Gram-negative bacteria and have a relevant role in bacteria-host interactions. Using 1D SDS-PAGE and highly sensitive LC-MS/MS analysis we identified in this study 192 EcN vesicular proteins with high confidence in three independent biological replicates. Of these proteins, 18 were encoded by strain-linked genes and 57 were common to pathogen-derived OMVs. These proteins may contribute to the ability of this probiotic to colonize the human gut as they fulfil functions related to adhesion, immune modulation or bacterial survival in host niches. This study describes the first global OMV proteome of a probiotic strain and provides evidence that probiotic-derived OMVs contain proteins that can target these vesicles to the host and mediate their beneficial effects on intestinal function. All MS data have been deposited in the ProteomeXchange with identifier PXD000367 (http://proteomecentral.proteomexchange.org/dataset/PXD000367). PMID:24307187

  3. Controlled magnetosomes: Embedding of magnetic nanoparticles into membranes of monodisperse lipid vesicles.

    PubMed

    Bixner, Oliver; Reimhult, Erik

    2016-03-15

    Magnetic nanoparticle-containing capsules have been proposed for many uses, including triggered drug delivery and imaging. Combining superparamagnetic iron oxide nanoparticles (SPIONs) with existing liposome drug delivery technology is an enticing near-future prospect, but it requires efficient methods of synthesis and formulation compatible with pharmaceutical applications. We report a facile way of producing large, unilamellar, and homogeneously sized magnetoliposomes with high content of monodisperse, hydrophobic SPIONs integrated in the lipid membrane by use of a solvent inversion technique. For low lipid concentrations, unilamellar and monodisperse vesicles were obtained that became increasingly multilamellar with higher lipid fraction. Both, the co-self-assembled structure and loading content were significantly influenced by the purity of the nanoparticle shell. SPIONs with homogeneous shells of nitrodopamine-anchored hydrophobic dispersants could be quantitatively loaded up to 20%w/w, while SPIONs also containing residual physisorbed oleic acid exhibited a loading cut-off around 10%w/w SPIONs accompanied by drastic changes in size distribution. Lipid acyl chain length crucially influenced the formation and resultant stability of the loaded assemblies. The formation of nanoparticle-loaded vesicles is exemplified in different biologically important media, yielding ready-to-use magnetoliposome formulations. PMID:26707773

  4. Apigenin and quercetin promote. Delta. pH-dependent accumulation of IAA in membrane vesicles

    SciTech Connect

    Woolard, D.D.; Clark, K.A. )

    1990-05-01

    Flavonoids may act as regulators of polar auxin transport. In the presence of a pH gradient (pH 8{sub in}/6{sub out}) the flavonoids quercetin and apigenin, as well as the synthetic herbicide napthylphthalamic acid (NPA), promote the accumulation of IAA in membrane vesicles from dark-grown zucchini hypocotyls. Simultaneous accumulation of {sup 3}H-IAA (10 nM) and {sup 14}C-butyric acid (5 {mu}M; included as a pH probe) was determined by a filtration assay after incubating the vesicles with 3 nM to 100 {mu}M quercetin, apigenin, NPA or unlabeled IAA. Maximal stimulation (% of Control) was observed with 3 {mu}M NPA (130%), 1 {mu}M quercetin (120%), or 3 {mu}M apigenin (115%); {Delta}pH was not affected by these concentrations. As reported by others, IAA uptake was saturable: 1 {mu}M unlabeled IAA eliminated {Delta}pH-dependent uptake of {sup 3}H-IAA without altering {Delta}pH. However, at 30 to 100 {mu}M, every compound tested collapsed the imposed pH gradient and therefore abolished specific {sup 3}H-IAA uptake.

  5. Bacterial Nanobioreactors--Directing Enzyme Packaging into Bacterial Outer Membrane Vesicles.

    PubMed

    Alves, Nathan J; Turner, Kendrick B; Daniele, Michael A; Oh, Eunkeu; Medintz, Igor L; Walper, Scott A

    2015-11-11

    All bacteria shed outer membrane vesicles (OMVs) loaded with a diverse array of small molecules, proteins, and genetic cargo. In this study we sought to hijack the bacterial cell export pathway to simultaneously produce, package, and release an active enzyme, phosphotriesterase (PTE). To accomplish this goal the SpyCatcher/SpyTag (SC/ST) bioconjugation system was utilized to produce a PTE-SpyCatcher (PTE-SC) fusion protein and a SpyTagged transmembrane porin protein (OmpA-ST), known to be abundant in OMVs. Under a range of physiological conditions the SpyTag and SpyCatcher domains interact with one another and form a covalent isopeptide bond driving packaging of PTE into forming OMVs. The PTE-SC loaded OMVs are characterized for size distribution, number of vesicles produced, cell viability, packaged PTE enzyme kinetics, OMV loading efficiency, and enzyme stability following iterative cycles of freezing and thawing. The PTE-loaded OMVs exhibit native-like enzyme kinetics when assayed with paraoxon as a substrate. PTE is often toxic to expression cultures and has a tendency to lose activity with improper handling. The coexpression of OmpA-ST with PTE-SC, however, greatly improved the overall PTE production levels by mitigating toxicity through exporting of the PTE-SC and greatly enhanced packaged enzyme stability against iterative cycles of freezing and thawing. PMID:26479678

  6. Bacterial Nanobioreactors--Directing Enzyme Packaging into Bacterial Outer Membrane Vesicles.

    PubMed

    Alves, Nathan J; Turner, Kendrick B; Daniele, Michael A; Oh, Eunkeu; Medintz, Igor L; Walper, Scott A

    2015-11-11

    All bacteria shed outer membrane vesicles (OMVs) loaded with a diverse array of small molecules, proteins, and genetic cargo. In this study we sought to hijack the bacterial cell export pathway to simultaneously produce, package, and release an active enzyme, phosphotriesterase (PTE). To accomplish this goal the SpyCatcher/SpyTag (SC/ST) bioconjugation system was utilized to produce a PTE-SpyCatcher (PTE-SC) fusion protein and a SpyTagged transmembrane porin protein (OmpA-ST), known to be abundant in OMVs. Under a range of physiological conditions the SpyTag and SpyCatcher domains interact with one another and form a covalent isopeptide bond driving packaging of PTE into forming OMVs. The PTE-SC loaded OMVs are characterized for size distribution, number of vesicles produced, cell viability, packaged PTE enzyme kinetics, OMV loading efficiency, and enzyme stability following iterative cycles of freezing and thawing. The PTE-loaded OMVs exhibit native-like enzyme kinetics when assayed with paraoxon as a substrate. PTE is often toxic to expression cultures and has a tendency to lose activity with improper handling. The coexpression of OmpA-ST with PTE-SC, however, greatly improved the overall PTE production levels by mitigating toxicity through exporting of the PTE-SC and greatly enhanced packaged enzyme stability against iterative cycles of freezing and thawing.

  7. Controlled magnetosomes: Embedding of magnetic nanoparticles into membranes of monodisperse lipid vesicles.

    PubMed

    Bixner, Oliver; Reimhult, Erik

    2016-03-15

    Magnetic nanoparticle-containing capsules have been proposed for many uses, including triggered drug delivery and imaging. Combining superparamagnetic iron oxide nanoparticles (SPIONs) with existing liposome drug delivery technology is an enticing near-future prospect, but it requires efficient methods of synthesis and formulation compatible with pharmaceutical applications. We report a facile way of producing large, unilamellar, and homogeneously sized magnetoliposomes with high content of monodisperse, hydrophobic SPIONs integrated in the lipid membrane by use of a solvent inversion technique. For low lipid concentrations, unilamellar and monodisperse vesicles were obtained that became increasingly multilamellar with higher lipid fraction. Both, the co-self-assembled structure and loading content were significantly influenced by the purity of the nanoparticle shell. SPIONs with homogeneous shells of nitrodopamine-anchored hydrophobic dispersants could be quantitatively loaded up to 20%w/w, while SPIONs also containing residual physisorbed oleic acid exhibited a loading cut-off around 10%w/w SPIONs accompanied by drastic changes in size distribution. Lipid acyl chain length crucially influenced the formation and resultant stability of the loaded assemblies. The formation of nanoparticle-loaded vesicles is exemplified in different biologically important media, yielding ready-to-use magnetoliposome formulations.

  8. Effect of surface and membrane potentials on IAA (indoleactic acid) uptake and binding by zucchini membrane vesicles

    SciTech Connect

    Clark, K.A.; Goldsmith, M.H.M.

    1986-08-01

    The polar transport of the endogenous hormone controlling extension growth of plant cells, indoleacetic acid (IAA), is thought to depend on transmembrane pH and electrical gradients resulting in part from the action of proton ATPases in the plasma membrane. Elements of this transport process are permeation of the membrane by the undissociated lipophilic indoleacetic acid (IAAH) from the acidic apoplast, followed by dissociation of the weak acid and accumulation of the IAA anion (IAA/sup -/) in the alkaline cytoplasm; a saturable symport of IAA/sup -/ with one or more protons; a carrier-mediated efflux of IAA/sup -/ down a considerable electrochemical gradient. The efflux is greater from the basal than the apical end of cells and is thought to be responsible for the overall polarity of the process. This step is also the site of action of napthylphthalamic acid (NPA) and herbicides that inhibit polar transport but stimulate net accumulation of auxin by tissues and cells. We are using membrane vesicles as a simplified system for studying the mechanisms involved in the transport and accumulation of auxin. In particular, we are interested in determining the involvement of the transmembrane pH (pH/sub o/ < pH/sub i/) and voltage gradients (K/sup +/ diffusion potential, (K/sup +/)/sub in/ > (K/sup +/)/sub out/) in IAA uptake. 19 refs., 6 figs.

  9. Multiple sodium-dependent nucleoside transport systems in bovine renal brush-border membrane vesicles.

    PubMed Central

    Williams, T C; Jarvis, S M

    1991-01-01

    Na(+)-dependent nucleoside transport was examined in bovine renal brush-border membrane vesicles. Two separate Na+/nucleoside cotransporters were shown to be present: (1) a system specific for purine nucleosides and uridine, designated as the N1 carrier, and (2) an Na(+)-dependent nucleoside transporter that accepts pyrimidine nucleosides, adenosine and analogues of adenosine, designated as the N2 system. Both systems exhibit a high affinity for nucleosides (apparent Km values approximately 10 microM), are insensitive to inhibition by facilitated-diffusion nucleoside transport inhibitors, are rheogenic and exhibit a high specificity for Na+. Na+ increases the affinity of the influx of guanosine and thymidine, nucleosides that serve as model permeants for the N1 and N2 nucleoside transporters respectively. The Na+/nucleoside coupling stoichiometry is consistent with 1:1 for both carriers. PMID:2001243

  10. Evaluation of a Burkholderia pseudomallei Outer Membrane Vesicle Vaccine in Nonhuman Primates.

    PubMed

    Petersen, Hailey; Nieves, Wildaliz; Russell-Lodrigue, Kasi; Roy, Chad J; Morici, Lisa A

    2014-01-01

    Burkholderia pseudomallei (Bps)is the causative agent of melioidosis and is endemic in regions of northern Australia and Southeast Asia. Bps is inherently resistant to multiple antibiotics and is considered a potential biological warfare agent by the U.S. DHHS. Therefore, effective vaccines are necessary to prevent natural infection and to safeguard against biological attack with this organism. In our previous work we have shown that immunization with naturally derived outer membrane vesicles (OMVs) from Bps provides significant protection against lethal aerosol and systemic infection in BALB/c mice. In this work, we evaluated the safety and immunogenicity of escalating doses of OMV vaccine in rhesus macaques. We show that immunization of rhesus macaques with Bps OMVs generates humoral immuneresponses to protective protein and polysaccharide antigens without any associated toxicity or reactogenicity. These results lay the groundwork for evaluation of protective efficacy of the OMV vaccine in the nonhuman primate model of melioidosis.

  11. Evaluation of a Burkholderia pseudomallei Outer Membrane Vesicle Vaccine in Nonhuman Primates

    PubMed Central

    Petersen, Hailey; Nieves, Wildaliz; Russell-Lodrigue, Kasi; Roy, Chad J.; Morici, Lisa A.

    2014-01-01

    Burkholderia pseudomallei (Bps)is the causative agent of melioidosis and is endemic in regions of northern Australia and Southeast Asia. Bps is inherently resistant to multiple antibiotics and is considered a potential biological warfare agent by the U.S. DHHS. Therefore, effective vaccines are necessary to prevent natural infection and to safeguard against biological attack with this organism. In our previous work we have shown that immunization with naturally derived outer membrane vesicles (OMVs) from Bps provides significant protection against lethal aerosol and systemic infection in BALB/c mice. In this work, we evaluated the safety and immunogenicity of escalating doses of OMV vaccine in rhesus macaques. We show that immunization of rhesus macaques with Bps OMVs generates humoral immuneresponses to protective protein and polysaccharide antigens without any associated toxicity or reactogenicity. These results lay the groundwork for evaluation of protective efficacy of the OMV vaccine in the nonhuman primate model of melioidosis. PMID:25165491

  12. Cellular Engineering with Membrane Fusogenic Liposomes to Produce Functionalized Extracellular Vesicles.

    PubMed

    Lee, Junsung; Lee, Hyoungjin; Goh, Unbyeol; Kim, Jiyoung; Jeong, Moonkyoung; Lee, Jean; Park, Ji-Ho

    2016-03-23

    Engineering of extracellular vesicles (EVs) without affecting biological functions remains a challenge, limiting the broad applications of EVs in biomedicine. Here, we report a method to equip EVs with various functional agents, including fluorophores, drugs, lipids, and bio-orthogonal chemicals, in an efficient and controlled manner by engineering parental cells with membrane fusogenic liposomes, while keeping the EVs intact. As a demonstration of how this method can be applied, we prepared EVs containing azide-lipids, and conjugated them with targeting peptides using copper-free click chemistry to enhance targeting efficacy to cancer cells. We believe that this liposome-based cellular engineering method will find utility in studying the biological roles of EVs and delivering therapeutic agents through their innate pathway. PMID:26954538

  13. Bacterial Division Proteins FtsZ and ZipA Induce Vesicle Shrinkage and Cell Membrane Invagination*

    PubMed Central

    Cabré, Elisa J.; Sánchez-Gorostiaga, Alicia; Carrara, Paolo; Ropero, Noelia; Casanova, Mercedes; Palacios, Pilar; Stano, Pasquale; Jiménez, Mercedes; Rivas, Germán; Vicente, Miguel

    2013-01-01

    Permeable vesicles containing the proto-ring anchoring ZipA protein shrink when FtsZ, the main cell division protein, polymerizes in the presence of GTP. Shrinkage, resembling the constriction of the cytoplasmic membrane, occurs at ZipA densities higher than those found in the cell and is modulated by the dynamics of the FtsZ polymer. In vivo, an excess of ZipA generates multilayered membrane inclusions within the cytoplasm and causes the loss of the membrane function as a permeability barrier. Overproduction of ZipA at levels that block septation is accompanied by the displacement of FtsZ and two additional division proteins, FtsA and FtsN, from potential septation sites to clusters that colocalize with ZipA near the membrane. The results show that elementary constriction events mediated by defined elements involved in cell division can be evidenced both in bacteria and in vesicles. PMID:23921390

  14. Cryptococcus neoformans cryoultramicrotomy and vesicle fractionation reveals an intimate association between membrane lipids and glucuronxylomannan

    PubMed Central

    Oliveira, Débora L.; Nimrichter, Leonardo; Miranda, Kildare; Frases, Susana; Faull, Kym F.; Casadevall, Arturo; Rodrigues, Marcio L.

    2009-01-01

    Cryptococcus neoformans is an encapsulated pathogenic fungus. The cryptococcal capsule is composed of polysaccharides and is necessary for virulence. It has been previously reported that glucuronoxylomannan (GXM), the major capsular component, is synthesized in cytoplasmic compartments and transported to the extracellular space in vesicles, but knowledge on the organelles involved in polysaccharide synthesis and traffic is extremely limited. In this paper we report the GXM distribution in C. neoformans cells sectioned by cryoultramicrotomy and visualized by transmission electron microscopy (TEM) and polysaccharide immunogold staining. Cryosections of fungal cells showed high preservation of intracellular organelles and cell wall structure. Incubation of cryosections with an antibody to GXM revealed that cytoplasmic structures associated to vesicular compartments and reticular membranes are in close proximity to the polysaccharide. GXM was generally found in association with the membrane of intracellular compartments and within different layers of the cell wall. Analysis of extracellular fractions from cryptococcal supernatants by transmission electron microscopy in combination with serologic, chromatographic and spectroscopic methods revealed fractions containing GXM and lipids. These results indicate an intimate association of GXM and lipids in both intracellular and extracellular spaces consistent with polysaccharide synthesis and transport in membrane-associated structures. PMID:19747978

  15. Cryptococcus neoformans cryoultramicrotomy and vesicle fractionation reveals an intimate association between membrane lipids and glucuronoxylomannan.

    PubMed

    Oliveira, Débora L; Nimrichter, Leonardo; Miranda, Kildare; Frases, Susana; Faull, Kym F; Casadevall, Arturo; Rodrigues, Marcio L

    2009-12-01

    Cryptococcus neoformans is an encapsulated pathogenic fungus. The cryptococcal capsule is composed of polysaccharides and is necessary for virulence. It has been previously reported that glucuronoxylomannan (GXM), the major capsular component, is synthesized in cytoplasmic compartments and transported to the extracellular space in vesicles, but knowledge on the organelles involved in polysaccharide synthesis and traffic is extremely limited. In this paper we report the GXM distribution in C. neoformans cells sectioned by cryoultramicrotomy and visualized by transmission electron microscopy (TEM) and polysaccharide immunogold staining. Cryosections of fungal cells showed high preservation of intracellular organelles and cell wall structure. Incubation of cryosections with an antibody to GXM revealed that cytoplasmic structures associated to vesicular compartments and reticular membranes are in close proximity to the polysaccharide. GXM was generally found in association with the membrane of intracellular compartments and within different layers of the cell wall. Analysis of extracellular fractions from cryptococcal supernatants by transmission electron microscopy in combination with serologic, chromatographic and spectroscopic methods revealed fractions containing GXM and lipids. These results indicate an intimate association of GXM and lipids in both intracellular and extracellular spaces consistent with polysaccharide synthesis and transport in membrane-associated structures.

  16. Ultrastructural antibody localization of alpha2-macroglobulin in membrane-limited vesicles in cultured cells.

    PubMed Central

    Willingham, M C; Yamada, S S; Pastan, I

    1978-01-01

    We have been developing a procedure for localizing intracellular antigens in cultured cells, by using peroxidase-labeled antibodies, that allows good morphologic preservation. Although useful, our previous technique did not preserve the morphology of membranes, and the location of the peroxidase reaction product was difficult to establish. In this paper, we report major improvements on the basic technique that markedly enhance the quality of localization and of morphology. Saponin is used to permeabilize membranes without destroying their morphology. The amount of reaction product is enhanced with a peroxidase-antiperoxidase label. The clarity of morphologic detail and contrast of reaction product density are increased by using postsectioning staining with the osmium/thiocarbohydrazide/osmium and uranyl acetate/lead citrate procedures. We have applied this technique to the ultrastructural localization of alpha2-macroglobulin and demonstrated that it is localized in membrane-limited vesicles. We have also used this method to improve the preservation of structures for localization by fluorescence microscopy. Images PMID:81488

  17. Production of Outer Membrane Vesicles by the Plague Pathogen Yersinia pestis

    PubMed Central

    Caulfield, Adam J.; Rangel, Stephanie M.; Lathem, Wyndham W.

    2014-01-01

    Many Gram-negative bacteria produce outer membrane vesicles (OMVs) during cell growth and division, and some bacterial pathogens deliver virulence factors to the host via the release of OMVs during infection. Here we show that Yersinia pestis, the causative agent of the disease plague, produces and releases native OMVs under physiological conditions. These OMVs, approximately 100 nm in diameter, contain multiple virulence-associated outer membrane proteins including the adhesin Ail, the F1 outer fimbrial antigen, and the protease Pla. We found that OMVs released by Y. pestis contain catalytically active Pla that is competent for plasminogen activation and α2-antiplasmin degradation. The abundance of OMV-associated proteins released by Y. pestis is significantly elevated at 37°C compared to 26°C and is increased in response to membrane stress and mutations in RseA, Hfq, and the major Braun lipoprotein (Lpp). In addition, we show that Y. pestis OMVs are able to bind to components of the extracellular matrix such as fibronectin and laminin. These data suggest that Y. pestis may produce OMVs during mammalian infection and we propose that dispersal of Pla via OMV release may influence the outcome of infection through interactions with Pla substrates such as plasminogen and Fas ligand. PMID:25198697

  18. Uptake of /sup 75/Se-selenite by brush border membrane vesicles from chick duodenum stimulated by vitamin D

    SciTech Connect

    Mykkanen, H.M.; Wasserman, R.H.

    1989-02-01

    Brush border membrane vesicles were isolated from mucosal homogenates of duodena from normal, rachitic and vitamin D-treated rachitic chicks using a discontinuous sucrose gradient, and further purified by glycerol gradient centrifugation. In vitro uptake of 75Se-selenite by purified brush border membrane vesicles was studied using a rapid filtration technique. The time course of 75Se uptake was non-linear; rapid initial binding was followed by a gradual decrease in the rate of uptake until an equilibrium value was reached at 60-120 min. The initial binding at 36 s was not affected by selenite concentration in the incubation buffer, while the fractional rate of uptake between the 36 s and 2 min time periods was clearly lower with 1 mM Se than with 4-100 microM Se. 75Se uptake did not show any dependency on the external Na-gradient, nor could it be inhibited by other anions (arsenate, phosphate). Treatment of rachitic chicks either with cholecalciferol (500 Iu, 72 h) or with 1,25(OH)2-cholecalciferol (0.5 microgram given 16 h prior to isolation of the vesicles) significantly enhanced 75Se uptake. A threefold excess of mannitol in the outside buffer reduced 75Se uptake by vesicles from vitamin D-deficient and D-treated chicks 60% and 35% respectively, but had no effect on vesicles from vitamin D-treated chicks preloaded with 75Se. Neither saponin treatment nor excess cold selenite could release the label from the vesicles preloaded with 75Se. These data are compatible with the hypothesis that selenite easily crosses the brush border membrane into the intravesicular space and, once inside, is tightly bound by the membrane.

  19. Osmotic and Chill Activation of Glycine Betaine Porter II in Listeria monocytogenes Membrane Vesicles

    PubMed Central

    Gerhardt, Paul N. M.; Tombras Smith, Linda; Smith, Gary M.

    2000-01-01

    Listeria monocytogenes is a foodborne pathogen known for its tolerance to conditions of osmotic and chill stress. Accumulation of glycine betaine has been found to be important in the organism's tolerance to both of these stresses. A procedure was developed for the purification of membranes from L. monocytogenes cells in which the putative ATP-driven glycine betaine permease glycine betaine porter II (Gbu) is functional. As is the case for the L. monocytogenes sodium-driven glycine betaine uptake system (glycine betaine porter I), uptake in this vesicle system was dependent on energization by ascorbate-phenazine methosulfate. Vesicles lacking the gbu gene product had no uptake activity. Transport by this porter did not require sodium ion and could be driven only weakly by artificial gradients. Uptake rates could be manipulated under conditions not affecting secondary transport but known to affect ATPase activity. The system was shown to be both osmotically activated and cryoactivated. Under conditions of osmotic activation, the system exhibited Arrhenius-type behavior although the uptake rates were profoundly affected by the physical state of the membrane, with breaks in Arrhenius curves at approximately 10 and 18°C. In the absence of osmotic activation, the permease could be activated by decreasing temperature within the range of 15 to 4°C. Kinetic analyses of the permease at 30°C revealed Km values for glycine betaine of 1.2 and 2.9 μM with Vmax values of 2,200 and 3,700 pmol/min · mg of protein under conditions of optimal osmotic activation as mediated by KCl and sucrose, respectively. PMID:10762257

  20. Insulin-stimulated plasma membrane fusion of Glut4 glucose transporter-containing vesicles is regulated by phospholipase D1.

    PubMed

    Huang, Ping; Altshuller, Yelena M; Hou, June Chunqiu; Pessin, Jeffrey E; Frohman, Michael A

    2005-06-01

    Insulin stimulates glucose uptake in fat and muscle by mobilizing Glut4 glucose transporters from intracellular membrane storage sites to the plasma membrane. This process requires the trafficking of Glut4-containing vesicles toward the cell periphery, docking at exocytic sites, and plasma membrane fusion. We show here that phospholipase D (PLD) production of the lipid phosphatidic acid (PA) is a key event in the fusion process. PLD1 is found on Glut4-containing vesicles, is activated by insulin signaling, and traffics with Glut4 to exocytic sites. Increasing PLD1 activity facilitates glucose uptake, whereas decreasing PLD1 activity is inhibitory. Diminished PA production does not substantially hinder trafficking of the vesicles or their docking at the plasma membrane, but it does impede fusion-mediated extracellular exposure of the transporter. The fusion block caused by RNA interference-mediated PLD1 deficiency is rescued by exogenous provision of a lipid that promotes fusion pore formation and expansion, suggesting that the step regulated by PA is late in the process of vesicle fusion. PMID:15772157

  1. Actin microfilaments play a critical role in endocytosis at the apical but not the basolateral surface of polarized epithelial cells

    PubMed Central

    1993-01-01

    Treatment with cytochalasin D, a drug that acts by inducing the depolymerization of the actin cytoskeleton, selectively blocked endocytosis of membrane bound and fluid phase markers from the apical surface of polarized MDCK cells without affecting the uptake from the basolateral surface. Thus, in MDCK cell transformants that express the VSV G protein, cytochalasin blocked the internalization of an anti-G mAb bound to apical G molecules, but did not reduce the uptake of antibody bound to the basolateral surface. The selective effect of cytochalasin D on apical endocytosis was also demonstrated by the failure of the drug to reduce the uptake of 125I-labeled transferrin, which occurs by receptor-mediated endocytosis, via clathrin-coated pits, almost exclusively from the basolateral surface. The actin cytoskeleton appears to play a critical role in adsorptive as well as fluid phase apical endocytic events, since treatment with cytochalasin D prevented the apical uptake of cationized ferritin, that occurs after the marker binds to the cell surface, as well as uptake of Lucifer yellow, a fluorescent soluble dye. Moreover, the drug efficiently blocked infection of the cells with influenza virus, when the viral inoculum was applied to the apical surface. On the other hand, it did not inhibit the basolateral uptake of Lucifer yellow, nor did it prevent infection with VSV from the basolateral surface, or with influenza when this virus was applied to monolayers in which the formation of tight junctions had been prevented by depletion of calcium ions. EM demonstrated that cytochalasin D leads to an increase in the number of coated pits in the apical surface where it suppresses the pinching off of coated vesicles. In addition, in drug-treated cells cationized ferritin molecules that were bound to microvilli were not cleared from the microvillar surface, as is observed in untreated cells. These findings indicate that there is a fundamental difference in the process by which

  2. Activation of Immune and Defense Responses in the Intestinal Mucosa by Outer Membrane Vesicles of Commensal and Probiotic Escherichia coli Strains.

    PubMed

    José Fábrega, María; Aguilera, Laura; Giménez, Rosa; Varela, Encarna; Alexandra Cañas, María; Antolín, María; Badía, Josefa; Baldomà, Laura

    2016-01-01

    The influence of microbiota in human health is well-known. Imbalances in microbiome structure have been linked to several diseases. Modulation of microbiota composition through probiotic therapy is an attempt to harness the beneficial effects of commensal microbiota. Although, there is wide knowledge of the responses induced by gut microbiota, the microbial factors that mediate these effects are not well-known. Gram-negative bacteria release outer membrane vesicles (OMVs) as a secretion mechanism of microbial factors, which have an important role in intercellular communication. Here, we investigated whether OMVs from the probiotic Escherichia coli strain Nissle 1917 (EcN) or the commensal E. coli strain ECOR12 trigger immune responses in various cellular models: (i) peripheral blood mononuclear cells (PBMCs) as a model of intestinal barrier disruption, (ii) apical stimulation of Caco-2/PMBCs co-culture as a model of intact intestinal mucosa, and (iii) colonic mucosa explants as an ex vivo model. Stimulations with bacterial lysates were also performed. Whereas, both OMVs and lysates activated expression and secretion of several cytokines and chemokines in PBMCs, only OMVs induced basolateral secretion and mRNA upregulation of these mediators in the co-culture model. We provide evidence that OMVs are internalized in polarized Caco-2 cells. The activated epithelial cells elicit a response in the underlying immunocompetent cells. The OMVs effects were corroborated in the ex vivo model. This experimental study shows that OMVs are an effective strategy used by beneficial gut bacteria to communicate with and modulate host responses, activating signaling events through the intestinal epithelial barrier. PMID:27242727

  3. Activation of Immune and Defense Responses in the Intestinal Mucosa by Outer Membrane Vesicles of Commensal and Probiotic Escherichia coli Strains

    PubMed Central

    José Fábrega, María; Aguilera, Laura; Giménez, Rosa; Varela, Encarna; Alexandra Cañas, María; Antolín, María; Badía, Josefa

    2016-01-01

    The influence of microbiota in human health is well-known. Imbalances in microbiome structure have been linked to several diseases. Modulation of microbiota composition through probiotic therapy is an attempt to harness the beneficial effects of commensal microbiota. Although, there is wide knowledge of the responses induced by gut microbiota, the microbial factors that mediate these effects are not well-known. Gram-negative bacteria release outer membrane vesicles (OMVs) as a secretion mechanism of microbial factors, which have an important role in intercellular communication. Here, we investigated whether OMVs from the probiotic Escherichia coli strain Nissle 1917 (EcN) or the commensal E. coli strain ECOR12 trigger immune responses in various cellular models: (i) peripheral blood mononuclear cells (PBMCs) as a model of intestinal barrier disruption, (ii) apical stimulation of Caco-2/PMBCs co-culture as a model of intact intestinal mucosa, and (iii) colonic mucosa explants as an ex vivo model. Stimulations with bacterial lysates were also performed. Whereas, both OMVs and lysates activated expression and secretion of several cytokines and chemokines in PBMCs, only OMVs induced basolateral secretion and mRNA upregulation of these mediators in the co-culture model. We provide evidence that OMVs are internalized in polarized Caco-2 cells. The activated epithelial cells elicit a response in the underlying immunocompetent cells. The OMVs effects were corroborated in the ex vivo model. This experimental study shows that OMVs are an effective strategy used by beneficial gut bacteria to communicate with and modulate host responses, activating signaling events through the intestinal epithelial barrier. PMID:27242727

  4. Heterotypic binding between neuronal membrane vesicles and glial cells is mediated by a specific cell adhesion molecule

    PubMed Central

    1984-01-01

    By means of a multistage quantitative assay, we have identified a new kind of cell adhesion molecule (CAM) on neuronal cells of the chick embryo that is involved in their adhesion to glial cells. The assay used to identify the binding component (which we name neuron-glia CAM or Ng-CAM) was designed to distinguish between homotypic binding (e.g., neuron to neuron) and heterotypic binding (e.g., neuron to glia). This distinction was essential because a single neuron might simultaneously carry different CAMs separately mediating each of these interactions. The adhesion of neuronal cells to glial cells in vitro was previously found to be inhibited by Fab' fragments prepared from antisera against neuronal membranes but not by Fab' fragments against N-CAM, the neural cell adhesion molecule. This suggested that neuron-glia adhesion is mediated by specific cell surface molecules different from previously isolated CAMs . To verify that this was the case, neuronal membrane vesicles were labeled internally with 6-carboxyfluorescein and externally with 125I-labeled antibodies to N-CAM to block their homotypic binding. Labeled vesicles bound to glial cells but not to fibroblasts during a 30-min incubation period. The specific binding of the neuronal vesicles to glial cells was measured by fluorescence microscopy and gamma spectroscopy of the 125I label. Binding increased with increasing concentrations of both glial cells and neuronal vesicles. Fab' fragments prepared from anti-neuronal membrane sera that inhibited binding between neurons and glial cells were also found to inhibit neuronal vesicle binding to glial cells. The inhibitory activity of the Fab' fragments was depleted by preincubation with neuronal cells but not with glial cells. Trypsin treatment of neuronal membrane vesicles released material that neutralized Fab' fragment inhibition; after chromatography, neutralizing activity was enriched 50- fold. This fraction was injected into mice to produce monoclonal

  5. Ectocytosis caused by sublytic autologous complement attack on human neutrophils. The sorting of endogenous plasma-membrane proteins and lipids into shed vesicles.

    PubMed Central

    Stein, J M; Luzio, J P

    1991-01-01

    During sublytic complement attack on human neutrophils, plasma-membrane vesicles are shed from the cell surface as a cell-protection mechanism. By using surface-iodinated neutrophils it was found that less than 2% of surface label was recovered in shed vesicles under conditions where 40% of complement component C9 was shed. SDS/PAGE of 125I-labelled shed vesicles and plasma membranes showed differences in iodination pattern, demonstrating the sorting of membrane proteins into the shed vesicles. Analysis of 32P-labelled phospholipids after labeling of neutrophils with [32P]Pi before sublytic complement attack showed the presence of phosphatidic acid, phosphatidylcholine, phosphatidyl-ethanolamine, phosphatidylinositol and polyphosphoinositides in shed vesicles. Quantitative analysis using [3H]acetic anhydride-labelling method showed that the molar proportions of phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and sphingomyelin were the same in shed vesicles as in plasma membranes. In contrast, the molar proportions of cholesterol and diacylglycerol relative to sphingomyelin were almost twice those found in plasma membranes. The data demonstrate the existence of protein and lipid sorting mechanisms during the formation of shed vesicles when neutrophils are subject to sublytic complement attack. The term 'ectocytosis' is proposed to describe triggered shedding of right-side-out membrane vesicles from the surface of eukaryotic cells. Images Fig. 2. Fig. 3. Fig. 4. PMID:1848755

  6. Common channels for water and protons at apical and basolateral cell membranes of frog skin and urinary bladder epithelia. Effects of oxytocin, heavy metals, and inhibitors of H(+)-adenosine triphosphatase

    SciTech Connect

    Harvey, B.; Lacoste, I.; Ehrenfeld, J. )

    1991-04-01

    We have compared the response of proton and water transport to oxytocin treatment in isolated frog skin and urinary bladder epithelia to provide further insights into the nature of water flow and H+ flux across individual apical and basolateral cell membranes. In isolated spontaneous sodium-transporting frog skin epithelia, lowering the pH of the apical solution from 7.4 to 6.4, 5.5, or 4.5 produced a fall in pHi in principal cells which was completely blocked by amiloride, indicating that apical Na+ channels are permeable to protons. When sodium transport was blocked by amiloride, the H+ permeability of the apical membranes of principal cells was negligible but increased dramatically after treatment with antidiuretic hormone (ADH). In the latter condition, lowering the pH of the apical solution caused a voltage-dependent intracellular acidification, accompanied by membrane depolarization, and an increase in membrane conductance and transepithelial current. These effects were inhibited by adding Hg2+ (100 microM) or dicyclohexylcarbodiimide (DCCD, 10(-5) M) to the apical bath. Net titratable H+ flux across frog skin was increased from 30 +/- 8 to 115 +/- 18 neq.h-1.cm-2 (n = 8) after oxytocin treatment (at apical pH 5.5 and serosal pH 7.4) and was completely inhibited by DCCD (10(-5) M). The basolateral membranes of the principal cells in frog skin epithelium were found to be spontaneously permeable to H+ and passive electrogenic H+ transport across this membrane was not affected by oxytocin. Lowering the pH of the basolateral bathing solution (pHb) produced an intracellular acidification and membrane depolarization (and an increase in conductance when the normal dominant K+ conductance of this membrane was abolished by Ba2+ 1 mM). These effects of low pHb were blocked by micromolar concentrations of heavy metals (Zn2+, Ni2+, Co2+, Cd2+, and Hg2+).

  7. Effect of chronic (4 weeks) ingestion of ethanol on transport of proline into intestinal brush border membrane vesicles

    SciTech Connect

    Beesley, R.C.; Jones, T.D.

    1986-03-01

    Hamsters were separated into two groups and fed liquid diets ad lib. Controls were given a diet similar to that described by DeCarli and Lieber while alcoholics received the same diet containing 5% ethanol isocalorically substituted for sucrose. The volume of diet consumed daily and the gain in body weights of alcoholics were not significantly different from those of controls. After four weeks the animals were sacrificed and the upper third of the small intestine was used to prepare brush border membrane vesicles. In the presence of a Na/sup +/ gradient, uptake of proline into vesicles prepared from both groups was rapid, reaching a maximum accumulation in 1 to 2 min and then decreasing to the equilibrium level. To normalize the results, the amount of proline take up at each time point was divided by the amount present at equilibrium. From the normalized data it was concluded that both the rate of uptake and the maximum accumulation of proline into brush border membrane vesicles isolated from alcoholics were significantly less than those obtained with vesicles from controls. These results suggest that chronic ingestion of ethanol results in a reduction in Na/sup +/-dependent transport of proline across the brush border membrane and, thus, may contribute to the malnutrition which is frequently associated with chronic alcoholism.

  8. Aboral changes in D-glucose transport by human intestinal brush-border membrane vesicles.

    PubMed Central

    Bluett, M K; Abumrad, N N; Arab, N; Ghishan, F K

    1986-01-01

    D-Glucose transport was investigated in isolated brush-border membrane vesicles from human small intestine. Characteristics of D-glucose transport from the jejunum were compared with that in the mid and terminal ileum. Jejunal and mid-ileal D-glucose transport was Na+-dependent and electrogenic. The transient overshoot of jejunal D-glucose transport was significantly greater than corresponding values in mid-ileum. The terminal ileum did not exhibit Na+-dependent D-glucose transport, but did exhibit Na+-dependent taurocholate transport. Na+-glucose co-transport activity as measured by tracer-exchange experiments was greatest in the jejunum, and diminished aborally. We conclude that D-glucose transport in man is Na+-dependent and electrogenic in the proximal intestine and directly related to the activity of D-glucose-Na+ transporters present in the brush-border membranes. D-Glucose transport in the terminal ileum resembles colonic transport of D-glucose. PMID:3800877

  9. Characterization and vaccine potential of outer membrane vesicles produced by Haemophilus parasuis

    DOE PAGES

    McCaig, William D.; Loving, Crystal L.; Hughes, Holly R.; Brockmeier, Susan L.; Charbit, Alain

    2016-03-01

    Haemophilus parasuis is a Gram-negative bacterium that colonizes the upper respiratory tract of swine and is capable of causing a systemic infection, resulting in high morbidity and mortality. H. parasuis isolates display a wide range of virulence and virulence factors are largely unknown. Commercial bacterins are often used to vaccinate swine against H. parasuis, though strain variability and lack of cross-reactivity can make this an ineffective means of protection. Outer membrane vesicles (OMV) are spherical structures naturally released from the membrane of bacteria and OMV are often enriched in toxins, signaling molecules and other bacterial components. Examination of OMV structuresmore » has led to identification of virulence factors in a number of bacteria and they have been successfully used as subunit vaccines. We have isolated OMV from both virulent and avirulent strains of H. parasuis, have examined their protein content and assessed their ability to induce an immune response in the host. Lastly, vaccination with purified OMV derived from the virulent H. parasuis Nagasaki strain provided protection against challenge with a lethal dose of the bacteria.« less

  10. Na-dependent L-proline transport by eel intestinal brush-border membrane vesicles

    SciTech Connect

    Vilella, S.; Ahearn, G.A.; Cassano, G.; Storelli, C. University of Hawaii at Manoa, Honolulu )

    1988-10-01

    L-({sup 3}H)proline uptake by brush-border membrane vesicles prepared from intestinal mucosa of the European eel, Anguilla anguilla, was stimulated by a transmembrane Na gradient (out > in.) Kinetic analysis of L-proline influx, under short-circuited membrane potential conditions, indicated the presence of an apparent single Na-dependent carrier process and a nonsaturable transfer component with an apparent diffusional permeability (P) of 1.53 {plus minus} 0.35 {mu}l{center dot}mg protein{sup {minus}1}{center dot}min{sup {minus}1}. An imposed transmembrane potential (inside negative) increased apparent L-proline binding affinity (lowered K{sub app}) without appreciably altering maximal amino acid influx (J{sub max}). Hill analysis of L-proline influx over a wide range of external Na concentrations indicated a 1:1 stoichiometry for Na-proline cotransport. Use of amino acid inhibitors of L-proline influx suggested that L-proline transfer may occur by either a classical Na-dependent A System with a wide substrate specificity or by the combination of Na-dependent PHE (phenylalanine preferring) and IMINO (proline, {alpha}-methylaminoisobutyric acid preferring) Systems.

  11. Heparin blocks /sup 125/I-calmodulin internalization by isolated rat renal brush border membrane vesicles

    SciTech Connect

    Meezan, E.; Elgavish, A.; Roden, L.; Wallace, R.W.

    1986-03-05

    /sup 125/I-Calmodulin is internalized by isolated rat renal brush border membrane vesicles (BBV) in a time, temperature and calcium dependent manner. Internalization of /sup 125/I-calmodulin into the osmotically sensitive space of BBV was distinguished from binding of the ligand to the outer BBV surface by examining the interaction of ligand and BBV at different medium osmolarities (300-1100 mosm), uptake was inversely proportional to medium osmolarity. Internalized /sup 125/I-calmodulin was intact and Western blots of solubilized BBV with /sup 125/I-calmodulin demonstrated the presence of several calmodulin-binding proteins of 143, 118, 50, 47.5, 46.5 and 35 kilodaltons which could represent potential intravesicular binding sites for the ligand. Heparin and the related glycosaminoglycan heparin sulfate both showed a dose-dependent inhibition (0.5-50 ..mu..g/ml) of /sup 125/I-calmodulin uptake by BBV, but other sulfated and nonsulfated glycosaminoglycans including chondroitin sulfates, keratan sulfate and hyaluronic acid showed little or no inhibitory effect. Desulfation of heparin virtually abolished the inhibition of uptake while depolymerization reduced it. Heparin did not block the binding of /sup 125/I-calmodulin to BBV proteins as assessed by Western blotting technique suggesting its effect was on internalization of the ligand rather than on its association with internal membrane proteins.

  12. Characterization and Vaccine Potential of Outer Membrane Vesicles Produced by Haemophilus parasuis

    PubMed Central

    McCaig, William D.; Loving, Crystal L.; Hughes, Holly R.; Brockmeier, Susan L.

    2016-01-01

    Haemophilus parasuis is a Gram-negative bacterium that colonizes the upper respiratory tract of swine and is capable of causing a systemic infection, resulting in high morbidity and mortality. H. parasuis isolates display a wide range of virulence and virulence factors are largely unknown. Commercial bacterins are often used to vaccinate swine against H. parasuis, though strain variability and lack of cross-reactivity can make this an ineffective means of protection. Outer membrane vesicles (OMV) are spherical structures naturally released from the membrane of bacteria and OMV are often enriched in toxins, signaling molecules and other bacterial components. Examination of OMV structures has led to identification of virulence factors in a number of bacteria and they have been successfully used as subunit vaccines. We have isolated OMV from both virulent and avirulent strains of H. parasuis, have examined their protein content and assessed their ability to induce an immune response in the host. Vaccination with purified OMV derived from the virulent H. parasuis Nagasaki strain provided protection against challenge with a lethal dose of the bacteria. PMID:26930282

  13. A novel mechanism for the biogenesis of outer membrane vesicles in Gram-negative bacteria

    PubMed Central

    Roier, Sandro; Zingl, Franz G.; Cakar, Fatih; Durakovic, Sanel; Kohl, Paul; Eichmann, Thomas O.; Klug, Lisa; Gadermaier, Bernhard; Weinzerl, Katharina; Prassl, Ruth; Lass, Achim; Daum, Günther; Reidl, Joachim; Feldman, Mario F.; Schild, Stefan

    2016-01-01

    Bacterial outer membrane vesicles (OMVs) have important biological roles in pathogenesis and intercellular interactions, but a general mechanism of OMV formation is lacking. Here we show that the VacJ/Yrb ABC (ATP-binding cassette) transport system, a proposed phospholipid transporter, is involved in OMV formation. Deletion or repression of VacJ/Yrb increases OMV production in two distantly related Gram-negative bacteria, Haemophilus influenzae and Vibrio cholerae. Lipidome analyses demonstrate that OMVs from VacJ/Yrb-defective mutants in H. influenzae are enriched in phospholipids and certain fatty acids. Furthermore, we demonstrate that OMV production and regulation of the VacJ/Yrb ABC transport system respond to iron starvation. Our results suggest a new general mechanism of OMV biogenesis based on phospholipid accumulation in the outer leaflet of the outer membrane. This mechanism is highly conserved among Gram-negative bacteria, provides a means for regulation, can account for OMV formation under all growth conditions, and might have important pathophysiological roles in vivo. PMID:26806181

  14. Hydrophobic ion interactions with membranes. Thermodynamic analysis of tetraphenylphosphonium binding to vesicles.

    PubMed Central

    Flewelling, R F; Hubbell, W L

    1986-01-01

    The thermodynamic properties for the interaction of the hydrophobic ion tetraphenylphosphonium (TPP+) with egg phosphatidylcholine vesicles were studied in detail by equilibrium dialysis and spin label techniques. A partition coefficient of beta = 4.2 + 0.4 x 10(-6) cm (K congruent to 100) was determined. Electrostatic saturation sets in at approximately 600 microM (about one absorbed TPP+ molecule per 100 lipids), and is not screened by salt. The temperature dependence of binding was determined, which reveals that the binding is entropy-driven with a positive (repulsive) enthalpy of binding, a result to be compared with hydrophobic anions in which the binding enthalpy is negative. The membrane dipole potential may be responsible for this binding difference. Activity coefficients are determined and shown to be significantly different from those of most common salts, an important result that should be considered in all hydrophobic ion studies. Comparison of the TPP+ results with those of its anionic structural analogue, tetraphenylboron (TPB-), permits a general analysis of hydrophobic ion interactions with membranes. A theoretical model consistent with the entire set of data is developed in an accompanying article. PMID:3006814

  15. Calcium reduces the sodium permeability of luminal membrane vesicles from toad bladder. Studies using a fast-reaction apparatus

    SciTech Connect

    Chase, H.S. Jr.; Al-Awqati, Q.

    1983-05-01

    Regulation of the sodium permeability of the luminal membrane is the major mechanism by which the net rate of sodium transport across tight epithelia is varied. Previous evidence has suggested that the permeability of the luminal membrane might be regulated by changes in intracellular sodium or calcium activities. To test this directly, we isolated a fraction of the plasma membrane from the toad urinary bladder, which contains a fast, amiloride-sensitive sodium flux with characteristics similar to those of the native luminal membrane. Using a flow-quench apparatus to measure the initial rate of sodium efflux from these vesicles in the millisecond time range, we have demonstrated that the isotope exchange permeability of these vesicles is very sensitive to calcium. Calcium reduces the sodium permeability, and the half-maximal inhibitory concentration is 0.5 microM, well within the range of calcium activity found in cells. Also, the permeability of the luminal membrane vesicles is little affected by the ambient sodium concentration. These results, when taken together with studies on whole tissue, suggest that cell calcium may be an important regulator of transepithelial sodium transport by its effect on luminal sodium permeability. The effect of cell sodium on permeability may be mediated by calcium rather than by sodium itself.

  16. In vitro effect of ethanol on sodium and glucose transport in rabbit renal brush border membrane vesicles.

    PubMed

    Parenti, P; Giordana, B; Hanozet, G M

    1991-11-18

    The effect of ethanol on sodium and glucose transport in rabbit renal brush border membrane vesicles was examined. When membrane vesicles were preincubated in the presence of ethanol the sodium-dependent D-glucose uptake was significantly inhibited. This effect, as suggested by O'Neill et al. (1986) FEBS Lett. 194, 183-188, may be due to a faster collapse of the Na+ gradient. As a matter of fact, the amiloride-insensitive sodium pathway was increased by ethanol in our brush border membrane preparation. However, sodium/D-glucose cotransport was inhibited by ethanol, although to a lesser degree, also in the absence of a sodium gradient. In addition, ethanol inhibited glucose-dependent sodium uptake, suggesting that a direct interaction with the translocator was involved. This conclusion was also supported by kinetic measurements showing a decrease of Vmax and an increase in Km for glucose in membrane vesicles treated with ethanol. Moreover, ethanol influenced the interaction of phlorizin with the cotransporter: uptake experiments performed in the presence of the two inhibitors demonstrated that phlorizin and ethanol behave as not mutually exclusive inhibitors of D-glucose transport. These data indicate that in rabbit renal brush border membranes ethanol not only affects the 'passive pathway', i.e. the sodium permeability, but it also directly interferes with carrier functions.

  17. Large-scale preparation of plasma membrane vesicles from PC-12 pheochromocytoma cells and their use in noradrenaline transport studies.

    PubMed

    Harder, R; Bönisch, H

    1984-08-01

    Plasma membranes were isolated from rat pheochromocytoma cells (PC-12) grown in spinner culture. The rapid and simple isolation procedure consisted of a differential and isopycnic centrifugation (in a linear sucrose gradient) with the aid of a high capacity fixed angle rotor equipped with siliconized centrifuge tubes. The isolated membranes were closed and osmotically active vesicles (about 0.3 micron in diameter) with a mean intravesicular water space of 1.84 microliters/mg protein. In the presence of an inward gradient of sodium chloride and an outward gradient of potassium, [3H]noradrenaline (50 nM) was taken up and accumulated 550-fold (at 31 degrees C). The uptake and accumulation of [3H]noradrenaline was temperature-sensitive and inhibited by the tricyclic antidepressant desipramine. Membrane vesicles isolated from PC-12 cells represent a useful model for the investigation of the molecular mechanism of the neuronal noradrenaline transport system. PMID:6466664

  18. Release of Membrane-Bound Vesicles and Inhibition of Tumor Cell Adhesion by the Peptide Neopetrosiamide A

    PubMed Central

    Austin, Pamela; Heller, Markus; Williams, David E.; McIntosh, Lawrence P.; Vogl, A. Wayne; Foster, Leonard J.; Andersen, Raymond J.; Roberge, Michel; Roskelley, Calvin D.

    2010-01-01

    Background Neopetrosiamide A (NeoA) is a 28-amino acid tricyclic peptide originally isolated from a marine sponge as a tumor cell invasion inhibitor whose mechanism of action is unknown. Methodology/Principal Findings We show that NeoA reversibly inhibits tumor cell adhesion, disassembles focal adhesions in pre-attached cells, and decreases the level of β1 integrin subunits on the cell surface. NeoA also induces the formation of dynamic, membrane-bound protrusions on the surface of treated cells and the release of membrane-bound vesicles into the culture medium. Proteomic analysis indicates that the vesicles contain EGF and transferrin receptors as well as a number of proteins involved in adhesion and migration including: β1 integrin and numerous α integrin subunits; actin and actin-binding proteins such as cofilin, moesin and myosin 1C; and membrane modulating eps15 homology domain (EHD) proteins. Surface labeling, trafficking inhibition, and real-time imaging experiments all suggest that β1 integrin-containing vesicles are released directly from NeoA-induced cell surface protrusions rather than from vesicles generated intracellularly. The biological activity of NeoA is dependent on its disulfide bond pattern and NMR spectroscopy indicates that the peptide is globular with a continuous ridge of hydrophobic groups flanked by charged amino acid residues that could facilitate a simultaneous interaction with lipids and proteins in the membrane. Conclusions/Significance NeoA is an anti-adhesive peptide that decreases cell surface integrin levels through a novel, yet to be elucidated, mechanism that involves the release of adhesion molecule-containing vesicles from the cell surface. PMID:20520768

  19. Quantitative Proteomics of the Neisseria Gonorrhoeae Cell Envelope and Membrane Vesicles for the Discovery of Potential Therapeutic Targets*

    PubMed Central

    Zielke, Ryszard A.; Wierzbicki, Igor H.; Weber, Jacob V.; Gafken, Philip R.; Sikora, Aleksandra E.

    2014-01-01

    Neisseria gonorrhoeae (GC) is a human-specific pathogen, and the agent of a sexually transmitted disease, gonorrhea. There is a critical need for new approaches to study and treat GC infections because of the growing threat of multidrug-resistant isolates and the lack of a vaccine. Despite the implied role of the GC cell envelope and membrane vesicles in colonization and infection of human tissues and cell lines, comprehensive studies have not been undertaken to elucidate their constituents. Accordingly, in pursuit of novel molecular therapeutic targets, we have applied isobaric tagging for absolute quantification coupled with liquid chromatography and mass spectrometry for proteome quantitative analyses. Mining the proteome of cell envelopes and native membrane vesicles revealed 533 and 168 common proteins, respectively, in analyzed GC strains FA1090, F62, MS11, and 1291. A total of 22 differentially abundant proteins were discovered including previously unknown proteins. Among those proteins that displayed similar abundance in four GC strains, 34 were found in both cell envelopes and membrane vesicles fractions. Focusing on one of them, a homolog of an outer membrane protein LptD, we demonstrated that its depletion caused loss of GC viability. In addition, we selected for initial characterization six predicted outer membrane proteins with unknown function, which were identified as ubiquitous in the cell envelopes derived from examined GC isolates. These studies entitled a construction of deletion mutants and analyses of their resistance to different chemical probes. Loss of NGO1985, in particular, resulted in dramatically decreased GC viability upon treatment with detergents, polymyxin B, and chloramphenicol, suggesting that this protein functions in the maintenance of the cell envelope permeability barrier. Together, these findings underscore the concept that the cell envelope and membrane vesicles contain crucial, yet under-explored determinants of GC

  20. Quantitative proteomics of the Neisseria gonorrhoeae cell envelope and membrane vesicles for the discovery of potential therapeutic targets.

    PubMed

    Zielke, Ryszard A; Wierzbicki, Igor H; Weber, Jacob V; Gafken, Philip R; Sikora, Aleksandra E

    2014-05-01

    Neisseria gonorrhoeae (GC) is a human-specific pathogen, and the agent of a sexually transmitted disease, gonorrhea. There is a critical need for new approaches to study and treat GC infections because of the growing threat of multidrug-resistant isolates and the lack of a vaccine. Despite the implied role of the GC cell envelope and membrane vesicles in colonization and infection of human tissues and cell lines, comprehensive studies have not been undertaken to elucidate their constituents. Accordingly, in pursuit of novel molecular therapeutic targets, we have applied isobaric tagging for absolute quantification coupled with liquid chromatography and mass spectrometry for proteome quantitative analyses. Mining the proteome of cell envelopes and native membrane vesicles revealed 533 and 168 common proteins, respectively, in analyzed GC strains FA1090, F62, MS11, and 1291. A total of 22 differentially abundant proteins were discovered including previously unknown proteins. Among those proteins that displayed similar abundance in four GC strains, 34 were found in both cell envelopes and membrane vesicles fractions. Focusing on one of them, a homolog of an outer membrane protein LptD, we demonstrated that its depletion caused loss of GC viability. In addition, we selected for initial characterization six predicted outer membrane proteins with unknown function, which were identified as ubiquitous in the cell envelopes derived from examined GC isolates. These studies entitled a construction of deletion mutants and analyses of their resistance to different chemical probes. Loss of NGO1985, in particular, resulted in dramatically decreased GC viability upon treatment with detergents, polymyxin B, and chloramphenicol, suggesting that this protein functions in the maintenance of the cell envelope permeability barrier. Together, these findings underscore the concept that the cell envelope and membrane vesicles contain crucial, yet under-explored determinants of GC

  1. Discovery of Salmonella Virulence Factors Translocated via Outer Membrane Vesicles to Murine Macrophages.

    SciTech Connect

    Yoon, Hyunjin; Ansong, Charles; Adkins, Joshua N.; Heffron, Fred

    2011-06-01

    We have previously shown that the regulators SpvR, FruR, IHF, PhoP/PhoQ, SsrA/SsrB, SlyA, Hnr, RpoE, SmpB, CsrA, RpoS, Crp, OmpR/EnvZ, and Hfq are essential for Salmonella Typhimurium virulence in mice. Here we use quantitative LC-MS-based proteomics profiling of in-frame deletion mutants of these 14 regulators to identify proteins that are coordinately regulated by these virulence regulators and are thus presumably novel factors contributing to Salmonella pathogenesis. Putative candidate proteins from proteomics analysis were determined, which exhibited similar abundance profiles to those of Salmonella pathogenicity island (SPI)-2 type III secretion system (TTSS) proteins. A subset of 5 proteins including STM0082, STM1548, PdgL, STM1633, and STM3595 was selected for further analysis. All 5 proteins were expressed inside macrophage cells and STM0082 (SrfN) was secreted into host cytoplasm. Furthermore, deletion of STM0082 attenuated virulence in mice when administered intraperitoneally as determined by competitive index. srfN transcription was positively regulated by SsrAB, however, secretion was independent of SPI-2 TTSS as well as SPI-1 TTSS and flagella. Proteins including PagK and STM2585A, which are positively regulated by PhoP/PhoQ, have sec signal peptides as predicted for SrfN and were secreted into macrophage cytoplasm regardless of SPI-2 TTSS. Isolation of outer membrane vesicles (OMVs) revealed the presence of SrfN, PagK, and STM2585A inside vesicle compartments. This result is the first case showing delivery of virulence effectors via OMVs in S. Typhimurium. Moreover, Hfq regulation of SrfN translation suggests that small non-coding RNAs may be responsible for regulating effector protein expression.

  2. Ontogeny of Na/H antiporter activity in rabbit renal brush border membrane vesicles.

    PubMed Central

    Beck, J C; Lipkowitz, M S; Abramson, R G

    1991-01-01

    The development of the Na/H antiporter was studied in renal brush border membrane vesicles (BBMV) from fetal and adult rabbits using isotopic and fluorescent techniques. The kinetics of the antiporter studied by 22Na+ uptake revealed that the Vmax was only 25% of that in the adult; however, the Km's for Na+ were not significantly different. These data were confirmed by a fluorescent assay using the pH-sensitive probe, acridine orange: the Vmax was significantly lower in the fetal BBMV. Conductive Na+ movement was estimated from amiloride-insensitive 22Na+ uptake and the rate of alkalinization induced by K+, an ion whose relative conductance was found to be similar to that of Na+. Although relative Na+ conductance was significantly greater in fetal BBMV, the lower Vmax in fetal vesicles could not be ascribed to this factor. Maternal administration of betamethasone (50 micrograms/kg intramuscularly) for 2 d before delivery significantly increased the Vmax of the antiporter to levels observed in the adult; Km was unaffected. Na/K ATPase activity increased fourfold after betamethasone, but the specific activities of four brush border marker enzymes and the kinetics of Na(+)-glucose cotransport were unchanged. These data indicate that there is a developmental increase in brush border Na/H exchange which is the result of an increase in the number and/or the turnover number of the carriers. Further, these data suggest that the postnatal increase in antiporter activity may be related to the surge in glucocorticoid concentration that occurs perinatally. Images PMID:1645751

  3. Gene Transfer Potential of Outer Membrane Vesicles of Acinetobacter baylyi and Effects of Stress on Vesiculation

    PubMed Central

    Fulsundar, Shweta; Harms, Klaus; Flaten, Gøril E.; Johnsen, Pål J.; Chopade, Balu Ananda

    2014-01-01

    Outer membrane vesicles (OMVs) are continually released from a range of bacterial species. Numerous functions of OMVs, including the facilitation of horizontal gene transfer (HGT) processes, have been proposed. In this study, we investigated whether OMVs contribute to the transfer of plasmids between bacterial cells and species using Gram-negative Acinetobacter baylyi as a model system. OMVs were extracted from bacterial cultures and tested for the ability to vector gene transfer into populations of Escherichia coli and A. baylyi, including naturally transformation-deficient mutants of A. baylyi. Anti-double-stranded DNA (anti-dsDNA) antibodies were used to determine the movement of DNA into OMVs. We also determined how stress affected the level of vesiculation and the amount of DNA in vesicles. OMVs were further characterized by measuring particle size distribution (PSD) and zeta potential. Transmission electron microscopy (TEM) and immunogold labeling were performed using anti-fluorescein isothiocyanate (anti-FITC)-conjugated antibodies and anti-dsDNA antibodies to track the movement of FITC-labeled and DNA-containing OMVs. Exposure to OMVs isolated from plasmid-containing donor cells resulted in HGT to A. baylyi and E. coli at transfer frequencies ranging from 10−6 to 10−8, with transfer efficiencies of approximately 103 and 102 per μg of vesicular DNA, respectively. Antibiotic stress was shown to affect the DNA content of OMVs as well as their hydrodynamic diameter and zeta potential. Morphological observations suggest that OMVs from A. baylyi interact with recipient cells in different ways, depending on the recipient species. Interestingly, the PSD measurements suggest that distinct size ranges of OMVs are released from A. baylyi. PMID:24657872

  4. Basolateral membrane H/OH/HCO3 transport in the rat cortical thick ascending limb. Evidence for an electrogenic Na/HCO3 cotransporter in parallel with a Na/H antiporter.

    PubMed Central

    Krapf, R

    1988-01-01

    Mechanisms involved in basolateral H/OH/HCO3 transport in the in vitro microperfused rat cortical thick ascending limb were examined by the microfluorometric determination of cell pH using (2',7')-bis-(carboxyethyl)-(5,6)-carboxyfluorescein. The mean cell pH in this segment perfused with 147 mM sodium and 25 mM HCO3 at pH 7.4 was 7.13 +/- 0.02 (n = 30). Lowering bath HCO3 from 25 to 5 mM (constant PCO2 of 40 mmHg) acidified the cells by 0.31 +/- 0.02 pH units at a rate of 0.56 +/- 0.08 pH units/min. Removal of bath sodium acidified the cells by 0.28 +/- 0.03 pH units at a rate of 0.33 +/- 0.04 pH units/min. The cell acidification was stilbene inhibitable and independent of chloride. There was no effect of bath sodium removal on cell pH in the absence of CO2/HCO3. Depolarization of the basolateral membrane (step increase in bath potassium) independent of the presence of chloride. Cell acidification induced by bath sodium removal persisted when the basolateral membrane was voltage clamped by high potassium/valinomycin. Although these results are consistent with a Na/(HCO3)n greater than 1 cotransporter, a Na/H antiporter was also suggested: 1 mM bath amiloride inhibited the cell pH defense against an acid load (rapid ammonia washout), both in the presence and absence of CO2/HCO3, and inhibited the cell acidification induced by bath sodium reduction from 50 to 0 mM. In conclusion, an electrogenic Na/(HCO3)n greater than 1 cotransporter in parallel with a Na/H antiporter exist on the basolateral membrane of the rat cortical thick ascending limb. PMID:2839547

  5. Membrane trafficking. The specificity of vesicle traffic to the Golgi is encoded in the golgin coiled-coil proteins.

    PubMed

    Wong, Mie; Munro, Sean

    2014-10-31

    The Golgi apparatus is a multicompartment central sorting station at the intersection of secretory and endocytic vesicular traffic. The mechanisms that permit cargo-loaded transport vesicles from different origins to selectively access different Golgi compartments are incompletely understood. We developed a rerouting and capture assay to investigate systematically the vesicle-tethering activities of 10 widely conserved golgin coiled-coil proteins. We find that subsets of golgins with distinct localizations on the Golgi surface have capture activities toward vesicles of different origins. These findings demonstrate that golgins act as tethers in vivo, and hence the specificity we find to be encoded in this tethering is likely to make a major contribution to the organization of membrane traffic at the Golgi apparatus.

  6. Electro-optics of membrane electroporation in diphenylhexatriene-doped lipid bilayer vesicles.

    PubMed

    Kakorin, S; Stoylov, S P; Neumann, E

    1996-01-16

    The electric (linear) dichroisms observed in the membrane electroporation of salt-filled lipid bilayer vesicles (diameter O = 2 alpha = 0.32 micron; inside [NaCl] = 0.2 M) in isotonic aqueous 0.284 M sucrose-0.2 mM NaCl solution indicate orientation changes of the anisotropic light scattering centers (lipid head groups) and of the optical transition moments of the membrane-inserted probe 1,6-diphenyl-1,3,5-hexatriene (DPH). Both the turbidity dichroism and DPH absorbance dichroism show peculiar features: (1) at external electric fields E > or = Esat the time course of the dichroism shows a maximum value (reversal): Esat = 4.0 (+/- 0.2) MV m-1, T = 293 K (20 degrees C), (2) this reversal value is independent of the field strength for E > or = Esat, (3) the dichroism amplitudes exhibit a maximum value Emax = 3.0 (+/- 0.5) MV m-1, (4) for the pulse duration of 10 microseconds there is one dominant visible normal mode, the relaxation rate increases up to tau-1 approximately 0.6 x 10(6) s-1 at Esat and then decreases for E > Esat. The data can be described in terms of local lipid phase transitions involving clusters Ln of n lipids in the pore edges according to the three-state scheme C<-->HO<-->HI, C being the closed bilayer state, HO the hydrophobic pore state and HI the hydrophilic or inverted pore state with rotated lipid and DPH molecules. At E > or = Esat, further transitions HO<-->HO* and HI<-->HI* are rapidly coupled to the C<-->HO transition, which is rate-limiting. The vesicle geometry conditions a cos theta dependence of the local membrane field effects relative to the E direction and the data reflect cos theta averages. The stationary induced transmembrane voltage delta phi (theta, lambda m) = -1.5 aEf(lambda m) magnitude of cos theta does not exceed the limiting value delta phi sat = -0.53 V, corresponding to the field strength Em,sat = -delta phi sat/d = 100 MV m-1 (10(3) kV cm-1), due to increasing membrane conductivity lambda m. At E = Esat, f(lambda m

  7. The Bif-1-Dynamin 2 membrane fission machinery regulates Atg9-containing vesicle generation at the Rab11-positive reservoirs

    PubMed Central

    Liu, Ying; Young, Megan M.; Serfass, Jacob; Tang, Zhenyuan; Abraham, Thomas; Wang, Hong-Gang

    2016-01-01

    Atg9 is a multispanning transmembrane protein that is required for autophagosome formation. During autophagy, vesicles containing Atg9 are generated through an unknown mechanism and delivered to the autophagosome formation sites. We have previously reported that Atg9-containing membranes undergo continuous tubulation and fission during nutrient starvation in a manner dependent on the curvature-inducing protein Bif-1/Sh3glb1. Here, we identify Dynamin 2 (DNM2) as a Bif-1-interacting protein that mediates the fission of Atg9-containing membranes during autophagy. The interaction of Bif-1 and DNM2 is enhanced upon nutrient starvation, and Bif-1 and DNM2 cooperatively induce the generation of Atg9-containing vesicles. Inhibition of the GTPase activity of DNM2 results in the accumulation of Atg9-positive tubular structures that originate from a Rab11-positive reservoir. Although Atg9 seems to be constitutively trafficked to the reservoir regardless of Bif-1 expression, membrane tubulation from the Atg9 reservoir is dependent on Bif-1 and is strongly induced upon nutrient starvation. These findings suggest that the generation of Atg9 vesicles from a Rab11-positive reservoir is tightly controlled by the Bif-1-DNM2 membrane fission machinery in response to cellular demand for autophagy. PMID:26980706

  8. Recruitment and SNARE-mediated fusion of vesicles in furrow membrane remodeling during cytokinesis in zebrafish embryos

    SciTech Connect

    Ming Liwai; Webb, Sarah E.; Lee, Karen W.; Miller, Andrew L. . E-mail: almiller@ust.hk

    2006-10-15

    Cytokinesis is the final stage in cell division that serves to partition cytoplasm and daughter nuclei into separate cells. Membrane remodeling at the cleavage plane is a required feature of cytokinesis in many species. In animal cells, however, the precise mechanisms and molecular interactions that mediate this process are not yet fully understood. Using real-time imaging in live, early stage zebrafish embryos, we demonstrate that vesicles labeled with the v-SNARE, VAMP-2, are recruited to the cleavage furrow during deepening in a microtubule-dependent manner. These vesicles then fuse with, and transfer their VAMP-2 fluorescent label to, the plasma membrane during both furrow deepening and subsequent apposition. This observation indicates that new membrane is being inserted during these stages of cytokinesis. Inhibition of SNAP-25 (a cognate t-SNARE of VAMP-2), using a monoclonal antibody, blocked VAMP-2 vesicle fusion and furrow apposition. Transient expression of mutant forms of SNAP-25 also produced defects in furrow apposition. SNAP-25 inhibition by either method, however, did not have any significant effect on furrow deepening. Thus, our data clearly indicate that VAMP-2 and SNAP-25 play an essential role in daughter blastomere apposition, possibly via the delivery of components that promote the cell-to-cell adhesion required for the successful completion of cytokinesis. Our results also support the idea that new membrane addition, which occurs during late stage cytokinesis, is not required for furrow deepening that results from contractile band constriction.

  9. Growth Conditions and Cell Cycle Phase Modulate Phase Transition Temperatures in RBL-2H3 Derived Plasma Membrane Vesicles.

    PubMed

    Gray, Erin M; Díaz-Vázquez, Gladys; Veatch, Sarah L

    2015-01-01

    Giant plasma membrane vesicle (GPMV) isolated from a flask of RBL-2H3 cells appear uniform at physiological temperatures and contain coexisting liquid-ordered and liquid-disordered phases at low temperatures. While a single GPMV transitions between these two states at a well-defined temperature, there is significant vesicle-to-vesicle heterogeneity in a single preparation of cells, and average transition temperatures can vary significantly between preparations. In this study, we explore how GPMV transition temperatures depend on growth conditions, and find that average transition temperatures are negatively correlated with average cell density over 15°C in transition temperature and nearly three orders of magnitude in average surface density. In addition, average transition temperatures are reduced by close to 10°C when GPMVs are isolated from cells starved of serum overnight, and elevated transition temperatures are restored when serum-starved cells are incubated in serum-containing media for 12 h. We also investigated variation in transition temperature of GPMVs isolated from cells synchronized at the G1/S border through a double Thymidine block and find that average transition temperatures are systematically higher in GPMVs produced from G1 or M phase cells than in GPMVs prepared from S or G1 phase cells. Reduced miscibility transition temperatures are also observed in GPMVs prepared from cells treated with TRAIL to induce apoptosis or sphingomyelinase, and in some cases a gel phase is observed at temperatures above the miscibility transition in these vesicles. We conclude that at least some variability in GPMV transition temperature arises from variation in the local density of cells and asynchrony of the cell cycle. It is hypothesized that GPMV transition temperatures are a proxy for the magnitude of lipid-mediated membrane heterogeneity in intact cell plasma membranes at growth temperatures. If so, these results suggest that cells tune their plasma membrane

  10. Engineered Asymmetric Synthetic Vesicles

    NASA Astrophysics Data System (ADS)

    Lu, Li; Chiarot, Paul

    2013-11-01

    Synthetic vesicles are small, fluid-filled spheres that are enclosed by a bilayer of lipid molecules. They can be used as models for investigating membrane biology and as delivery vehicles for pharmaceuticals. In practice, it is difficult to simultaneously control membrane asymmetry, unilamellarity, vesicle size, vesicle-to-vesicle uniformity, and luminal content. Membrane asymmetry, where each leaflet of the bilayer is composed of different lipids, is of particular importance as it is a feature of most natural membranes. In this study, we leverage microfluidic technology to build asymmetric vesicles at high-throughput. We use the precise flow control offered by microfluidic devices to make highly uniform emulsions, with controlled internal content, that serve as templates to build the synthetic vesicles. Flow focusing, dielectrophoretic steering, and interfacial lipid self-assembly are critical procedures performed on-chip to produce the vesicles. Fluorescent and confocal microscopy are used to evaluate the vesicle characteristics.

  11. Coexistence of intracytoplasmic lumens and membrane-bound vesicles in an invasive carcinoma arising in a cystosarcoma phyllodes.

    PubMed

    Gilks, B; Tavassoli, F A

    1988-01-01

    An unusual invasive breast carcinoma, arising in a cystosarcoma phyllodes and characterized by a variable cytoplasmic appearance and mucin content, was evaluated to determine the nature of the secretory material within the cells as well as the type of secretory organelle at the ultrastructural level. Histochemical studies revealed both acidic (sialic acid) and neutral mucin within the tumor cells. Ultrastructural analysis revealed secretory material within membrane-bound vesicles in some cells and within intracytoplasmic lumens in others; some cells contained both membrane-bound vesicles and intracytoplasmic lumens simultaneously. The Golgi derivation of the intracytoplasmic lumens was supported by their presence within or near hyperplastic Golgi complexes. The histochemical characteristics of the secretory material is correlated with their ultrastructural site of accumulation.

  12. In vitro binding of the asialoglycoprotein receptor to the beta adaptin of plasma membrane coated vesicles.

    PubMed Central

    Beltzer, J P; Spiess, M

    1991-01-01

    The asialoglycoprotein (ASGP) receptor was used to probe total clathrin-coated vesicle proteins and purified adaptor proteins (APs) which had been fractionated by gel electrophoresis and transferred to nitrocellulose. The receptor was found to interact with proteins of approximately 100 kDa. The cytoplasmic domain of the ASGP receptor subunit H1 fused to dihydrofolate reductase competed for receptor binding to the 100 kDa polypeptide in the plasma membrane-type AP complexes (AP-2). A fusion protein containing the cytoplasmic domain of the endocytic mutant haemagglutinin HA-Y543 also competed, but a protein with the wild-type haemagglutinin sequence did not. This indicates that the observed interaction is specific for the cytoplasmic domain of the receptor and involves the tyrosine signal for endocytosis. When fractionated by gel electrophoresis in the presence of urea, the ASGP receptor binding polypeptide displayed a characteristic shift in electrophoretic mobility identifying it as the beta adaptin. Partial proteolysis of the AP-2 preparation followed by the receptor binding assay revealed that the aminoterminal domain of the beta adaptin contains the binding site for receptors. Images PMID:1935897

  13. Differential Responses of Pattern Recognition Receptors to Outer Membrane Vesicles of Three Periodontal Pathogens

    PubMed Central

    Lenzo, Jason C.; Holden, James A.; Chen, Yu-Yen; Singleton, William; Gause, Katelyn T.; Yan, Yan; Caruso, Frank; Reynolds, Eric C.

    2016-01-01

    Highly purified outer membrane vesicles (OMVs) of the periodontal pathogens, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia were produced using tangential flow ultrafiltration, ultracentrifugation and Optiprep density gradient separation. Cryo-TEM and light scattering showed OMVs to be single lipid-bilayers with modal diameters of 75 to 158 nm. Enumeration of OMVs by nanoparticle flow-cytometry at the same stage of late exponential culture indicated that P. gingivalis was the most prolific OMV producer. P. gingivalis OMVs induced strong TLR2 and TLR4-specific responses and moderate responses in TLR7, TLR8, TLR9, NOD1 and NOD2 expressing-HEK-Blue cells. Responses to T. forsythia OMVs were less than those of P. gingivalis and T. denticola OMVs induced only weak responses. Compositional analyses of OMVs from the three pathogens demonstrated differences in protein, fatty acids, lipopolysaccharide, peptidoglycan fragments and nucleic acids. Periodontal pathogen OMVs induced differential pattern recognition receptor responses that have implications for their role in chronic periodontitis. PMID:27035339

  14. Direct effect of vanadium on citrate uptake by rat renal brush border membrane vesicles (BBMV).

    PubMed

    Sato, Kazuhiro; Kusaka, Yukinori; Akino, Hironobu; Kanamaru, Hiroshi; Okada, Kenichiro

    2002-07-01

    Vanadium pentoxide is used as a catalyst and a ferrovanadium alloy ingredient in automotive steels and in jet engines and airframes. In addition, vanadium is found in fuel oils. Thus, occupational exposures to vanadium pentoxide and trioxide may occur during the cleaning of oil-fired ship boilers, and from oil-fired power station boilers. Occupational exposure to vanadium pentoxide induces green tongue, asthmatic symptoms and albuminuria with cast. Urinary citrate is freely filtered at the glomerulus, and its reabsorption in the proximal tubule is the major determinant of the rate of renal excretion. In this study, we exposed rat renal brush border membrane vesicles (BBMV) to vanadium pentoxide and examined their citrate uptake characteristics. The preincubation of BBMV with 1 mM V2O5 for 8 hours significantly inhibited citrate uptake compared with that of BBMV without V2O5, preincubation. These findings indicate that the preincubation of BBMV with vanadium pentoxide results in a time-dependent inhibition of citrate uptake by BBMV. These findings might contribute to nephrotoxicity in vanadium exposure. PMID:12141377

  15. Protecting enzymatic function through directed packaging into bacterial outer membrane vesicles.

    PubMed

    Alves, Nathan J; Turner, Kendrick B; Medintz, Igor L; Walper, Scott A

    2016-01-01

    Bacteria possess innate machinery to transport extracellular cargo between cells as well as package virulence factors to infect host cells by secreting outer membrane vesicles (OMVs) that contain small molecules, proteins, and genetic material. These robust proteoliposomes have evolved naturally to be resistant to degradation and provide a supportive environment to extend the activity of encapsulated cargo. In this study, we sought to exploit bacterial OMV formation to package and maintain the activity of an enzyme, phosphotriesterase (PTE), under challenging storage conditions encountered for real world applications. Here we show that OMV packaged PTE maintains activity over free PTE when subjected to elevated temperatures (>100-fold more activity after 14 days at 37 °C), iterative freeze-thaw cycles (3.4-fold post four-cycles), and lyophilization (43-fold). We also demonstrate how lyophilized OMV packaged PTE can be utilized as a cell free reagent for long term environmental remediation of pesticide/chemical warfare contaminated areas. PMID:27117743

  16. CALM regulates clathrin-coated vesicle size and maturation by directly sensing and driving membrane curvature.

    PubMed

    Miller, Sharon E; Mathiasen, Signe; Bright, Nicholas A; Pierre, Fabienne; Kelly, Bernard T; Kladt, Nikolay; Schauss, Astrid; Merrifield, Christien J; Stamou, Dimitrios; Höning, Stefan; Owen, David J

    2015-04-20

    The size of endocytic clathrin-coated vesicles (CCVs) is remarkably uniform, suggesting that it is optimized to achieve the appropriate levels of cargo and lipid internalization. The three most abundant proteins in mammalian endocytic CCVs are clathrin and the two cargo-selecting, clathrin adaptors, CALM and AP2. Here we demonstrate that depletion of CALM causes a substantial increase in the ratio of "open" clathrin-coated pits (CCPs) to "necked"/"closed" CCVs and a doubling of CCP/CCV diameter, whereas AP2 depletion has opposite effects. Depletion of either adaptor, however, significantly inhibits endocytosis of transferrin and epidermal growth factor. The phenotypic effects of CALM depletion can be rescued by re-expression of wild-type CALM, but not with CALM that lacks a functional N-terminal, membrane-inserting, curvature-sensing/driving amphipathic helix, the existence and properties of which are demonstrated. CALM is thus a major factor in controlling CCV size and maturation and hence in determining the rates of endocytic cargo uptake.

  17. Outer Membrane Vesicle-Mediated Export of Processed PrtV Protease from Vibrio cholerae

    PubMed Central

    Duperthuy, Marylise; Johnson, Tanya L.; Åhlund, Monika; Lundmark, Richard; Oscarsson, Jan; Sandkvist, Maria; Uhlin, Bernt Eric; Wai, Sun Nyunt

    2015-01-01

    Background Outer membrane vesicles (OMVs) are known to release from almost all Gram-negative bacteria during normal growth. OMVs carry different biologically active toxins and enzymes into the surrounding environment. We suggest that OMVs may therefore be able to transport bacterial proteases into the target host cells. We present here an analysis of the Vibrio cholerae OMV-associated protease PrtV. Methodology/Principal Findings In this study, we demonstrated that PrtV was secreted from the wild type V. cholerae strain C6706 via the type II secretion system in association with OMVs. By immunoblotting and electron microscopic analysis using immunogold labeling, the association of PrtV with OMVs was examined. We demonstrated that OMV-associated PrtV was biologically active by showing altered morphology and detachment of cells when the human ileocecum carcinoma (HCT8) cells were treated with OMVs from the wild type V. cholerae strain C6706 whereas cells treated with OMVs from the prtV isogenic mutant showed no morphological changes. Furthermore, OMV-associated PrtV protease showed a contribution to bacterial resistance towards the antimicrobial peptide LL-37. Conclusion/Significance Our findings suggest that OMVs released from V. cholerae can deliver a processed, biologically active form of PrtV that contributes to bacterial interactions with target host cells. PMID:26222047

  18. Immunoproteomic Profiling of Bordetella pertussis Outer Membrane Vesicle Vaccine Reveals Broad and Balanced Humoral Immunogenicity.

    PubMed

    Raeven, René H M; van der Maas, Larissa; Tilstra, Wichard; Uittenbogaard, Joost P; Bindels, Tim H E; Kuipers, Betsy; van der Ark, Arno; Pennings, Jeroen L A; van Riet, Elly; Jiskoot, Wim; Kersten, Gideon F A; Metz, Bernard

    2015-07-01

    The current resurgence of whooping cough is alarming, and improved pertussis vaccines are thought to offer a solution. Outer membrane vesicle vaccines (omvPV) are potential vaccine candidates, but omvPV-induced humoral responses have not yet been characterized in detail. The purpose of this study was to determine the antigen composition of omvPV and to elucidate the immunogenicity of the individual antigens. Quantitative proteome analysis revealed the complex composition of omvPV. The omvPV immunogenicity profile in mice was compared to those of classic whole cell vaccine (wPV), acellular vaccine (aPV), and pertussis infection. Pertussis-specific antibody levels, antibody isotypes, IgG subclasses, and antigen specificity were determined after vaccination or infection by using a combination of multiplex immunoassays, two-dimensional immunoblotting, and mass spectrometry. The vaccines and infection raised strong antibody responses, but large quantitative and qualitative differences were measured. The highest antibody levels were obtained by omvPV. All IgG subclasses (IgG1/IgG2a/IgG2b/IgG3) were elicited by omvPV and in a lower magnitude by wPV, but not by aPV (IgG1) or infection (IgG2a/b). The majority of omvPV-induced antibodies were directed against Vag8, BrkA, and LPS. The broad and balanced humoral response makes omvPV a promising pertussis vaccine candidate.

  19. Fluorescent BODIPY Rotor: Viscometer for Cellular Organelles and Membrane-Mimicking Vesicles

    NASA Astrophysics Data System (ADS)

    Kimball, J.; Raut, S.; Fudala, R.; Doan, H.; Maliwal, B.; Sabnis, N.; Lacko, A.; Gryczynski, I.; Dzyuba, S.; Gryczynski, Z.

    2015-03-01

    Many cellular processes, such as mass and signal transport, metabolism and protein-protein interactions are governed in part by diffusion, and thus affected by their local microviscosity. Changes in this microviscosity has also been linked to various diseases, including atherosclerosis, Alzheimer's disease and diabetes. Therefore, directly measuring the heterogeneous viscosity of cellular constitutes can lead to greater understanding of these processes. To this effect, a novel homodiemeric BODIPY dye was evaluated as a fluorescent rotor probe for this application. A linear dependence on viscosity in the range of typical cellular microviscosity was established for steady-state and time-resolved properties of the dye. It was then embedded in vitro to membrane-mimicking lipid vesicles (DPPC, POPC, and POPC plus cholesterol) and results indicated it to be a viable sensor for lifetime-based determination of microviscosity. The BODIPY dye was lastly endocytosed by SKOV3 cells and Fluorescence Lifetime Imaging Microscopy (FLIM) was performed, successfully mapping the viscosity of internal cell components. This work was supported by the NIH Grant R01EB12003, the NSF Grant CBET-1264608, and the INFOR Grant from TCU.

  20. Modulation of GABA-stimulated chloride influx into membrane vesicles from rat cerebral cortex by triazolobenzodiazepines

    SciTech Connect

    Obata, T.; Yamamura, H.I.

    1988-01-01

    The effects of triazolobenzodiazepines of GABA-stimulated /sup 36/Cl/sup -/ uptake by membrane vesicles from rat cerebral cortex were examined. Triazolam and alprazolam showed a significant enhancement of GABA-stimulated /sup 36/Cl/sup -/ uptake at 0.01-10 uM. On the other hand, adinazolam showed a small enhancement at 0.1-1 uM followed by a significant inhibition of GABA-stimulated /sup 36/Cl/sup -/ uptake at 100 uM. The enhancement of GABA-stimulated /sup 36/Cl/sup -/ uptake by 1 uM alprazolam was antagonized by Ro15-1788, a benzodiazepine antagonist, but the inhibition of this response by 30 uM adinazolam was not antagonized by Ro15-1788. These results indicate that triazolobenzodiazepines enhanced GABA-stimulated /sup 36/Cl/sup -/ uptake through benzodiazepine receptors. High concentrations of adinazolam inhibit GABA-stimulated /sup 36/Cl/sup -/ uptake which may be due to the direct blockade of GABA-gated chloride channel. 23 references, 4 figures.

  1. Identification and Characterization of Outer Membrane Vesicle-Associated Proteins in Salmonella enterica Serovar Typhimurium

    PubMed Central

    Bai, Jaewoo; Kim, Seul I; Ryu, Sangryeol

    2014-01-01

    Salmonella enterica serovar Typhimurium is a primary cause of enteric diseases and has acquired a variety of virulence factors during its evolution into a pathogen. Secreted virulence factors interact with commensal flora and host cells and enable Salmonella to survive and thrive in hostile environments. Outer membrane vesicles (OMVs) released from many Gram-negative bacteria function as a mechanism for the secretion of complex mixtures, including virulence factors. We performed a proteomic analysis of OMVs that were isolated under standard laboratory and acidic minimal medium conditions and identified 14 OMV-associated proteins that were observed in the OMV fraction isolated only under the acidic minimal medium conditions, which reproduced the nutrient-deficient intracellular milieu. The inferred roles of these 14 proteins were diverse, including transporter, enzyme, and transcriptional regulator. The absence of these proteins influenced Salmonella survival inside murine macrophages. Eleven of these proteins were predicted to possess secretion signal sequences at their N termini, and three (HupA, GlnH, and PhoN) of the proteins were found to be translocated into the cytoplasm of host cells. The comparative proteomic profiling of OMVs performed in this study revealed different protein compositions in the OMVs isolated under the two different conditions, which indicates that the OMV cargo depends on the growth conditions and provides a deeper insight into how Salmonella utilizes OMVs to adapt to environmental changes. PMID:24935973

  2. Auxin regulation of a proton translocating ATPase in pea root plasma membrane vesicles. [Pisum sativum. L

    SciTech Connect

    Gabathuler, R.; Cleland, R.E.

    1985-12-01

    Pea root microsomal vesicles have been fractionated on a Dextran step gradient to give three fractions, each of which carries out ATP-dependent proton accumulation as measured by fluorescence quenching of quinacrine. The fraction at the 4/6% Dextran interface is enriched in plasma membrane, as determined by UDPG sterol glucosyltransferase and vanadate-inhibited ATPase. The vanadate-sensitive phosphohydrolase is not specific for ATP, has a K/sub m/ of about 0.23 millimolar for MgATP, is only slightly affected by K/sup +/ or Cl/sup -/ and is insensitive to auxin. Proton transport, on the other hand, is more specific for ATP, enhanced by anions (NO/sub 3//sup -/ > Cl/sup -/) and has a K/sub m/ of about 0.7 millimolar. Auxins decrease the K/sub m/ to about 0.35 millimolar, with no significant effect on the V/sub max/, while antiauxins or weak acids have no such effect. It appears that auxin has the ability to alter the efficiency of the ATP-driven proton transport.

  3. A Shigella flexneri virulence plasmid encoded factor controls production of outer membrane vesicles.

    PubMed

    Sidik, Saima; Kottwitz, Haila; Benjamin, Jeremy; Ryu, Julie; Jarrar, Ameer; Garduno, Rafael; Rohde, John R

    2014-12-01

    Shigella spp. use a repertoire of virulence plasmid-encoded factors to cause shigellosis. These include components of a Type III Secretion Apparatus (T3SA) that is required for invasion of epithelial cells and many genes of unknown function. We constructed an array of 99 deletion mutants comprising all genes encoded by the virulence plasmid (excluding those known to be required for plasmid maintenance) of Shigella flexneri. We screened these mutants for their ability to bind the dye Congo red: an indicator of T3SA function. This screen focused our attention on an operon encoding genes that modify the cell envelope including virK, a gene of partially characterized function. We discovered that virK is required for controlled release of proteins to the culture supernatant. Mutations in virK result in a temperature-dependent overproduction of outer membrane vesicles (OMVs). The periplasmic chaperone/protease DegP, a known regulator of OMV production in Escherichia coli (encoded by a chromosomal gene), was found to similarly control OMV production in S. flexneri. Both virK and degP show genetic interactions with mxiD, a structural component of the T3SA. Our results are consistent with a model in which VirK and DegP relieve the periplasmic stress that accompanies assembly of the T3SA. PMID:25378474

  4. A Shigella flexneri Virulence Plasmid Encoded Factor Controls Production of Outer Membrane Vesicles

    PubMed Central

    Sidik, Saima; Kottwitz, Haila; Benjamin, Jeremy; Ryu, Julie; Jarrar, Ameer; Garduno, Rafael; Rohde, John R.

    2014-01-01

    Shigella spp. use a repertoire of virulence plasmid-encoded factors to cause shigellosis. These include components of a Type III Secretion Apparatus (T3SA) that is required for invasion of epithelial cells and many genes of unknown function. We constructed an array of 99 deletion mutants comprising all genes encoded by the virulence plasmid (excluding those known to be required for plasmid maintenance) of Shigella flexneri. We screened these mutants for their ability to bind the dye Congo red: an indicator of T3SA function. This screen focused our attention on an operon encoding genes that modify the cell envelope including virK, a gene of partially characterized function. We discovered that virK is required for controlled release of proteins to the culture supernatant. Mutations in virK result in a temperature-dependent overproduction of outer membrane vesicles (OMVs). The periplasmic chaperone/protease DegP, a known regulator of OMV production in Escherichia coli (encoded by a chromosomal gene), was found to similarly control OMV production in S. flexneri. Both virK and degP show genetic interactions with mxiD, a structural component of the T3SA. Our results are consistent with a model in which VirK and DegP relieve the periplasmic stress that accompanies assembly of the T3SA. PMID:25378474

  5. Vaccines against meningococcal serogroup B disease containing outer membrane vesicles (OMV)

    PubMed Central

    Holst, Johan; Oster, Philipp; Arnold, Richard; Tatley, Michael V.; Næss, Lisbeth M.; Aaberge, Ingeborg S.; Galloway, Yvonne; McNicholas, Anne; O’Hallahan, Jane; Rosenqvist, Einar; Black, Steven

    2013-01-01

    The utility of wild-type outer membrane vesicle (wtOMV) vaccines against serogroup B (MenB) meningococcal disease has been explored since the 1970s. Public health interventions in Cuba, Norway and New Zealand have demonstrated that these protein-based vaccines can prevent MenB disease. Data from large clinical studies and retrospective statistical analyses in New Zealand give effectiveness estimates of at least 70%. A consistent pattern of moderately reactogenic and safe vaccines has been seen with the use of approximately 60 million doses of three different wtOMV vaccine formulations. The key limitation of conventional wtOMV vaccines is their lack of broad protective activity against the large diversity of MenB strains circulating globally. The public health intervention in New Zealand (between 2004–2008) when MeNZB was used to control a clonal MenB epidemic, provided a number of new insights regarding international and public-private collaboration, vaccine safety surveillance, vaccine effectiveness estimates and communication to the public. The experience with wtOMV vaccines also provide important information for the next generation of MenB vaccines designed to give more comprehensive protection against multiple strains. PMID:23857274

  6. Flocculation of Escherichia coli Cells in Association with Enhanced Production of Outer Membrane Vesicles.

    PubMed

    Ojima, Yoshihiro; Nguyen, Minh Hong; Yajima, Reiki; Taya, Masahito

    2015-09-01

    Microbial flocculation is a phenomenon of aggregation of dispersed bacterial cells in the form of flocs or flakes. In this study, the mechanism of spontaneous flocculation of Escherichia coli cells by overexpression of the bcsB gene was investigated. The flocculation induced by overexpression of bcsB was consistent among the various E. coli strains examined, including the K-12, B, and O strains, with flocs that resembled paper scraps in structure being about 1 to 2 mm. The distribution of green fluorescent protein-labeled E. coli cells within the floc structure was investigated by three-dimensional confocal laser scanning microscopy. Flocs were sensitive to proteinase K, indicating that the main component of the flocs was proteinous. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nano-liquid chromatography tandem mass spectrometry analyses of the flocs strongly suggested the involvement of outer membrane vesicles (OMVs) in E. coli flocculation. The involvement of OMVs in flocculation was supported by transmission electron microscopy observation of flocs. Furthermore, bcsB-induced E. coli flocculation was greatly suppressed in strains with hypovesiculation phenotypes (ΔdsbA and ΔdsbB strains). Thus, our results demonstrate the strong correlation between spontaneous flocculation and enhanced OMV production of E. coli cells.

  7. Differential Responses of Pattern Recognition Receptors to Outer Membrane Vesicles of Three Periodontal Pathogens.

    PubMed

    Cecil, Jessica D; O'Brien-Simpson, Neil M; Lenzo, Jason C; Holden, James A; Chen, Yu-Yen; Singleton, William; Gause, Katelyn T; Yan, Yan; Caruso, Frank; Reynolds, Eric C

    2016-01-01

    Highly purified outer membrane vesicles (OMVs) of the periodontal pathogens, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia were produced using tangential flow ultrafiltration, ultracentrifugation and Optiprep density gradient separation. Cryo-TEM and light scattering showed OMVs to be single lipid-bilayers with modal diameters of 75 to 158 nm. Enumeration of OMVs by nanoparticle flow-cytometry at the same stage of late exponential culture indicated that P. gingivalis was the most prolific OMV producer. P. gingivalis OMVs induced strong TLR2 and TLR4-specific responses and moderate responses in TLR7, TLR8, TLR9, NOD1 and NOD2 expressing-HEK-Blue cells. Responses to T. forsythia OMVs were less than those of P. gingivalis and T. denticola OMVs induced only weak responses. Compositional analyses of OMVs from the three pathogens demonstrated differences in protein, fatty acids, lipopolysaccharide, peptidoglycan fragments and nucleic acids. Periodontal pathogen OMVs induced differential pattern recognition receptor responses that have implications for their role in chronic periodontitis.

  8. Differential Responses of Pattern Recognition Receptors to Outer Membrane Vesicles of Three Periodontal Pathogens.

    PubMed

    Cecil, Jessica D; O'Brien-Simpson, Neil M; Lenzo, Jason C; Holden, James A; Chen, Yu-Yen; Singleton, William; Gause, Katelyn T; Yan, Yan; Caruso, Frank; Reynolds, Eric C

    2016-01-01

    Highly purified outer membrane vesicles (OMVs) of the periodontal pathogens, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia were produced using tangential flow ultrafiltration, ultracentrifugation and Optiprep density gradient separation. Cryo-TEM and light scattering showed OMVs to be single lipid-bilayers with modal diameters of 75 to 158 nm. Enumeration of OMVs by nanoparticle flow-cytometry at the same stage of late exponential culture indicated that P. gingivalis was the most prolific OMV producer. P. gingivalis OMVs induced strong TLR2 and TLR4-specific responses and moderate responses in TLR7, TLR8, TLR9, NOD1 and NOD2 expressing-HEK-Blue cells. Responses to T. forsythia OMVs were less than those of P. gingivalis and T. denticola OMVs induced only weak responses. Compositional analyses of OMVs from the three pathogens demonstrated differences in protein, fatty acids, lipopolysaccharide, peptidoglycan fragments and nucleic acids. Periodontal pathogen OMVs induced differential pattern recognition receptor responses that have implications for their role in chronic periodontitis. PMID:27035339

  9. Multifunctional Transmembrane Protein Ligands for Cell-Specific Targeting of Plasma Membrane-Derived Vesicles.

    PubMed

    Zhao, Chi; Busch, David J; Vershel, Connor P; Stachowiak, Jeanne C

    2016-07-01

    Liposomes and nanoparticles that bind selectively to cell-surface receptors can target specific populations of cells. However, chemical conjugation of ligands to these particles is difficult to control, frequently limiting ligand uniformity and complexity. In contrast, the surfaces of living cells are decorated with highly uniform populations of sophisticated transmembrane proteins. Toward harnessing cellular capabilities, here it is demonstrated that plasma membrane vesicles (PMVs) derived from donor cells can display engineered transmembrane protein ligands that precisely target cells on the basis of receptor expression. These multifunctional targeting proteins incorporate (i) a protein ligand, (ii) an intrinsically disordered protein spacer to make the ligand sterically accessible, and (iii) a fluorescent protein domain that enables quantification of the ligand density on the PMV surface. PMVs that display targeting proteins with affinity for the epidermal growth factor receptor (EGFR) bind at increasing concentrations to breast cancer cells that express increasing levels of EGFR. Further, as an example of the generality of this approach, PMVs expressing a single-domain antibody against green fluorescence protein (eGFP) bind to cells expressing eGFP-tagged receptors with a selectivity of ≈50:1. The results demonstrate the versatility of PMVs as cell targeting systems, suggesting diverse applications from drug delivery to tissue engineering. PMID:27294846

  10. p23, a major COPI-vesicle membrane protein, constitutively cycles through the early secretory pathway.

    PubMed

    Nickel, W; Sohn, K; Bünning, C; Wieland, F T

    1997-10-14

    A novel type I transmembrane protein of COPI-coated vesicles, p23, has been demonstrated to be localized mainly to the Golgi complex. This protein and p24, another member of the p24 family, have been shown to bind coatomer via their short cytoplasmic tails. Here we demonstrate that p23 continuously cycles through the early secretory pathway. The cytoplasmic tail of p23 is shown to act as a functional retrieval signal as it confers endoplasmic reticulum (ER) residence to a CD8-p23 fusion protein. This ER localization is, at least in part, a result of retrieval from post-ER compartments because CD8-p23 fusion proteins receive post-ER modifications. In contrast, the cytoplasmic tail of p24 has been shown not to retrieve a CD8-p24 fusion protein. The coatomer binding motifs FF and KK in the cytoplasmic tail of p23 are reported to influence the steady-state localization of the CD8-p23 fusion protein within the ER-Golgi recycling pathway. It appears that the steady-state Golgi localization of endogenous p23 is maintained by its lumenal domain, as a fusion protein with the lumenal domain of CD8, and the membrane span as well as the cytoplasmic tail of p23 is no longer detected in the Golgi. PMID:9326620

  11. Characterization of Outer Membrane Vesicles from Brucella melitensis and Protection Induced in Mice

    PubMed Central

    Avila-Calderón, Eric Daniel; Lopez-Merino, Ahidé; Jain, Neeta; Peralta, Humberto; López-Villegas, Edgar Oliver; Sriranganathan, Nammalwar; Boyle, Stephen M.; Witonsky, Sharon; Contreras-Rodríguez, Araceli

    2012-01-01

    The outer membrane vesicles (OMVs) from smooth B. melitensis 16 M and a derived rough mutant, VTRM1 strain, were purified and characterized with respect to protein content and induction of immune responses in mice. Proteomic analysis showed 29 proteins present in OMVs from B. melitensis 16 M; some of them are well-known Brucella immunogens such as SOD, GroES, Omp31, Omp25, Omp19, bp26, and Omp16. OMVs from a rough VTRM1 induced significantly higher expression of IL-12, TNFα, and IFNγ genes in bone marrow dendritic cells than OMVs from smooth strain 16 M. Relative to saline control group, mice immunized intramuscularly with rough and smooth OMVs were protected from challenge with virulent strain B. melitensis 16 M just as well as the group immunized with live strain B. melitensis Rev1 (P < 0.005). Additionally, the levels of serum IgG2a increased in mice vaccinated with OMVs from rough strain VTRM1 consistent with the induction of cell-mediated immunity. PMID:22242036

  12. Flocculation of Escherichia coli Cells in Association with Enhanced Production of Outer Membrane Vesicles.

    PubMed

    Ojima, Yoshihiro; Nguyen, Minh Hong; Yajima, Reiki; Taya, Masahito

    2015-09-01

    Microbial flocculation is a phenomenon of aggregation of dispersed bacterial cells in the form of flocs or flakes. In this study, the mechanism of spontaneous flocculation of Escherichia coli cells by overexpression of the bcsB gene was investigated. The flocculation induced by overexpression of bcsB was consistent among the various E. coli strains examined, including the K-12, B, and O strains, with flocs that resembled paper scraps in structure being about 1 to 2 mm. The distribution of green fluorescent protein-labeled E. coli cells within the floc structure was investigated by three-dimensional confocal laser scanning microscopy. Flocs were sensitive to proteinase K, indicating that the main component of the flocs was proteinous. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nano-liquid chromatography tandem mass spectrometry analyses of the flocs strongly suggested the involvement of outer membrane vesicles (OMVs) in E. coli flocculation. The involvement of OMVs in flocculation was supported by transmission electron microscopy observation of flocs. Furthermore, bcsB-induced E. coli flocculation was greatly suppressed in strains with hypovesiculation phenotypes (ΔdsbA and ΔdsbB strains). Thus, our results demonstrate the strong correlation between spontaneous flocculation and enhanced OMV production of E. coli cells. PMID:26092467

  13. CALM Regulates Clathrin-Coated Vesicle Size and Maturation by Directly Sensing and Driving Membrane Curvature

    PubMed Central

    Miller, Sharon E.; Mathiasen, Signe; Bright, Nicholas A.; Pierre, Fabienne; Kelly, Bernard T.; Kladt, Nikolay; Schauss, Astrid; Merrifield, Christien J.; Stamou, Dimitrios; Höning, Stefan; Owen, David J.

    2015-01-01

    Summary The size of endocytic clathrin-coated vesicles (CCVs) is remarkably uniform, suggesting that it is optimized to achieve the appropriate levels of cargo and lipid internalization. The three most abundant proteins in mammalian endocytic CCVs are clathrin and the two cargo-selecting, clathrin adaptors, CALM and AP2. Here we demonstrate that depletion of CALM causes a substantial increase in the ratio of “open” clathrin-coated pits (CCPs) to “necked”/“closed” CCVs and a doubling of CCP/CCV diameter, whereas AP2 depletion has opposite effects. Depletion of either adaptor, however, significantly inhibits endocytosis of transferrin and epidermal growth factor. The phenotypic effects of CALM depletion can be rescued by re-expression of wild-type CALM, but not with CALM that lacks a functional N-terminal, membrane-inserting, curvature-sensing/driving amphipathic helix, the existence and properties of which are demonstrated. CALM is thus a major factor in controlling CCV size and maturation and hence in determining the rates of endocytic cargo uptake. PMID:25898166

  14. Protecting enzymatic function through directed packaging into bacterial outer membrane vesicles

    PubMed Central

    Alves, Nathan J.; Turner, Kendrick B.; Medintz, Igor L.; Walper, Scott A.

    2016-01-01

    Bacteria possess innate machinery to transport extracellular cargo between cells as well as package virulence factors to infect host cells by secreting outer membrane vesicles (OMVs) that contain small molecules, proteins, and genetic material. These robust proteoliposomes have evolved naturally to be resistant to degradation and provide a supportive environment to extend the activity of encapsulated cargo. In this study, we sought to exploit bacterial OMV formation to package and maintain the activity of an enzyme, phosphotriesterase (PTE), under challenging storage conditions encountered for real world applications. Here we show that OMV packaged PTE maintains activity over free PTE when subjected to elevated temperatures (>100-fold more activity after 14 days at 37 °C), iterative freeze-thaw cycles (3.4-fold post four-cycles), and lyophilization (43-fold). We also demonstrate how lyophilized OMV packaged PTE can be utilized as a cell free reagent for long term environmental remediation of pesticide/chemical warfare contaminated areas. PMID:27117743

  15. Mechanism of mercurial inhibition of sodium-coupled alanine uptake in liver plasma membrane vesicles from Raja erinacea

    SciTech Connect

    Sellinger, M.; Ballatori, N.; Boyer, J.L. )

    1991-02-01

    In mammalian hepatocytes the L-alanine carrier contains a sulfhydryl group that is essential for its activity and is inhibited by mercurials. In hepatocytes of the evolutionarily primitive little skate (Raja erinacea), HgCl2 inhibits Na(+)-dependent alanine uptake and Na+/K(+)-ATPase and increase K+ permeability. To distinguish between direct effects of HgCl2 on the Na(+)-alanine cotransporter and indirect effects on membrane permeability, (3H)alanine transport was studied in plasma membrane vesicles. (3H)Alanine uptake was stimulated by an out-to-in Na+ but not K+ gradient and was saturable confirming the presence of Na(+)-alanine cotransport in liver plasma membranes from this species. Preincubation of the vesicles with HgCl2 for 5 min reduced initial rates of Na(+)-dependent but not Na(+)-independent alanine uptake in a dose-dependent manner (10-200 microM). In the presence of equal concentrations of NaCl or KCl inside and outside of the vesicles, 75 microM HgCl2 directly inhibited sodium-dependent alanine-(3H)alanine exchange, demonstrating that HgCl2 directly affected the alanine cotransporter. Inhibition of Na(+)-dependent alanine uptake by 30 microM HgCl2 was reversed by dithiothreitol (1 mM). HgCl2 (10-30 microM) also increased initial rates of 22Na uptake (at 5 sec), whereas 22Na uptake rates were decreased at HgCl2 concentrations greater than 50 microM. Higher concentrations of HgCl2 (100-200 microM) produced nonspecific effects on vesicle integrity. These studies indicate that HgCl2 inhibits Na(+)-dependent alanine uptake in skate hepatocytes by three different concentration-dependent mechanisms: direct interaction with the transporters, dissipation of the driving force (Na+ gradient), and loss of membrane integrity.

  16. Golgi-derived vesicles from developing epithelial cells bind actin filaments and possess myosin-I as a cytoplasmically oriented peripheral membrane protein

    PubMed Central

    1993-01-01

    In the intestinal brush border, the mechanoenzyme myosin-I links the microvillus core actin filaments with the plasma membrane. Previous immunolocalization shows that myosin-I is associated with vesicles in mature enterocytes (Drenckhahn, D., and R. Dermietzel. 1988. J. Cell Biol. 107:1037-1048) suggesting a potential role mediating vesicle motility. We now report that myosin-I is associated with Golgi-derived vesicles isolated from cells that are rapidly assembling brush borders in intestinal crypts. Crypt cells were isolated in hyperosmotic buffer, homogenized, and fractionated using differential- and equilibrium- density centrifugation. Fractions containing 50-100-nm vesicles, a similar size to those observed in situ, were identified by EM and were shown to contain myosin-I as demonstrated by immunoblotting and immunolabel negative staining. Galactosyltransferase, a marker enzyme for trans-Golgi membranes was present in these fractions, as was alkaline phosphatase, which is an apical membrane targeted enzyme. Galactosyltransferase was also present in vesicles immuno-purified with antibodies to myosin-I. Villin, a marker for potential contamination from fragmented microvilli, was absent. Myosin-I was found to reside on the vesicle "outer" or cytoplasmic surface for it was accessible to exogenous proteases and intact vesicles could be immunolabeled with myosin-I antibodies in solution. The bound myosin-I could be extracted from the vesicles using NaCl, KI and Na2CO3, suggesting that it is a vesicle peripheral membrane protein. These vesicles were shown to bundle actin filaments in an ATP-dependent manner. These results are consistent with a role for myosin-I as an apically targeted motor for vesicle translocation in epithelial cells. PMID:8416982

  17. Klebsiella pneumoniae O antigen loss alters the outer membrane protein composition and the selective packaging of proteins into secreted outer membrane vesicles.

    PubMed

    Cahill, Bethaney K; Seeley, Kent W; Gutel, Dedra; Ellis, Terri N

    2015-11-01

    Klebsiella pneumoniae is a nosocomial pathogen which naturally secretes lipopolysaccharide (LPS) and cell envelope associated proteins into the environment through the production of outer membrane vesicles (OMVs). The loss of the LPS O antigen has been demonstrated in other bacterial species to significantly alter the composition of OMVs. Therefore, this study aimed to comprehensively analyze the impact of O antigen loss on the sub-proteomes of both the outer membrane and secreted OMVs from K. pneumoniae. As determined by LC-MS/MS, OMVs were highly enriched with outer membrane proteins involved in cell wall, membrane, and envelope biogenesis as compared to the source cellular outer membrane. Deletion of wbbO, the enzyme responsible for O antigen attachment to LPS, decreased but did not eliminate this enrichment effect. Additionally, loss of O antigen resulted in OMVs with increased numbers of proteins involved in post-translational modification, protein turnover, and chaperones as compared to secreted vesicles from the wild type. This alteration of OMV composition may be a compensatory mechanism to deal with envelope stress. This comprehensive analysis confirms the highly distinct protein composition of OMVs as compared to their source membrane, and provides evidence for a selective sorting mechanism that involves LPS polysaccharides. These data support the hypothesis that modifications to LPS alters both the mechanics of protein sorting and the contents of secreted OMVs and significantly impacts the protein composition of the outer membrane.

  18. A comparison of the haemagglutinating and enzymic activities of Bacteroides fragilis whole cells and outer membrane vesicles.

    PubMed

    Patrick, S; McKenna, J P; O'Hagan, S; Dermott, E

    1996-04-01

    The haemagglutinating and enzymic activities of the obligately anaerobic pathogenic bacterium Bacteroides fragilis were examined. Outer membrane vesicles are released from the surface of B. fragilis. They can be detected by electron microscopy in ultrathin sections and bacterial suspensions after negative staining. Electron microscopy and immunogold labelling with a MAb specific for surface polysaccharide of B. fragilis confirmed that the vesicles carried outer membrane associated epitopes. The haemagglutinating activity of whole cells from populations of B. fragilis strains NCTC9343, BE3 and LS66 enriched by Percoll density gradient centrifugation for a large capsule (LC), electron dense layer (EDL); non-capsulate by light microscopy) and outer membrane vesicles (OMV) which had been purified by centrifugation from EDL-enriched populations were compared using human and horse erythrocytes. The enzymic activity of OMV, LC- and EDL-enriched populations, as detected by the API ZYM kit, was compared for strains NCTC 9343 and BE3. Purified OMV from the strains examined exhibited both haemagglutinating and enzymatic activity. Haemagglutination by the EDL-enriched population was sensitive to treatment with sodium periodate. The LC-enriched population haemagglutinated only after ultrasonic removal of the capsule. This indicates that the LC masks a haemagglutinin. The results suggest a potential role for OMV in the virulence of B. fragilis. PMID:8737489

  19. A Splice-Isoform of Vesicle-associated Membrane Protein-1 (VAMP-1) Contains a Mitochondrial Targeting Signal

    PubMed Central

    Isenmann, Sandra; Khew-Goodall, Yeesim; Gamble, Jennifer; Vadas, Mathew; Wattenberg, Binks W.

    1998-01-01

    Screening of a library derived from primary human endothelial cells revealed a novel human isoform of vesicle-associated membrane protein-1 (VAMP-1), a protein involved in the targeting and/or fusion of transport vesicles to their target membrane. We have termed this novel isoform VAMP-1B and designated the previously described isoform VAMP-1A. VAMP-1B appears to be an alternatively spliced form of VAMP-1. A similar rat splice variant of VAMP-1 (also termed VAMP-1B) has recently been reported. Five different cultured cell lines, from different lineages, all contained VAMP-1B but little or no detectable VAMP-1A mRNA, as assessed by PCR. In contrast, brain mRNA contained VAMP-1A but no VAMP-1B. The VAMP-1B sequence encodes a protein identical to VAMP-1A except for the carboxy-terminal five amino acids. VAMP-1 is anchored in the vesicle membrane by a carboxy-terminal hydrophobic sequence. In VAMP-1A the hydrophobic anchor is followed by a single threonine, which is the carboxy-terminal amino acid. In VAMP-1B the predicted hydrophobic membrane anchor is shortened by four amino acids, and the hydrophobic sequence is immediately followed by three charged amino acids, arginine-arginine-aspartic acid. Transfection of human endothelial cells with epitope-tagged VAMP-1B demonstrated that VAMP-1B was targeted to mitochondria whereas VAMP-1A was localized to the plasma membrane and endosome-like structures. Analysis of C-terminal mutations of VAMP-1B demonstrated that mitochondrial targeting depends both on the addition of positive charge at the C terminus and a shortened hydrophobic membrane anchor. These data suggest that mitochondria may be integrated, at least at a mechanistic level, to the vesicular trafficking pathways that govern protein movement between other organelles of the cell. PMID:9658161

  20. Membrane Vesicles Released by Intestinal Epithelial Cells Infected with Rotavirus Inhibit T-Cell Function

    PubMed Central

    Barreto, Alfonso; Rodríguez, Luz-Stella; Rojas, Olga Lucía; Wolf, Marie; Greenberg, Harry B.; Franco, Manuel A.

    2010-01-01

    Abstract Rotavirus (RV) predominantly replicates in intestinal epithelial cells (IEC), and “danger signals” released by these cells may modulate viral immunity. We have recently shown that human model IEC (Caco-2 cells) infected with rhesus-RV release a non-inflammatory group of immunomodulators that includes heat shock proteins (HSPs) and TGF-β1. Here we show that both proteins are released in part in association with membrane vesicles (MV) obtained from filtrated Caco-2 supernatants concentrated by ultracentrifugation. These MV express markers of exosomes (CD63 and others), but not of the endoplasmic reticulum (ER) or nuclei. Larger quantities of proteins associated with MV were released by RV-infected cells than by non-infected cells. VP6 co-immunoprecipitated with CD63 present in these MV, and VP6 co-localized with CD63 in RV-infected cells, suggesting that this viral protein is associated with the MV, and that this association occurs intracellularly. CD63 present in MV preparations from stool samples from 36 children with gastroenteritis due or not due to RV were analyzed. VP6 co-immunoprecipitated with CD63 in 3/8 stool samples from RV-infected children, suggesting that these MV are released by RV-infected cells in vivo. Moreover, fractions that contained MV from RV-infected cells induced death and inhibited proliferation of CD4+ T cells to a greater extent than fractions from non-infected cells. These effects were in part due to TGF-β, because they were reversed by treatment of the T cells with the TGF-β-receptor inhibitor ALK5i. MV from RV-infected and non-infected cells were heterogeneous, with morphologies and typical flotation densities described for exosomes (between 1.10 and 1.18 g/mL), and denser vesicles (>1.24 g/mL). Both types of MV from RV-infected cells were more efficient at inhibiting T-cell function than were those from non-infected cells. We propose that RV infection of IEC releases MV that modulate viral immunity. PMID:21142445

  1. Localization of blood-group A and I antigenic sites on inside-out and rightside-out human erythrocyte membrane vesicles.

    PubMed Central

    Schenkel-Brunner, H; Cartron, J P; Doinel, C

    1979-01-01

    Investigations of the fixation of 125I-labelled anti-A and anti-I antibodies onto rightside-out and inside-out membrane vesicles prepared from human A1 and OI erythrocytes, respectively, showed that both antibodies were bound to the rightside-out vesicles, giving clear evidence that blood group A and I antigenic sites are exclusively localized on the external surface of the membrane. PMID:84784

  2. Light-driven amino acid uptake in Streptococcus cremoris or Clostridium acetobutylicum membrane vesicles fused with liposomes containing bacterial reaction centers

    SciTech Connect

    Crielaard, W.; Driessen, A.J.; Molenaar, D.; Hellingwerf, K.J.; Konings, W.N.

    1988-04-01

    Reaction centers of the phototrophic bacterium Rhodopseudomonas palustris were introduced as proton motive force-generating systems in membrane vesicles of two anaerobic bacteria. Liposomes containing reaction center-light-harvesting complex I pigment protein complexes were fused with membrane vesicles of Streptococcus cremoris or Clostridium acetobutylicum by freeze-thawing and sonication. Illumination of these fused membranes resulted in the generation of a proton motive force of approximately -110 mV. The magnitude of the proton motive force in these membranes could be varied by changing the light intensity. As a result of this proton motive force, amino acid transport into the fused membranes could be observed. The initial rate of leucine transport by membrane vesicles of S. cremoris increased exponentially with the proton motive force. An H+/leucine stoichiometry of 0.8 was determined from the steady-state level of leucine accumulation and the proton motive force, and this stoichiometry was found to be independent of the magnitude of the proton motive force. These results indicate that the introduction of bacterial reaction centers in membrane vesicles by the fusion procedure yields very attractive model systems for the study of proton motive force-consuming processes in membrane vesicles of (strict) anaerobic bacteria.

  3. Effects of chronic ethanol ingestion on the vasoactive intestinal peptide receptor-effector system from rat seminal vesicle membranes.

    PubMed

    Juarranz, M G; Marinero, M J; Bodega, G; Prieto, J C; Guijarro, L G

    1999-02-01

    We studied the modifications of the vasoactive intestinal peptide (VIP) receptor/effector system from the rat seminal vesicle after chronic ethanol ingestion. Ethanol treatment resulted in a decreased height of the secretory epithelium of seminal vesicle as well as in a weight loss of this gland. These morphological changes were accompanied by an increase of immunoreactive vasoactive intestinal peptide (VIP) levels and a decrease of the stimulatory effect of VIP adenylate cyclase activity in the seminal vesicle. The loss of sensitivity of the enzyme to VIP was conceivably related to a decrease in the affinity of VIP receptors rather than to a decrease in their number. The changes in the affinity of the VIP receptors were accompanied with a lower sensitivity of VIP binding to GTP, which suggest an uncoupling between the receptor and the transductor molecules. However, chronic exposure to ethanol did not modify either the levels of G-protein subunits (alpha(s) and alpha(i1/2)) or the GTPase activity from seminal vesicle membranes. Moreover, ethanol feeding did not affect adenylate cyclase activity stimulated by forskolin or by Gpp(NH)p. Thus, ethanol-induced changes in the sensitivity of adenylate cyclase to VIP appear to be attributed to an alteration in the VIP-receptor/G-protein interphase rather than in the G-protein/adenylate cyclase connection. PMID:10069562

  4. Isolation of plasma membrane vesicles from mouse placenta at term and measurement of system A and system beta amino acid transporter activity.

    PubMed

    Kusinski, L C; Jones, C J P; Baker, P N; Sibley, C P; Glazier, J D

    2010-01-01

    Placental amino acid transport is essential for optimal fetal growth and development, with a reduced fetal provision of amino acids being implicated as a potential cause of fetal growth restriction (FGR). Understanding placental insufficiency related FGR has been aided by the development of mouse models that have features of the human disease. However, to take maximal advantage of these, methods are required to study placental function in the mouse. Here, we report a method to isolate plasma membrane vesicles from mouse placenta near-term and have used these to investigate two amino acid transporters, systems A and beta, the activities of which are reduced in human placental microvillous plasma membrane (MVM) vesicles from FGR pregnancies. Plasma membrane vesicles were isolated at embryonic day 18 by a protocol involving homogenisation, MgCl(2) precipitation and centrifugation. Vesicles were enriched 11.3+/-0.5-fold in alkaline phosphatase activity as compared to initial homogenate, with minimal intracellular organelle contamination as judged by marker analyses. Cytochemistry revealed alkaline phosphatase was localised between trophoblast layers I and II, with intense reaction product deposited on the maternal-facing plasma membrane of layer II, suggesting that vesicles were derived from this trophoblast membrane. System A and system beta activity in mouse placental vesicles, measured as Na(+)-dependent uptake of (14)C-methylaminoisobutyric acid (MeAIB) and (3)H-taurine respectively confirmed localisation of these transporters to the maternal-facing plasma membrane of layer II. Comparison to human placental MVM showed that system A activity was comparable at initial rate between species whilst system beta activity was significantly lower in mouse. This mirrored the lower expression of TAUT observed in mouse placental vesicles. We conclude that syncytiotrophoblast layer II-derived plasma membrane vesicles can be isolated and used to examine transporter function.

  5. A vacuolar-type proton pump in a vesicle fraction enriched with potassium transporting plasma membranes from tobacco hornworm midgut

    SciTech Connect

    Wieczorek, H.; Weerth, S.; Schindlbeck, M.; Klein, U.

    1989-07-05

    Mg-ATP dependent electrogenic proton transport, monitored with fluorescent acridine orange, 9-aminoacridine, and oxonol V, was investigated in a fraction enriched with potassium transporting goblet cell apical membranes of Manduca sexta larval midgut. Proton transport and the ATPase activity from the goblet cell apical membrane exhibited similar substrate specificity and inhibitor sensitivity. ATP and GTP were far better substrates than UTP, CTP, ADP, and AMP. Azide and vanadate did not inhibit proton transport, whereas 100 microM N,N'-dicyclohexylcarbodiimide and 30 microM N-ethylmaleimide were inhibitors. The pH gradient generated by ATP and limiting its hydrolysis was 2-3 pH units. Unlike the ATPase activity, proton transport was not stimulated by KCl. In the presence of 20 mM KCl, a proton gradient could not be developed or was dissipated. Monovalent cations counteracted the proton gradient in an order of efficacy like that for stimulation of the membrane-bound ATPase activity: K+ = Rb+ much greater than Li+ greater than Na+ greater than choline (chloride salts). Like proton transport, the generation of an ATP dependent and azide- and vanadate-insensitive membrane potential (vesicle interior positive) was prevented largely by 100 microM N,N'-dicyclohexylcarbodiimide and 30 microM N-ethylmaleimide. Unlike proton transport, the membrane potential was not affected by 20 mM KCl. In the presence of 150 mM choline chloride, the generation of a membrane potential was suppressed, whereas the pH gradient increased 40%, indicating an anion conductance in the vesicle membrane. Altogether, the results led to the following new hypothesis of electrogenic potassium transport in the lepidopteran midgut. A vacuolar-type electrogenic ATPase pumps protons across the apical membrane of the goblet cell, thus energizing electroneutral proton/potassium antiport. The result is a net active and electrogenic potassium flux.

  6. Legionella pneumophila-Derived Outer Membrane Vesicles Promote Bacterial Replication in Macrophages

    PubMed Central

    Jung, Anna Lena; Stoiber, Cornelia; Herkt, Christina E.; Schulz, Christine; Bertrams, Wilhelm; Schmeck, Bernd

    2016-01-01

    The formation and release of outer membrane vesicles (OMVs) is a phenomenon of Gram-negative bacteria. This includes Legionella pneumophila (L. pneumophila), a causative agent of severe pneumonia. Upon its transmission into the lung, L. pneumophila primarily infects and replicates within macrophages. Here, we analyzed the influence of L. pneumophila OMVs on macrophages. To this end, differentiated THP-1 cells were incubated with increasing doses of Legionella OMVs, leading to a TLR2-dependent classical activation of macrophages with the release of pro-inflammatory cytokines. Inhibition of TLR2 and NF-κB signaling reduced the induction of pro-inflammatory cytokines. Furthermore, treatment of THP-1 cells with OMVs prior to infection reduced replication of L. pneumophila in THP-1 cells. Blocking of TLR2 activation or heat denaturation of OMVs restored bacterial replication in the first 24 h of infection. With prolonged infection-time, OMV pre-treated macrophages became more permissive for bacterial replication than untreated cells and showed increased numbers of Legionella-containing vacuoles and reduced pro-inflammatory cytokine induction. Additionally, miRNA-146a was found to be transcriptionally induced by OMVs and to facilitate bacterial replication. Accordingly, IRAK-1, one of miRNA-146a’s targets, showed prolonged activation-dependent degradation, which rendered THP-1 cells more permissive for Legionella replication. In conclusion, L. pneumophila OMVs are initially potent pro-inflammatory stimulators of macrophages, acting via TLR2, IRAK-1, and NF-κB, while at later time points, OMVs facilitate L. pneumophila replication by miR-146a-dependent IRAK-1 suppression. OMVs might thereby promote spreading of L. pneumophila in the host. PMID:27105429

  7. Meningococcal Outer Membrane Vesicle Composition-Dependent Activation of the Innate Immune Response.

    PubMed

    Zariri, Afshin; Beskers, Joep; van de Waterbeemd, Bas; Hamstra, Hendrik Jan; Bindels, Tim H E; van Riet, Elly; van Putten, Jos P M; van der Ley, Peter

    2016-10-01

    Meningococcal outer membrane vesicles (OMVs) have been extensively investigated and successfully implemented as vaccines. They contain pathogen-associated molecular patterns, including lipopolysaccharide (LPS), capable of triggering innate immunity. However, Neisseria meningitidis contains an extremely potent hexa-acylated LPS, leading to adverse effects when its OMVs are applied as vaccines. To create safe OMV vaccines, detergent treatment is generally used to reduce the LPS content. While effective, this method also leads to loss of protective antigens such as lipoproteins. Alternatively, genetic modification of LPS can reduce its toxicity. In the present study, we have compared the effects of standard OMV isolation methods using detergent or EDTA with those of genetic modifications of LPS to yield a penta-acylated lipid A (lpxL1 and pagL) on the in vitro induction of innate immune responses. The use of detergent decreased both Toll-like receptor 4 (TLR4) and TLR2 activation by OMVs, while the LPS modifications reduced only TLR4 activation. Mutational removal of PorB or lipoprotein factor H binding protein (fHbp), two proteins known to trigger TLR2 signaling, had no effect, indicating that multiple TLR2 ligands are removed by detergent treatment. Detergent-treated OMVs and lpxL1 OMVs showed similar reductions of cytokine profiles in the human monocytic cell line MM6 and human dendritic cells (DCs). OMVs with the alternative penta-acylated LPS structure obtained after PagL-mediated deacylation showed reduced induction of proinflammatory cytokines interleukin-6 (IL-6) and IL-1β but not of IP-10, a typical TRIF-dependent chemokine. Taken together, these data show that lipid A modification can be used to obtain OMVs with reduced activation of innate immunity, similar to what is found after detergent treatment. PMID:27481244

  8. A combined vaccine approach against Vibrio cholerae and ETEC based on outer membrane vesicles

    PubMed Central

    Leitner, Deborah R.; Lichtenegger, Sabine; Temel, Philipp; Zingl, Franz G.; Ratzberger, Desiree; Roier, Sandro; Schild-Prüfert, Kristina; Feichter, Sandra; Reidl, Joachim; Schild, Stefan

    2015-01-01

    Enteric infections induced by pathogens like Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) remain a massive burden in developing countries with increasing morbidity and mortality rates. Previously, we showed that the immunization with genetically detoxified outer membrane vesicles (OMVs) derived from V. cholerae elicits a protective immune response based on the generation of O antigen antibodies, which effectively block the motility by binding to the sheathed flagellum. In this study, we investigated the potential of lipopolysaccharide (LPS)-modified and toxin negative OMVs isolated from V. cholerae and ETEC as a combined OMV vaccine candidate. Our results indicate that the immunization with V. cholerae or ETEC OMVs induced a species-specific immune response, whereas the combination of both OMV species resulted in a high-titer, protective immune response against both pathogens. Interestingly, the immunization with V. cholerae OMVs alone resulted in a so far uncharacterized and cholera toxin B-subunit (CTB) independent protection mechanism against an ETEC colonization. Furthermore, we investigated the potential use of V. cholerae OMVs as delivery vehicles for the heterologously expression of the ETEC surface antigens, CFA/I, and FliC. Although we induced a detectable immune response against both heterologously expressed antigens, none of these approaches resulted in an improved protection compared to a simple combination of V. cholerae and ETEC OMVs. Finally, we expanded the current protection model from V. cholerae to ETEC by demonstrating that the inhibition of motility via anti-FliC antibodies represents a relevant protection mechanism of an OMV-based ETEC vaccine candidate in vivo. PMID:26322032

  9. A combined vaccine approach against Vibrio cholerae and ETEC based on outer membrane vesicles.

    PubMed

    Leitner, Deborah R; Lichtenegger, Sabine; Temel, Philipp; Zingl, Franz G; Ratzberger, Desiree; Roier, Sandro; Schild-Prüfert, Kristina; Feichter, Sandra; Reidl, Joachim; Schild, Stefan

    2015-01-01

    Enteric infections induced by pathogens like Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) remain a massive burden in developing countries with increasing morbidity and mortality rates. Previously, we showed that the immunization with genetically detoxified outer membrane vesicles (OMVs) derived from V. cholerae elicits a protective immune response based on the generation of O antigen antibodies, which effectively block the motility by binding to the sheathed flagellum. In this study, we investigated the potential of lipopolysaccharide (LPS)-modified and toxin negative OMVs isolated from V. cholerae and ETEC as a combined OMV vaccine candidate. Our results indicate that the immunization with V. cholerae or ETEC OMVs induced a species-specific immune response, whereas the combination of both OMV species resulted in a high-titer, protective immune response against both pathogens. Interestingly, the immunization with V. cholerae OMVs alone resulted in a so far uncharacterized and cholera toxin B-subunit (CTB) independent protection mechanism against an ETEC colonization. Furthermore, we investigated the potential use of V. cholerae OMVs as delivery vehicles for the heterologously expression of the ETEC surface antigens, CFA/I, and FliC. Although we induced a detectable immune response against both heterologously expressed antigens, none of these approaches resulted in an improved protection compared to a simple combination of V. cholerae and ETEC OMVs. Finally, we expanded the current protection model from V. cholerae to ETEC by demonstrating that the inhibition of motility via anti-FliC antibodies represents a relevant protection mechanism of an OMV-based ETEC vaccine candidate in vivo. PMID:26322032

  10. Bacillus thuringiensis delta-endotoxin binding to brush border membrane vesicles of rice stem borers.

    PubMed

    Alcantara, Edwin P; Aguda, Remedios M; Curtiss, April; Dean, Donald H; Cohen, Michael B

    2004-04-01

    The receptor binding step in the molecular mode of action of five delta-endotoxins (Cry1Ab, Cry1Ac, Cry1C, Cry2A, and Cry9C) from Bacillus thuringiensis was examined to find toxins with different receptor sites in the midgut of the striped stem borer (SSB) Chilo suppressalis (Walker) and yellow stem borer (YSB) Scirpophaga incertulas (Walker) (Lepidoptera: Pyralidae). Homologous competition assays were used to estimate binding affinities (K(com)) of (125)I-labelled toxins to brush border membrane vesicles (BBMV). The SSB BBMV affinities in decreasing order was: Cry1Ab = Cry1Ac > Cry9C > Cry2A > Cry1C. In YSB, the order of decreasing affinities was: Cry1Ac > Cry1Ab > Cry9C = Cry2A > Cry1C. The number of binding sites (B(max)) estimated by homologous competition binding among the Cry toxins did not affect toxin binding affinity (K(com)) to both insect midgut BBMVs. Results of the heterologous competition binding assays suggest that Cry1Ab and Cry1Ac compete for the same binding sites in SSB and YSB. Other toxins bind with weak (Cry1C, Cry2A) or no affinity (Cry9C) to Cry1Ab and Cry1Ac binding sites in both species. Cry2A had the lowest toxicity to 10-day-old SSB and Cry1Ab and Cry1Ac were the most toxic. Taken together, the results of this study show that Cry1Ab or Cry1Ac could be combined with either Cry1C, Cry2A, or Cry9C for more durable resistance in transgenic rice. Cry1Ab should not be used together with Cry1Ac because a mutation in one receptor site could diminish binding of both toxins. PMID:15027071

  11. Leucine transport in brush border membrane vesicles from freshwater insect larvae.

    PubMed

    Forcella, Matilde; Berra, Elisa; Giacchini, Roberto; Parenti, Paolo

    2006-11-01

    Leucine transport across brush border membrane vesicles prepared from four insect species common to European freshwater streams has been characterized. The species studied were: Ephemera danica (Ephemeroptera: Ephemeridae), Isoperla grammatica (Plecoptera: Perlodidae), Hydropsyche pellucidula (Trichoptera: Hydropsychidae), and Hybomitra bimaculata (Diptera: Tabanidae). The transport differed among the studied taxa for several features, including pH and sodium dependence, substrate affinity and specificity, and efficiency. In H. pellucidula and E. danica, leucine uptake was higher at pH 7.4 than at more alkaline or acidic pH values, whereas in I. grammatica and H. bimaculata, the uptake was rather constant when pH varied from 5.0 to 7.4, then strongly decreased at pH 8.8. All but E. danica displayed a transient intravescicular leucine accumulation in the presence of sodium, suggesting the existence of a cation-leucine symport mechanism. The sodium dependence ranged according to the following order: H. pellucidula > I. grammatica > H. bimaculata > E. danica. Moreover, in H. pellucidula and I. grammatica, the sodium-dependence was stronger at pH 8.8 than at pH 7.4. In E. danica, leucine uptake was sodium-independent at all pH values. The highest value of V(max) (45.3 pmol.s(-1).mg proteins(-1)) was in E. danica, which, however, displayed the lowest affinity (K(m) 137 muM) when compared to the kinetic parameters of other taxa. The V(max) and K(m) values were: 40 and 52.5, 32.1 and 12.5, and 4.5 and 230 for H. bimaculata, H. pellucidula, and I. grammatica, respectively. The obtained results are discussed within our current knowledge of amino acid transport systems in insects.

  12. Comparative proteomic analysis of outer membrane vesicles from Shigella flexneri under different culture conditions

    SciTech Connect

    Chen, Yong; Liu, Liguo; Fu, Hua; Wei, Candong Jin, Qi

    2014-10-31

    Highlights: • We utilized mTRAQ-based quantification to study protein changes in Congo red-induced OMVs. • A total of 148 proteins were identified in S. flexneri-derived OMVs. • Twenty-eight and five proteins are significantly up- and down-regulated in the CR-induced OMV, respectively. • The result implied that a special sorting mechanism of particular proteins into OMVs may exist. • Key node proteins in the protein interaction network might be important for pathogenicity. - Abstract: The production of outer membrane vesicles (OMVs) is a common and regulated process of gram-negative bacteria. Nonetheless, the processes of Shigella flexneri OMV production still remain unclear. S. flexneri is the causative agent of endemic shigellosis in developing countries. The Congo red binding of strains is associated with increased infectivity of S. flexneri. Therefore, understanding the modulation pattern of OMV protein expression induced by Congo red will help to elucidate the bacterial pathogenesis. In the present study, we investigated the proteomic composition of OMVs and the change in OMV protein expression induced by Congo red using mTRAQ-based quantitative comparative proteomics. mTRAQ labelling increased the confidence in protein identification, and 148 total proteins were identified in S. flexneri-derived OMVs. These include a variety of important virulence factors, including Ipa proteins, TolC family, murein hydrolases, and members of the serine protease autotransporters of Enterobacteriaceae (SPATEs) family. Among the identified proteins, 28 and five proteins are significantly up- and down-regulated in the Congo red-induced OMV, respectively. Additionally, by comprehensive comparison with previous studies focused on DH5a-derived OMV, we identified some key node proteins in the protein–protein interaction network that may be involved in OMV biogenesis and are common to all gram-negative bacteria.

  13. Xylella fastidiosa outer membrane vesicles modulate plant colonization by blocking attachment to surfaces.

    PubMed

    Ionescu, Michael; Zaini, Paulo A; Baccari, Clelia; Tran, Sophia; da Silva, Aline M; Lindow, Steven E

    2014-09-16

    Outer membrane vesicles (OMVs) of Gram-negative bacteria have been studied intensively in recent years, primarily in their role in delivering virulence factors and antigens during pathogenesis. However, the near ubiquity of their production suggests that they may play other roles, such as responding to envelope stress or trafficking various cargoes to prevent dilution or degradation by other bacterial species. Here we show that OMVs produced by Xylella fastidiosa, a xylem-colonizing plant pathogenic bacterium, block its interaction with various surfaces such as the walls of xylem vessels in host plants. The release of OMVs was suppressed by the diffusible signal factor-dependent quorum-sensing system, and a X. fastidiosa ΔrpfF mutant in which quorum signaling was disrupted was both much more virulent to plants and less adhesive to glass and plant surfaces than the WT strain. The higher virulence of the ΔrpfF mutant was associated with fivefold higher numbers of OMVs recovered from xylem sap of infected plants. The frequency of attachment of X. fastidiosa to xylem vessels was 20-fold lower in the presence of OMVs than in their absence. OMV production thus is a strategy used by X. fastidiosa cells to adjust attachment to surfaces in its transition from adhesive cells capable of insect transmission to an "exploratory" lifestyle for systemic spread within the plant host which would be hindered by attachment. OMV production may contribute to the movement of other bacteria in porous environments by similarly reducing their contact with environmental constituents. PMID:25197068

  14. Outer Membrane Vesicles Mediate Transport of Biologically Active Vibrio cholerae Cytolysin (VCC) from V. cholerae Strains

    PubMed Central

    Elluri, Sridhar; Enow, Constance; Vdovikova, Svitlana; Rompikuntal, Pramod K.; Dongre, Mitesh; Carlsson, Sven; Pal, Amit; Uhlin, Bernt Eric; Wai, Sun Nyunt

    2014-01-01

    Background Outer membrane vesicles (OMVs) released from Gram-negative bacteria can serve as vehicles for the translocation of virulence factors. Vibrio cholerae produce OMVs but their putative role in translocation of effectors involved in pathogenesis has not been well elucidated. The V. cholerae cytolysin (VCC), is a pore-forming toxin that lyses target eukaryotic cells by forming transmembrane oligomeric β-barrel channels. It is considered a potent toxin that contributes to V. cholerae pathogenesis. The mechanisms involved in the secretion and delivery of the VCC have not been extensively studied. Methodology/Principal Findings OMVs from V. cholerae strains were isolated and purified using a differential centrifugation procedure and Optiprep centrifugation. The ultrastructure and the contents of OMVs were examined under the electron microscope and by immunoblot analyses respectively. We demonstrated that VCC from V. cholerae strain V:5/04 was secreted in association with OMVs and the release of VCC via OMVs is a common feature among V. cholerae strains. The biological activity of OMV-associated VCC was investigated using contact hemolytic assay and epithelial cell cytotoxicity test. It showed toxic activity on both red blood cells and epithelial cells. Our results indicate that the OMVs architecture might play a role in stability of VCC and thereby can enhance its biological activities in comparison with the free secreted VCC. Furthermore, we tested the role of OMV-associated VCC in host cell autophagy signalling using confocal microscopy and immunoblot analysis. We observed that OMV-associated VCC triggered an autophagy response in the target cell and our findings demonstrated for the first time that autophagy may operate as a cellular defence mechanism against an OMV-associated bacterial virulence factor. Conclusion/Significance Biological assays of OMVs from the V. cholerae strain V:5/04 demonstrated that OMV-associated VCC is indeed biologically active and

  15. Energization of amino acid transport in energy-depleted Ehrlich cells and plasma membrane vesicles.

    PubMed

    Ohsawa, M; Kilberg, M S; Kimmel, G; Christensen, H N

    1980-06-20

    We redirect attention to contributions to the energization, of the active transport of amino acids in the Ehrlich cell, beyond the known energization, by down-gradient comigration of Na+, beyond possible direct energization by coupling to ATP breakdown, and beyond known energization by exchange with prior accumulations of amino acids. We re-emphasize the uphill operation of System L, and by prior depletion of cellular amino acids show that this system must receive energy beyond that made available by their coupled exodus. After this depletion the Na+-indepdendent accumulation of the norbornane amino acid, 2-aminobicycloheptane-2-carboxylic acid becomes strongly subject to stimulation by incubation with glucose. Energy transfer between Systems A and L through the mutual substrate action of ordinary amino acids was minimized although not entirely avoided by the use of amino acid analogs specific to each system. When 2,4-dinitrophenol was included in the depleting treatment, and pyruvate, phenazine methosulfate, or glucose used for restoration, recovery of uptake of the norbornane amino acid was independent of external Na+ or K+ levels. Restoration or the uptake of 2-(methylamino)isobutyric acid was, however, decreased by omission of external K+. Contrary to an earlier finding, restoration of uptake of each of these amino acids was associated with distinct and usually correlated rises in cellular ATP levels. ATP addition failed to stimulate exodus of the norbornane amino acid from plasma membrane vesicles, although either NADH or phenazine methosulfate did stimulate exodus. ATP production and use is thus associated with transport energization although evidence for a direct role failed to appear.

  16. Uptake and binding of /sup 125/I-calmodulin by isolated rat renal brush border membrane vesicles

    SciTech Connect

    Meezan, E.; Elgavish, A.; Wallace, R.W.

    1986-05-01

    The authors have investigated the interaction of /sup 125/I-calmodulin with isolated rat renal brush border membrane vesicles (BBV) using an experimental protocol which allows us to distinguish between ligand binding to the outside of the vesicles vs. uptake and possible binding to the vesicle interior. By examining the association of /sup 125/I-calmodulin with BBV as a function of medium osmolarity (300-1100 mosm) to alter intravesicular space, virtually all ligand interaction with BBV was found to represent uptake of intact /sup 125/I-calmodulin into the intravesicular space. Uptake appeared specific by the following criteria: (1) it was largely calcium dependent (2) it was inhibited in a dose dependent fashion by calmodulin and the homologous protein troponin C, but not by unrelated proteins (lysozyme, cytochrome C, insulin) (3) it was inhibited by known calmodulin antagonists (calmidazolium, mellitin, trifluoperazine). Calmodulin uptake may be followed by binding of /sup 125/I-calmodulin to intravesicular BBV proteins; calmodulin-binding proteins in BBV with molecular weights of 143K, 118K, 50K, 47.5K, 46.5K and 35K were identified by Western blotting techniques. The specific association of /sup 125/I-calmodulin with isolated BBV is of interest in regard to the possible role of this calcium regulatory protein in the protein reabsorptive and ion transport functions of this renal tubular membrane fraction.

  17. Tubular Membrane Formation of Binary Giant Unilamellar Vesicles Composed of Cylinder and Inverse-Cone-Shaped Lipids

    PubMed Central

    Sakuma, Yuka; Taniguchi, Takashi; Kawakatsu, Toshihiro; Imai, Masayuki

    2013-01-01

    We have succeeded in controlling tubular membrane formations in binary giant unilamellar vesicles (GUVs) using a simple temperature changing between the homogeneous one-phase region and the two-phase coexistence region. The binary GUV is composed of inverse-cone (bulky hydrocarbon chains and a small headgroup) and cylinder-shaped lipids. When the temperature was set in the two-phase coexistence region, the binary GUV had a spherical shape with solidlike domains. By increasing the temperature to the homogeneous one-phase region, the excess area created by the chain melting of the lipid produced tubes inside the GUV. The tubes had a radius on the micrometer scale and were stable in the one-phase region. When we again decreased the temperature to the two-phase coexisting region, the tubes regressed and the GUVs recovered their phase-separated spherical shape. We infer that the tubular formation was based on the mechanical balance of the vesicle membrane (spontaneous tension) coupled with the asymmetric distribution of the inverse-cone-shaped lipids between the inner and outer leaflets of the vesicle (lipid sorting). PMID:24209852

  18. Local membrane length conservation in two-dimensional vesicle simulation using a multicomponent lattice Boltzmann equation method

    NASA Astrophysics Data System (ADS)

    Halliday, I.; Lishchuk, S. V.; Spencer, T. J.; Pontrelli, G.; Evans, P. C.

    2016-08-01

    We present a method for applying a class of velocity-dependent forces within a multicomponent lattice Boltzmann equation simulation that is designed to recover continuum regime incompressible hydrodynamics. This method is applied to the problem, in two dimensions, of constraining to uniformity the tangential velocity of a vesicle membrane implemented within a recent multicomponent lattice Boltzmann simulation method, which avoids the use of Lagrangian boundary tracers. The constraint of uniform tangential velocity is carried by an additional contribution to an immersed boundary force, which we derive here from physical arguments. The result of this enhanced immersed boundary force is to apply a physically appropriate boundary condition at the interface between separated lattice fluids, defined as that region over which the phase-field varies most rapidly. Data from this enhanced vesicle boundary method are in agreement with other data obtained using related methods [e.g., T. Krüger, S. Frijters, F. Günther, B. Kaoui, and J. Harting, Eur. Phys. J. 222, 177 (2013), 10.1140/epjst/e2013-01834-y] and underscore the importance of a correct vesicle membrane condition.

  19. Local membrane length conservation in two-dimensional vesicle simulation using a multicomponent lattice Boltzmann equation method.

    PubMed

    Halliday, I; Lishchuk, S V; Spencer, T J; Pontrelli, G; Evans, P C

    2016-08-01

    We present a method for applying a class of velocity-dependent forces within a multicomponent lattice Boltzmann equation simulation that is designed to recover continuum regime incompressible hydrodynamics. This method is applied to the problem, in two dimensions, of constraining to uniformity the tangential velocity of a vesicle membrane implemented within a recent multicomponent lattice Boltzmann simulation method, which avoids the use of Lagrangian boundary tracers. The constraint of uniform tangential velocity is carried by an additional contribution to an immersed boundary force, which we derive here from physical arguments. The result of this enhanced immersed boundary force is to apply a physically appropriate boundary condition at the interface between separated lattice fluids, defined as that region over which the phase-field varies most rapidly. Data from this enhanced vesicle boundary method are in agreement with other data obtained using related methods [e.g., T. Krüger, S. Frijters, F. Günther, B. Kaoui, and J. Harting, Eur. Phys. J. 222, 177 (2013)10.1140/epjst/e2013-01834-y] and underscore the importance of a correct vesicle membrane condition. PMID:27627411

  20. L-alanine uptake in membrane vesicles from Mytilus edulis gills

    SciTech Connect

    Pajor, A.M.; Wright, S.H.

    1986-03-05

    Previous studies have shown that gills from M. edulis can accumulate L-alanine from seawater by a saturable process specific for ..cap alpha..-neutral amino acids. This uptake occurs against chemical gradients in excess of 10/sup 6/ to 1. To further characterize this uptake, membrane vesicles were prepared from M. edulis gill tissue by differential centrifugation. Enrichments of putative enzyme markers (relative to that in combined initial fractions) were as follows: ..gamma..-Glutamyltranspeptidase, 25-30x; Alkaline Phosphatase, 5-6x; K/sup +/-dependent para-Nitrophenyl Phosphatase, 3-5x; Succinate Dehydrogenase 0.1-0.2x. These results suggest that the preparation is enriched in plasma membranes, although histochemical studies will be needed to verify this. The time course of /sup 14/C-L-alanine uptake in the presence of inwardly-directed Na/sup +/ gradient showed a transient overshoot (3-5 fold) at 10 minutes which decreased to equilibrium after six hours. The size of the overshoot and early uptake rates depended on the size of the inwardly-directed Na/sup +/ gradient. No overshoot was seen in the presence of inwardly-directed gradients of LiCl or choline-Cl, or with equilibrium concentrations NaCl or mannitol. A reduced overshoot was seen with a gradient of NaSCN. A small overshoot was seen with an inwardly-directed gradient of KCl. Transport of L-alanine included saturable and diffusive components. Uptake of 6 ..mu..M L-alanine was inhibited more than 80% by 100 ..mu..M ..cap alpha..-zwitterionic amino acids (alanine, leucine, glycine); by 30 to 75% by proline, aspartate and lysine; and less than 20% by a ..beta..-amino acid, taurine. The results of these experiments agree with those from intact gill studies and support the hypothesis that L-alanine is transported into gill epithelial cells by a secondary active transport process involving Na/sup +/.

  1. Effect of toluene on Pseudomonas stutzeri ST-9 morphology - plasmolysis, cell size, and formation of outer membrane vesicles.

    PubMed

    Michael, Esti; Nitzan, Yeshayahu; Langzam, Yakov; Luboshits, Galia; Cahan, Rivka

    2016-08-01

    Isolated toluene-degrading Pseudomonas stutzeri ST-9 bacteria were grown in a minimal medium containing toluene (100 mg·L(-1)) (MMT) or glucose (MMG) as the sole carbon source, with specific growth rates of 0.019 h(-1) and 0.042 h(-1), respectively. Scanning (SEM) as well as transmission (TEM) electron microscope analyses showed that the bacterial cells grown to mid-log phase in the presence of toluene possess a plasmolysis space. TEM analysis revealed that bacterial cells that were grown in MMT were surrounded by an additional "material" with small vesicles in between. Membrane integrity was analyzed by leakage of 260 nm absorbing material and demonstrated only 7% and 8% leakage from cultures grown in MMT compared with MMG. X-ray microanalysis showed a 4.3-fold increase in Mg and a 3-fold increase in P in cells grown in MMT compared with cells grown in MMG. Fluorescence-activated cell sorting (FACS) analysis indicated that the permeability of the membrane to propidium iodide was 12.6% and 19.6% when the cultures were grown in MMG and MMT, respectively. The bacterial cell length increased by 8.5% ± 0.1% and 17% ± 2%, as measured using SEM images and FACS analysis, respectively. The results obtained in this research show that the presence of toluene led to morphology changes, such as plasmolysis, cell size, and formation of outer membrane vesicles. However, it does not cause significant damage to membrane integrity.

  2. Cadmium inhibition of L-alanine transport into renal brush border membrane vesicles isolated from the winter flounder (Pseudopleuronectes americanus)

    SciTech Connect

    Bevan, C.; Kinne-Saffran, E.; Foulkes, E.C.; Kinne, R.K. )

    1989-12-01

    Using isolated brush border membrane vesicles from the kidney of the winter flounder (Pseudopleuronectes americanus), we have studied the effect of cadmium on L-alanine transport. Pretreatment of vesicles with 0.1 mM Cd{sup 2+} resulted in inhibition of L-alanine uptake in the presence of a NaCl (but not KCl) gradient. Inhibition was due to a specific interaction with the sodium-alanine cotransport system and not a change in the driving forces for alanine transport, since Cd{sup 2+} did not affect sodium-dependent D-glucose uptake. The effect of Cd{sup 2+} on Na{sup +}-alanine cotransport showed mixed-type inhibition which is only partially reversible by EDTA. Cd{sup 2+} uptake itself was shown to be time and temperature dependent, resulting in binding to both sides of the membrane. No direct correlation was possible between inhibition of L-alanine transport and the amount of Cd{sup 2+} taken up by the membranes. Nevertheless, the striking time dependence of the effect of Cd{sup 2+} on sodium-dependent L-alanine uptake and the inability of EDTA to reverse the inhibitory action of Cd{sup 2+} suggest that Cd{sup 2+} inhibits Na+-alanine cotransport at the cytoplasmic side of the membrane.

  3. Proteins of the exocytotic core complex mediate platelet alpha-granule secretion. Roles of vesicle-associated membrane protein, SNAP-23, and syntaxin 4.

    PubMed

    Flaumenhaft, R; Croce, K; Chen, E; Furie, B; Furie, B C

    1999-01-22

    To understand the molecular basis of granule release from platelets, we examined the role of vesicle-associated membrane protein, SNAP-23, and syntaxin 4 in alpha-granule secretion. A vesicle-associated membrane protein, SNAP-23, and syntaxin 4 were detected in platelet lysate. These proteins form a SDS-resistant complex that disassembles upon platelet activation. To determine whether these proteins are involved in alpha-granule secretion, we developed a streptolysin O-permeabilized platelet model of alpha-granule secretion. Streptolysin O-permeabilized platelets released alpha-granules, as measured by surface expression of P-selectin, in response to Ca2+ up to 120 min after permeabilization. Incubation of streptolysin O-permeabilized platelets with an antibody directed against vesicle-associated membrane protein completely inhibited Ca2+-induced alpha-granule release. Tetanus toxin cleaved platelet vesicle-associated membrane protein and inhibited Ca2+-induced alpha-granule secretion from streptolysin O-permeabilized platelets. An antibody to syntaxin 4 also inhibited Ca2+-induced alpha-granule release by approximately 75% in this system. These results show that vesicle-associated membrane protein, SNAP-23, and syntaxin 4 form a heterotrimeric complex in platelets that disassembles with activation and demonstrate that alpha-granule release is dependent on vesicle SNAP receptor-target SNAP receptor (vSNARE-tSNARE) interactions. PMID:9891020

  4. Active auxin uptake by zucchini membrane vesicles: quantitation using ESR volume and delta pH determinations

    SciTech Connect

    Lomax, T.L.; Mehlhorn, R.J.; Briggs, W.R.

    1985-10-01

    Closed and pH-tight membrane vesicles prepared from hypocotyls of 5-day-old dark-grown seedlings of Cucurbita pepo accumulate the plant growth hormone indole-3-acetic acid along an imposed proton gradient (pH low outside, high inside). The use of electron paramagnetic spin probes permitted quantitation both of apparent vesicle volume and magnitude of the pH gradient. Under the experimental conditions used, hormone accumulation was at minimum 20-fold, a value 4 times larger than what one would predict if accumulation reflected only diffusional equilibrium at the measured pH gradient. It is concluded that hormone uptake is an active process, with each protonated molecule of hormone accompanied by an additional proton. Experiments with ionophores confirm that it is the pH gradient itself which drives the uptake.

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  7. Chloride/bicarbonate exchanger SLC26A7 is localized in endosomes in medullary collecting duct cells and is targeted to the basolateral membrane in hypertonicity and potassium depletion.

    PubMed

    Xu, Jie; Worrell, Roger T; Li, Hong C; Barone, Sharon L; Petrovic, Snezana; Amlal, Hassane; Soleimani, Manoocher

    2006-04-01

    SLC26A7 is a Cl(-)/HCO(3)(-) exchanger that is expressed on the basolateral membrane and in the cytoplasm of two distinct acid-secreting epithelial cells: The A-intercalated cells in the kidney outer medullary collecting duct and the gastric parietal cells. The intracellular localization of SLC26A7 suggests the possibility of trafficking between cell membrane and intracellular compartments. For testing this hypothesis, full-length human SLC26A7 cDNA was fused with green fluorescence protein and transiently expressed in MDCK epithelial cells. In monolayer cells in isotonic medium, SLC26A7 showed punctate distribution throughout the cytoplasm. However, in medium that was made hypertonic for 16 h, SLC26A7 was detected predominantly in the plasma membrane. The presence of mitogen-activated protein kinase inhibitors blocked the trafficking of SLC26A7 to the plasma membrane. Double-labeling studies demonstrated the localization of SLC26A7 to the transferrin receptor-positive endosomes. A chimera that was composed of the amino terminal fragment of SLC26A7 and the carboxyl terminal fragment of SLC26A1, and a C-terminal-truncated SLC26A7 were retained in the cytoplasm in hypertonicity. In separate studies, SLC26A7 showed predominant localization in plasma membrane in potassium-depleted isotonic medium (0.5 or 2 mEq/L KCl) versus cytoplasmic distribution in normal potassium isotonic medium (4 mEq/L). It is concluded that SLC26A7 is present in endosomes, and its targeting to the basolateral membrane is increased in hypertonicity and potassium depletion. The trafficking to the cell surface suggests novel functional upregulation of SLC26A7 in states that are associated with hypokalemia or increased medullary tonicity. Additional studies are needed to ascertain the role of SLC26A7 in enhanced bicarbonate absorption in outer medullary collecting duct in hypokalemia and in acid-base regulation in conditions that are associated with increased medullary tonicity. PMID:16524946

  8. Modifier role of internal H+ in activating the Na+-H+ exchanger in renal microvillus membrane vesicles.

    PubMed

    Aronson, P S; Nee, J; Suhm, M A

    1982-09-01

    The intracellular pH in animal cells in generally maintained at a higher level than would be expected if H+ were passively distributed across the plasma membrane. In a wide variety of cells including sea urchin eggs, skeletal muscle, renal and intestinal epithelial cells, and neuroblastoma cells, plasma membrane Na+-H+ exchangers mediate the uphill extrusion of H+ coupled to, and thus energized by, the downhill entry of Na+. Plasma membrane vesicles isolated from the luminal (microvillus, brush border) surface of renal proximal tubular cells possess a Na+-H+ exchanger that seems to be representative of the Na+-H+ exchangers found in other tissues. For example, the renal microvillus membrane Na+-H+ exchanger, like other Na+-H+ exchangers, mediates electroneutral cation exchange, is sensitive to inhibition by the diuretic drug amiloride, and has affinity for Li+ in addition to Na+ and H+ (refs 5, 9). Here we have examined the effect of internal H+ on the activity of the Na+-H+ exchanger in renal microvillus membrane vesicles. Our results suggest that internal H+, independent of its role as a substrate for exchange with external independent of its role as a substrate for exchange with external independent of its role as a substrate for exchange with external Na+, has an important modifier role as an allosteric activator of the Na+-H+ exchanger. Allosteric behaviour with respect to internal H+ is a property that would enhance the ability of plasma membrane Na+-H+ exchangers to extrude intracellular acid loads and thereby contribute to the regulation of intracellular pH.

  9. Lysates of turkey-grown Pasteurella multocida: effects of solubilizing agents on the immunologic properties of membrane vesicles.

    PubMed

    Brogden, K A; Rimler, R B

    1983-03-01

    Membrane vesicles from lysed suspensions of turkey-grown Pasteurella multocida were treated with various solubilizing agents to release protein that may contain cross-protection factor. Potassium thiocyanate, NaOH-glycine, lithium diiodosalicylate, guanidine hydrochloride, n-butanol, dimethyl sulfoxide, Triton X-100, and sodium lauryl sarcosinate were each tested as solubilizing agents. Vaccines made from combining solubilized membrane vesicles with complete lysate supernatant fluid produced various degrees of protection against challenge exposure with a heterologous serotype of P multocida in turkeys. Only vaccines prepared from membranes that were solubilized with potassium thiocyanate and sodium lauryl sarcosinate protected as well as complete lysate from turkey-grown P multocida. The amount of protein in each vaccine did not relate to protection. Distinct chemical differences were observed between lysates prepared from turkey-grown P multocida and lysates prepared from 41 C broth-grown P multocida. The external morphology of P multocida, after treatment with lysozyme and EDTA, was similar whether grown in broth or in turkeys.

  10. Damage in spherical cellular membrane generated by the shock waves: Coarse-grained molecular dynamics simulation of lipid vesicle

    NASA Astrophysics Data System (ADS)

    Sliozberg, Yelena; Chantawansri, Tanya

    2014-11-01

    Traumatic Brain Injury is a major health issue that is hard to diagnose since it often occurs without signs of external injuries. While it is well known that exposure of biological cells to shock waves causes damage to the cell membrane, it is currently unknown by which mechanisms damage is caused, and how it depends on physical parameters such as shock wave velocity, shock pulse duration, or shock pulse shape. In this computational study, we use a coarse-grained model of the lipid vesicle as a generic model of a cell membrane to elucidate the general principles of the cellular damage induced by the shock wave direct passage through the cranium. Results indicate that the extent of the liposome compression does not strongly depend on the pressure pulse and that liposome extension is very sensitive to the change in the negative pressure phase. The structural integrity of the vesicle is altered as pores form in the lipid membrane at overall pressure impulses generated by supersonic shock waves, which are greater than 5 Pa.s at single or repetitive exposure. Consequently, these permeability changes may lead to changes in the influx of sodium, potassium, and calcium ions.

  11. Role of tetanus neurotoxin insensitive vesicle-associated membrane protein (TI-VAMP) in vesicular transport mediating neurite outgrowth.

    PubMed

    Martinez-Arca, S; Alberts, P; Zahraoui, A; Louvard, D; Galli, T

    2000-05-15

    How vesicular transport participates in neurite outgrowth is still poorly understood. Neurite outgrowth is not sensitive to tetanus neurotoxin thus does not involve synaptobrevin-mediated vesicular transport to the plasma membrane of neurons. Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is a vesicle-SNARE (soluble N-ethylmaleimide-sensitive fusion protein [NSF] attachment protein [SNAP] receptor), involved in transport to the apical plasma membrane in epithelial cells, a tetanus neurotoxin-resistant pathway. Here we show that TI-VAMP is essential for vesicular transport-mediating neurite outgrowth in staurosporine-differentiated PC12 cells. The NH(2)-terminal domain, which precedes the SNARE motif of TI-VAMP, inhibits the association of TI-VAMP with synaptosome-associated protein of 25 kD (SNAP25). Expression of this domain inhibits neurite outgrowth as potently as Botulinum neurotoxin E, which cleaves SNAP25. In contrast, expression of the NH(2)-terminal deletion mutant of TI-VAMP increases SNARE complex formation and strongly stimulates neurite outgrowth. These results provide the first functional evidence for the role of TI-VAMP in neurite outgrowth and point to its NH(2)-terminal domain as a key regulator in this process.

  12. Role of Tetanus Neurotoxin Insensitive Vesicle-Associated Membrane Protein (Ti-Vamp) in Vesicular Transport Mediating Neurite Outgrowth

    PubMed Central

    Martinez-Arca, Sonia; Alberts, Philipp; Zahraoui, Ahmed; Louvard, Daniel; Galli, Thierry

    2000-01-01

    How vesicular transport participates in neurite outgrowth is still poorly understood. Neurite outgrowth is not sensitive to tetanus neurotoxin thus does not involve synaptobrevin-mediated vesicular transport to the plasma membrane of neurons. Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is a vesicle-SNARE (soluble N-ethylmaleimide-sensitive fusion protein [NSF] attachment protein [SNAP] receptor), involved in transport to the apical plasma membrane in epithelial cells, a tetanus neurotoxin-resistant pathway. Here we show that TI-VAMP is essential for vesicular transport-mediating neurite outgrowth in staurosporine-differentiated PC12 cells. The NH2-terminal domain, which precedes the SNARE motif of TI-VAMP, inhibits the association of TI-VAMP with synaptosome-associated protein of 25 kD (SNAP25). Expression of this domain inhibits neurite outgrowth as potently as Botulinum neurotoxin E, which cleaves SNAP25. In contrast, expression of the NH2-terminal deletion mutant of TI-VAMP increases SNARE complex formation and strongly stimulates neurite outgrowth. These results provide the first functional evidence for the role of TI-VAMP in neurite outgrowth and point to its NH2-terminal domain as a key regulator in this process. PMID:10811829

  13. Rab6 Is Required for Multiple Apical Transport Pathways but Not the Basolateral Transport Pathway in Drosophila Photoreceptors.

    PubMed

    Iwanami, Nozomi; Nakamura, Yuri; Satoh, Takunori; Liu, Ziguang; Satoh, Akiko K

    2016-02-01

    Polarized membrane trafficking is essential for the construction and maintenance of multiple plasma membrane domains of cells. Highly polarized Drosophila photoreceptors are an excellent model for studying polarized transport. A single cross-section of Drosophila retina contains many photoreceptors with 3 clearly differentiated plasma membrane domains: a rhabdomere, stalk, and basolateral membrane. Genome-wide high-throughput ethyl methanesulfonate screening followed by precise immunohistochemical analysis identified a mutant with a rare phenotype characterized by a loss of 2 apical transport pathways with normal basolateral transport. Rapid gene identification using whole-genome resequencing and single nucleotide polymorphism mapping identified a nonsense mutation of Rab6 responsible for the apical-specific transport deficiency. Detailed analysis of the trafficking of a major rhabdomere protein Rh1 using blue light-induced chromophore supply identified Rab6 as essential for Rh1 to exit the Golgi units. Rab6 is mostly distributed from the trans-Golgi network to a Golgi-associated Rab11-positive compartment that likely recycles endosomes or transport vesicles going to recycling endosomes. Furthermore, the Rab6 effector, Rich, is required for Rab6 recruitment in the trans-Golgi network. Moreover, a Rich null mutation phenocopies the Rab6 null mutant, indicating that Rich functions as a guanine nucleotide exchange factor for Rab6. The results collectively indicate that Rab6 and Rich are essential for the trans-Golgi network-recycling endosome transport of cargoes destined for 2 apical domains. However, basolateral cargos are sorted and exported from the trans-Golgi network in a Rab6-independent manner.

  14. Rab6 Is Required for Multiple Apical Transport Pathways but Not the Basolateral Transport Pathway in Drosophila Photoreceptors

    PubMed Central

    Liu, Ziguang; Satoh, Akiko K.

    2016-01-01

    Polarized membrane trafficking is essential for the construction and maintenance of multiple plasma membrane domains of cells. Highly polarized Drosophila photoreceptors are an excellent model for studying polarized transport. A single cross-section of Drosophila retina contains many photoreceptors with 3 clearly differentiated plasma membrane domains: a rhabdomere, stalk, and basolateral membrane. Genome-wide high-throughput ethyl methanesulfonate screening followed by precise immunohistochemical analysis identified a mutant with a rare phenotype characterized by a loss of 2 apical transport pathways with normal basolateral transport. Rapid gene identification using whole-genome resequencing and single nucleotide polymorphism mapping identified a nonsense mutation of Rab6 responsible for the apical-specific transport deficiency. Detailed analysis of the trafficking of a major rhabdomere protein Rh1 using blue light-induced chromophore supply identified Rab6 as essential for Rh1 to exit the Golgi units. Rab6 is mostly distributed from the trans-Golgi network to a Golgi-associated Rab11-positive compartment that likely recycles endosomes or transport vesicles going to recycling endosomes. Furthermore, the Rab6 effector, Rich, is required for Rab6 recruitment in the trans-Golgi network. Moreover, a Rich null mutation phenocopies the Rab6 null mutant, indicating that Rich functions as a guanine nucleotide exchange factor for Rab6. The results collectively indicate that Rab6 and Rich are essential for the trans-Golgi network–recycling endosome transport of cargoes destined for 2 apical domains. However, basolateral cargos are sorted and exported from the trans-Golgi network in a Rab6-independent manner. PMID:26890939

  15. Rab6 Is Required for Multiple Apical Transport Pathways but Not the Basolateral Transport Pathway in Drosophila Photoreceptors.

    PubMed

    Iwanami, Nozomi; Nakamura, Yuri; Satoh, Takunori; Liu, Ziguang; Satoh, Akiko K

    2016-02-01

    Polarized membrane trafficking is essential for the construction and maintenance of multiple plasma membrane domains of cells. Highly polarized Drosophila photoreceptors are an excellent model for studying polarized transport. A single cross-section of Drosophila retina contains many photoreceptors with 3 clearly differentiated plasma membrane domains: a rhabdomere, stalk, and basolateral membrane. Genome-wide high-throughput ethyl methanesulfonate screening followed by precise immunohistochemical analysis identified a mutant with a rare phenotype characterized by a loss of 2 apical transport pathways with normal basolateral transport. Rapid gene identification using whole-genome resequencing and single nucleotide polymorphism mapping identified a nonsense mutation of Rab6 responsible for the apical-specific transport deficiency. Detailed analysis of the trafficking of a major rhabdomere protein Rh1 using blue light-induced chromophore supply identified Rab6 as essential for Rh1 to exit the Golgi units. Rab6 is mostly distributed from the trans-Golgi network to a Golgi-associated Rab11-positive compartment that likely recycles endosomes or transport vesicles going to recycling endosomes. Furthermore, the Rab6 effector, Rich, is required for Rab6 recruitment in the trans-Golgi network. Moreover, a Rich null mutation phenocopies the Rab6 null mutant, indicating that Rich functions as a guanine nucleotide exchange factor for Rab6. The results collectively indicate that Rab6 and Rich are essential for the trans-Golgi network-recycling endosome transport of cargoes destined for 2 apical domains. However, basolateral cargos are sorted and exported from the trans-Golgi network in a Rab6-independent manner. PMID:26890939

  16. Improving the immunogenicity of a trivalent Neisseria meningitidis native outer membrane vesicle vaccine by genetic modification.

    PubMed

    Zhang, Lan; Wen, Zhiyun; Lin, Jing; Xu, Hui; Herbert, Paul; Wang, Xin-Min; Mehl, John T; Ahl, Patrick L; Dieter, Lance; Russell, Ryann; Kosinski, Mike J; Przysiecki, Craig T

    2016-07-29

    Trivalent native outer membrane vesicles (nOMVs) derived from three genetically modified Neisseria meningitidis serogroup B strains have been previously evaluated immunologically in mice and rabbits. This nOMV vaccine elicited serum bactericidal activity (SBA) against multiple N. meningitidis serogroup B strains as well as strains from serogroups C, Y, W, and X. In this study, we used trivalent nOMVs isolated from the same vaccine strains and evaluated their immunogenicity in an infant Rhesus macaque (IRM) model whose immune responses to the vaccine are likely to be more predictive of the responses in human infants. IRMs were immunized with trivalent nOMV vaccines and sera were evaluated for exogenous human serum complement-dependent SBA (hSBA). Antibody responses to selected hSBA generating antigens contained within the trivalent nOMVs were also measured and we found that antibody titers against factor H binding protein variant 2 (fHbpv2) were very low in the sera from animals immunized with these original nOMV vaccines. To increase the fHbp content in the nOMVs, the vaccine strains were further genetically altered by addition of another fHbp gene copy into the porB locus. Trivalent nOMVs from the three new vaccine strains had higher fHbp antigen levels and generated higher anti-fHbp antibody responses in immunized mice and IRMs. As expected, fHbp insertion into the porB locus resulted in no PorB expression. Interestingly, higher expression of PorA, an hSBA generating antigen, was observed for all three modified vaccine strains. Compared to the trivalent nOMVs from the original strains, higher PorA levels in the improved nOMVs resulted in higher anti-PorA antibody responses in mice and IRMs. In addition, hSBA titers against other strains with PorA as the only hSBA antigen in common with the vaccine strains also increased. PMID:27269057

  17. Assessment of outer membrane vesicles of periodontopathic bacterium Porphyromonas gingivalis as possible mucosal immunogen.

    PubMed

    Nakao, Ryoma; Hasegawa, Hideki; Dongying, Bai; Ohnishi, Makoto; Senpuku, Hidenobu

    2016-08-31

    Periodontitis is the most prevalent infectious disease and related to oral and systemic health, therefore novel prophylaxis to prevent the disease is highly desirable. Here, we assessed the outer membrane vesicles (OMVs) of a keystone periodontal pathogen, Porphyromonas gingivalis, as a candidate mucosal immunogen and adjuvant for a periodontitis vaccine. The structural and functional stability of OMVs, demonstrated by proteinase K resistance and ability to withstand long-term storage, are considered advantageous for carrying the OMV components into the host immune system. Intranasal vaccination of OMVs in mice elicited production of P. gingivalis-specific antibodies in blood and saliva by OMVs in a dose-dependent manner, which was dramatically enhanced by addition of a TLR3 agonist, Poly(I:C). Serum samples from mice immunized with OMVs plus Poly(I:C) adjuvant [OMV+Poly(I:C)] showed significant inhibition of gingipain proteolytic activity of not only the vaccine strain, but also heterologous strains. The viability of P. gingivalis was also decreased by preincubation with OMV+Poly(I:C)-immunized sera, while the killing effect was partially blocked by heat-inactivation of the sera. Saliva samples from mice immunized with OMV+Poly(I:C) enhanced bacterial agglutination of both the vaccine and heterologous strains. In an oral infection mouse model, the numbers of P. gingivalis in the oral cavity were significantly decreased in mice intranasally immunized with OMV+Poly(I:C) as compared to mock (only Poly[I:C])-immunized mice. The high levels of serum IgG (including IgG1 and IgG2a) and salivary S-IgA were elicited in mice intranasally immunized with OMV+Poly(I:C), which were maintained for at least 28 and 18weeks, respectively, after immunization. An experiment examining the accumulation of OMVs after intranasal immunization in proximal organs and an intracerebral injection experiment confirmed the safety of OMVs. Based on our results, we propose that intranasal

  18. Improving the immunogenicity of a trivalent Neisseria meningitidis native outer membrane vesicle vaccine by genetic modification.

    PubMed

    Zhang, Lan; Wen, Zhiyun; Lin, Jing; Xu, Hui; Herbert, Paul; Wang, Xin-Min; Mehl, John T; Ahl, Patrick L; Dieter, Lance; Russell, Ryann; Kosinski, Mike J; Przysiecki, Craig T

    2016-07-29

    Trivalent native outer membrane vesicles (nOMVs) derived from three genetically modified Neisseria meningitidis serogroup B strains have been previously evaluated immunologically in mice and rabbits. This nOMV vaccine elicited serum bactericidal activity (SBA) against multiple N. meningitidis serogroup B strains as well as strains from serogroups C, Y, W, and X. In this study, we used trivalent nOMVs isolated from the same vaccine strains and evaluated their immunogenicity in an infant Rhesus macaque (IRM) model whose immune responses to the vaccine are likely to be more predictive of the responses in human infants. IRMs were immunized with trivalent nOMV vaccines and sera were evaluated for exogenous human serum complement-dependent SBA (hSBA). Antibody responses to selected hSBA generating antigens contained within the trivalent nOMVs were also measured and we found that antibody titers against factor H binding protein variant 2 (fHbpv2) were very low in the sera from animals immunized with these original nOMV vaccines. To increase the fHbp content in the nOMVs, the vaccine strains were further genetically altered by addition of another fHbp gene copy into the porB locus. Trivalent nOMVs from the three new vaccine strains had higher fHbp antigen levels and generated higher anti-fHbp antibody responses in immunized mice and IRMs. As expected, fHbp insertion into the porB locus resulted in no PorB expression. Interestingly, higher expression of PorA, an hSBA generating antigen, was observed for all three modified vaccine strains. Compared to the trivalent nOMVs from the original strains, higher PorA levels in the improved nOMVs resulted in higher anti-PorA antibody responses in mice and IRMs. In addition, hSBA titers against other strains with PorA as the only hSBA antigen in common with the vaccine strains also increased.

  19. High affinity 3H-Phe uptake by brush border membrane vesicles from whole larvae of Aedes aegypti (AaBMVw)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Brush border membrane vesicles from whole Aedes aegypti larvae (AaBBMVw) are confirmed to be valid preparations for membrane transport studies. The Abdul-Rauf and Ellar method was used to isolate AaBBMVw that were frozen, stored for several months, transported to a distant site, thawed and used to s...

  20. Cysteine depletion causes oxidative stress and triggers outer membrane vesicle release by Neisseria meningitidis; implications for vaccine development.

    PubMed

    van de Waterbeemd, Bas; Zomer, Gijsbert; van den Ijssel, Jan; van Keulen, Lonneke; Eppink, Michel H; van der Ley, Peter; van der Pol, Leo A

    2013-01-01

    Outer membrane vesicles (OMV) contain immunogenic proteins and contribute to in vivo survival and virulence of bacterial pathogens. The first OMV vaccines successfully stopped Neisseria meningitidis serogroup B outbreaks but required detergent-extraction for endotoxin removal. Current vaccines use attenuated endotoxin, to preserve immunological properties and allow a detergent-free process. The preferred process is based on spontaneously released OMV (sOMV), which are most similar to in vivo vesicles and easier to purify. The release mechanism however is poorly understood resulting in low yield. This study with N. meningitidis demonstrates that an external stimulus, cysteine depletion, can trigger growth arrest and sOMV release in sufficient quantities for vaccine production (±1500 human doses per liter cultivation). Transcriptome analysis suggests that cysteine depletion impairs iron-sulfur protein assembly and causes oxidative stress. Involvement of oxidative stress is confirmed by showing that addition of reactive oxygen species during cysteine-rich growth also triggers vesiculation. The sOMV in this study are similar to vesicles from natural infection, therefore cysteine-dependent vesiculation is likely to be relevant for the in vivo pathogenesis of N. meningitidis. PMID:23372704

  1. Use of membrane vesicles as a simplified system for studying transport of auxin. Progress report

    SciTech Connect

    Goldsmith, M.H.

    1985-01-01

    The accumulation of indoleacetic acid (IAA) inside microsomal vesicles depends on the presence of a pH gradient and is reversible when the ..delta..pH is collapsed by ionosphores. Accumulation is stimulated by either napthylphthalamic acid or TIBA. The accumulation of IAA by the vesicles can be saturated. At concentrations of 1 ..mu..M or less, IAA, synthetic auxins, or auxin antagonists do not affect the pH gradient, but decrease the accumulation of /sup 3/H-IAA, and therefore compete specifically for uptake. Concentrations of 10 ..mu..M and above, uptake of either the auxins or weak acids is sufficient to overcome the buffering capacity of the solution within the vesicles. The collapse of the pH gradient by such high concentrations affects uptake of either /sup 3/H-IAA or /sup 14/C-BA to similar extents and thus is nonspecific. 3 refs.

  2. Molecular mechanisms involved in secretory vesicle recruitment to the plasma membrane in beta-cells.

    PubMed

    Varadi, Aniko; Ainscow, E K; Allan, V J; Rutter, G A

    2002-04-01

    Glucose stimulates the release of insulin in part by activating the recruitment of secretory vesicles to the cell surface. While this movement is known to be microtubule-dependent, the molecular motors involved are undefined. Active kinesin was found to be essential for vesicle translocation in live beta-cells, since microinjection of cDNA encoding dominant-negative KHC(mut) (motor domain of kinesin heavy chain containing a Thr(93)-->Asn point mutation) blocked vesicular movements. Moreover, expression of KHC(mut) strongly inhibited the sustained, but not acute, stimulation of secretion by glucose. Thus, vesicles released during the first phase of insulin secretion exist largely within a translocation-independent pool. Kinesin-driven anterograde movement of vesicles is then necessary for the sustained (second phase) of insulin release. Kinesin may, therefore, represent a novel target for increases in intracellular ATP concentrations in response to elevated extracellular glucose and may be involved in the ATP-sensitive K+channel-independent stimulation of secretion by the sugar.

  3. Clostridium difficile toxin CDT hijacks microtubule organization and reroutes vesicle traffic to increase pathogen adherence.

    PubMed

    Schwan, Carsten; Kruppke, Anna S; Nölke, Thilo; Schumacher, Lucas; Koch-Nolte, Friedrich; Kudryashev, Mikhail; Stahlberg, Henning; Aktories, Klaus

    2014-02-11

    Clostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis by the actions of Rho-glucosylating toxins A and B. Recently identified hypervirulent strains, which are associated with increased morbidity and mortality, additionally produce the actin-ADP-ribosylating toxin C. difficile transferase (CDT). CDT depolymerizes actin, causes formation of microtubule-based protrusions, and increases pathogen adherence. Here we show that CDT-induced protrusions allow vesicle traffic and contain endoplasmic reticulum tubules, connected to microtubules via the calcium sensor Stim1. The toxin reroutes Rab11-positive vesicles containing fibronectin, which is involved in bacterial adherence, from basolateral to the apical membrane sides in a microtubule- and Stim1-dependent manner. The data yield a model of C. difficile adherence regulated by actin depolymerization, microtubule restructuring, subsequent Stim1-dependent Ca(2+) signaling, vesicle rerouting, and secretion of ECM proteins to increase bacterial adherence.

  4. Multi-serotype outer membrane vesicles of Shigellae confer passive protection to the neonatal mice against shigellosis.

    PubMed

    Mitra, Soma; Chakrabarti, Manoj K; Koley, Hemanta

    2013-06-28

    Recently, we have demonstrated, immunization of adult female mice with outer membrane vesicles (OMVs) of Shigella boydii type 4 protected their offspring passively from shigellosis. In our present study, we have advanced our research by formulating multi-serotype outer membrane vesicles (MOMVs), mixing the OMVs of Shigella dysenteriae 1 Δstx, Shigella flexneri 2a, 3a and 6, S. boydii type 4 and Shigella sonnei to achieve a broad spectrum protection against shigellosis. Adult mice were immunized orally with 50 μg of MOMVs, four times at weekly intervals. Immunological parameters were observed at various time points, before, during and after immunization, in adult mice. Passive protection was examined in their offspring by measuring protective efficacy and studying intestinal colonization, after challenging with various Shigella strains. Immunized dams exhibited a consistent broad spectrum antibody response. 3-4 day-old offspring of immunized dams showed significant long term passive protection against wild type S. flexneri 2a, 3a, and 6, S. boydii type 2 and S. dysenteriae 1. Their stomach extracts, essentially containing mother's milk, have also exhibited significant levels of anti-MOMVs immunoglobulins. In conclusion, MOMVs formulation represents an easy, safe immunization strategy that was found suitable to provide complete passive protection to the neonatal mice against all four serogroups of Shigellae. It could be exploited for the development of a novel non-living vaccine against human shigellosis in near future. PMID:23684822

  5. Characterization and pathogenic role of outer membrane vesicles produced by the fish pathogen Piscirickettsia salmonis under in vitro conditions.

    PubMed

    Oliver, Cristian; Valenzuela, Karla; Hernández, Mauricio; Sandoval, Rodrigo; Haro, Ronie E; Avendaño-Herrera, Ruben; Cárcamo, Juan G; Villar, Maite T; Artigues, Antonio; Garduño, Rafael; Yáñez, Alejandro J

    2016-02-29

    Piscirickettsia salmonis is one of the major fish pathogens affecting Chilean aquaculture. This Gram-negative bacterium is highly infectious and is the etiological agent of Piscirickettsiosis. Little is currently known about how the virulence factors expressed by P. salmonis are delivered to host cells. However, it is known that several Gram-negative microorganisms constitutively release outer membrane vesicles (OMVs), which have been implicated in the delivery of virulence factors to host cells. In this study, OMVs production by P. salmonis was observed during infection in CHSE-214 cells and during normal growth in liquid media. The OMVs were spherical vesicles ranging in size between 25 and 145 nm. SDS-PAGE analysis demonstrated that the protein profile of the OMVs was similar to the outer membrane protein profile of P. salmonis. Importantly, the bacterial chaperonin Hsp60 was found in the OMVs of P. salmonis by Western-blot and LC-MS/MS analyses. Finally, in vitro infection assays showed that purified OMVs generated a cytopathic effect on CHSE-214 cells, suggesting a role in pathogenesis. Therefore, OMVs might be an important vehicle for delivering effector molecules to host cells during P. salmonis infection. PMID:26854350

  6. Immunization with Escherichia coli outer membrane vesicles protects bacteria-induced lethality via Th1 and Th17 cell responses.

    PubMed

    Kim, Oh Youn; Hong, Bok Sil; Park, Kyong-Su; Yoon, Yae Jin; Choi, Seng Jin; Lee, Won Hee; Roh, Tae-Young; Lötvall, Jan; Kim, Yoon-Keun; Gho, Yong Song

    2013-04-15

    Outer membrane vesicles (OMVs), secreted from Gram-negative bacteria, are spherical nanometer-sized proteolipids enriched with outer membrane proteins. OMVs, also known as extracellular vesicles, have gained interests for use as nonliving complex vaccines and have been examined for immune-stimulating effects. However, the detailed mechanism on how OMVs elicit the vaccination effect has not been studied extensively. In this study, we investigated the immunological mechanism governing the protective immune response of OMV vaccines. Immunization with Escherichia coli-derived OMVs prevented bacteria-induced lethality and OMV-induced systemic inflammatory response syndrome. As verified by adoptive transfer and gene-knockout studies, the protective effect of OMV immunization was found to be primarily by the stimulation of T cell immunity rather than B cell immunity, especially by the OMV-Ag-specific production of IFN-γ and IL-17 from T cells. By testing the bacteria-killing ability of macrophages, we also demonstrated that IFN-γ and IL-17 production is the main factor promoting bacterial clearances. Our findings reveal that E. coli-derived OMV immunization effectively protects bacteria-induced lethality and OMV-induced systemic inflammatory response syndrome primarily via Th1 and Th17 cell responses. This study therefore provides a new perspective on the immunological detail regarding OMV vaccination. PMID:23514742

  7. Outer membrane vesicles induce immune responses to virulence proteins and protect against colonization by enterotoxigenic Escherichia coli.

    PubMed

    Roy, Koushik; Hamilton, David J; Munson, George P; Fleckenstein, James M

    2011-11-01

    Enterotoxigenic Escherichia coli (ETEC) strains are a heterogeneous group of pathogens that produce heat-labile (LT) and/or heat-stable (ST) enterotoxins. Collectively, these pathogens are responsible for hundreds of thousands of deaths annually in developing countries, particularly in children under the age of 5 years. The heterogeneity of previously investigated molecular targets and the lack of complete sustained protection afforded by antitoxin immunity have impeded progress to date toward a broadly protective vaccine. Many pathogens, including ETEC, have the capacity to form outer membrane vesicles (OMV), which often contain one or more virulence proteins. Prompted by recent studies that identified several immunogenic virulence proteins in outer membrane vesicles of ETEC, we sought to examine the immunogenicity and protective efficacy of these structures in a murine model of infection. Here we demonstrate that immunization with OMV impairs ETEC colonization of the small intestine and stimulates antibodies that recognize the heat-labile toxin and two additional putative virulence proteins, the EtpA adhesin and CexE. Similar to earlier studies with EtpA, vaccination with LT alone also inhibited intestinal colonization. Together, these findings suggest that OMV could be exploited to deliver protective antigens relevant to development of ETEC vaccines. PMID:21900530

  8. Effect of toluene on Pseudomonas stutzeri ST-9 morphology - plasmolysis, cell size, and formation of outer membrane vesicles.

    PubMed

    Michael, Esti; Nitzan, Yeshayahu; Langzam, Yakov; Luboshits, Galia; Cahan, Rivka

    2016-08-01

    Isolated toluene-degrading Pseudomonas stutzeri ST-9 bacteria were grown in a minimal medium containing toluene (100 mg·L(-1)) (MMT) or glucose (MMG) as the sole carbon source, with specific growth rates of 0.019 h(-1) and 0.042 h(-1), respectively. Scanning (SEM) as well as transmission (TEM) electron microscope analyses showed that the bacterial cells grown to mid-log phase in the presence of toluene possess a plasmolysis space. TEM analysis revealed that bacterial cells that were grown in MMT were surrounded by an additional "material" with small vesicles in between. Membrane integrity was analyzed by leakage of 260 nm absorbing material and demonstrated only 7% and 8% leakage from cultures grown in MMT compared with MMG. X-ray microanalysis showed a 4.3-fold increase in Mg and a 3-fold increase in P in cells grown in MMT compared with cells grown in MMG. Fluorescence-activated cell sorting (FACS) analysis indicated that the permeability of the membrane to propidium iodide was 12.6% and 19.6% when the cultures were grown in MMG and MMT, respectively. The bacterial cell length increased by 8.5% ± 0.1% and 17% ± 2%, as measured using SEM images and FACS analysis, respectively. The results obtained in this research show that the presence of toluene led to morphology changes, such as plasmolysis, cell size, and formation of outer membrane vesicles. However, it does not cause significant damage to membrane integrity. PMID:27256870

  9. Complexin 2 modulates vesicle-associated membrane protein (VAMP) 2-regulated zymogen granule exocytosis in pancreatic acini.

    PubMed

    Falkowski, Michelle A; Thomas, Diana D H; Groblewski, Guy E

    2010-11-12

    Complexins are soluble proteins that regulate the activity of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes necessary for vesicle fusion. Neuronal specific complexin 1 has inhibitory and stimulatory effects on exocytosis by clamping trans-SNARE complexes in a prefusion state and promoting conformational changes to facilitate membrane fusion following cell stimulation. Complexins are unable to bind to monomeric SNARE proteins but bind with high affinity to ternary SNARE complexes and with lower affinity to target SNARE complexes. Far less is understood about complexin function outside the nervous system. Pancreatic acini express the complexin 2 isoform by RT-PCR and immunoblotting. Immunofluorescence microscopy revealed complexin 2 localized along the apical plasma membrane consistent with a role in secretion. Accordingly, complexin 2 was found to interact with vesicle-associated membrane protein (VAMP) 2, syntaxins 3 and 4, but not with VAMP 8 or syntaxin 2. Introduction of recombinant complexin 2 into permeabilized acini inhibited Ca(2+)-stimulated secretion in a concentration-dependent manner with a maximal inhibition of nearly 50%. Mutations of the central α-helical domain reduced complexin 2 SNARE binding and concurrently abolished its inhibitory activity. Surprisingly, mutation of arginine 59 to histidine within the central α-helical domain did not alter SNARE binding and moreover, augmented Ca(2+)-stimulated secretion by 130% of control. Consistent with biochemical studies, complexin 2 colocalized with VAMP 2 along the apical plasma membrane following cholecystokinin-8 stimulation. These data demonstrate a functional role for complexin 2 outside the nervous system and indicate that it participates in the Ca(2+)-sensitive regulatory pathway for zymogen granule exocytosis.

  10. Complexin 2 Modulates Vesicle-associated Membrane Protein (VAMP) 2-regulated Zymogen Granule Exocytosis in Pancreatic Acini*

    PubMed Central

    Falkowski, Michelle A.; Thomas, Diana D. H.; Groblewski, Guy E.

    2010-01-01

    Complexins are soluble proteins that regulate the activity of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes necessary for vesicle fusion. Neuronal specific complexin 1 has inhibitory and stimulatory effects on exocytosis by clamping trans-SNARE complexes in a prefusion state and promoting conformational changes to facilitate membrane fusion following cell stimulation. Complexins are unable to bind to monomeric SNARE proteins but bind with high affinity to ternary SNARE complexes and with lower affinity to target SNARE complexes. Far less is understood about complexin function outside the nervous system. Pancreatic acini express the complexin 2 isoform by RT-PCR and immunoblotting. Immunofluorescence microscopy revealed complexin 2 localized along the apical plasma membrane consistent with a role in secretion. Accordingly, complexin 2 was found to interact with vesicle-associated membrane protein (VAMP) 2, syntaxins 3 and 4, but not with VAMP 8 or syntaxin 2. Introduction of recombinant complexin 2 into permeabilized acini inhibited Ca2+-stimulated secretion in a concentration-dependent manner with a maximal inhibition of nearly 50%. Mutations of the central α-helical domain reduced complexin 2 SNARE binding and concurrently abolished its inhibitory activity. Surprisingly, mutation of arginine 59 to histidine within the central α-helical domain did not alter SNARE binding and moreover, augmented Ca2+-stimulated secretion by 130% of control. Consistent with biochemical studies, complexin 2 colocalized with VAMP 2 along the apical plasma membrane following cholecystokinin-8 stimulation. These data demonstrate a functional role for complexin 2 outside the nervous system and indicate that it participates in the Ca2+-sensitive regulatory pathway for zymogen granule exocytosis. PMID:20829354

  11. Secreted Oral Epithelial Cell Membrane Vesicles Induce Epstein-Barr Virus Reactivation in Latently Infected B Cells

    PubMed Central

    Lin, Zhen; Swan, Kenneth; Zhang, Xin; Cao, Subing; Brett, Zoe; Drury, Stacy; Fewell, Claire; Puetter, Adriane; Wang, Xia; Ferris, MaryBeth; Sullivan, Deborah E.; Li, Li

    2016-01-01

    ABSTRACT In the oral epithelium, peripheral stores of Epstein-Barr virus (EBV) are transmitted from infiltrating B cells to epithelial cells. Once the virus is transmitted to epithelial cells, the highly permissive nature of this cell type for lytic replication allows virus amplification and exchange to other hosts. Since the initial transfer of EBV from B cells to epithelial cells requires transitioning of the B-cell to a state that induces virus reactivation, we hypothesized that there might be epithelium-specific signals that allow the infiltrating B cells to sense the appropriate environment to initiate reactivation and begin this exchange process. We previously found that the epithelium-specific miR-200 family of microRNAs promotes EBV lytic replication. Here we show that there are high levels of miR-200 family members in oral and tonsillar epithelia and in saliva. Analysis of cultured oral epithelial cells (OKF6) showed that they actively secrete membrane vesicles (exosomes) that are enriched with miR-200 family members. Coculturing of EBV-positive B cells with OKF6 cells induced viral reactivation. Further, treatment of EBV-positive B cells with OKF6 cell-derived membrane vesicles promoted reactivation. Using a cell system that does not naturally express miR-200 family members, we found that enforced expression of a miR-200 family member produced membrane vesicles that were able to induce the lytic cascade in EBV-positive B cells. We propose that membrane vesicles secreted by oral and tonsillar epithelial cells may serve as a tissue-specific environmental cue that initiates reactivation in B cells, promoting the transfer of virus from peripheral B-cell stores to the oral epithelium to facilitate virus amplification and exchange to other hosts. IMPORTANCE Epstein-Barr virus (EBV) is an important human pathogen that is causally associated with several lymphomas and carcinomas. The switch from latency to the lytic cycle is critical for successful host infection

  12. K⁺-dependent ³H-D-glucose transport by hepatopancreatic brush border membrane vesicles of a marine shrimp.

    PubMed

    Obi, Ijeoma E; Sterling, Kenneth M; Ahearn, Gregory A

    2013-01-01

    The effects of sodium, potassium, sugar inhibitors, and membrane potential on ³H-D-glucose uptake by hepatopancreatic epithelial brush border membrane vesicles (BBMV) of the Atlantic marine shrimp, Litopenaeus setiferus, were investigated. Brush border membrane vesicles were prepared using a MgCl₂/EGTA precipitation method and uptake experiments were conducted using a high speed filtration technique. ³H-D-Glucose uptake was stimulated by both sodium and potassium and these transport rates were almost doubled in the presence of an inside-negative-induced membrane potential. Kinetics of ³H-D-glucose influx were hyperbolic functions of both external Na⁺ or K⁺, and an induced membrane potential increased influx J(max) and lowered K(m) in both salts. ³H-D-Glucose influx versus [glucose] in both Na⁺ or K⁺ media also displayed Michaelis-Menten properties that were only slightly affected by induced membrane potential. Phloridzin was a poor inhibitor of 0.5 mM ³H-D-glucose influx, requiring at least 5 mM in NaCl and 10 mM in KCl to significantly reduce hexose transport. Several sugars (D-galactose, α-methyl-D-gluco-pyranoside, unlabeled D-glucose, D-fructose, and D-mannose) were used at 75 mM as potential inhibitors of 0.1 mM ³H-D-glucose influx. Only unlabeled D-glucose, D-fructose, and D-mannose significantly (p < 0.05) reduced labeled glucose transport. An additional experiment using increasing concentrations of D-mannose (0, 10, 25, 75, and 100 mM) showed this hexose to be an effective inhibitor of 0.1 mM ³H-D-glucose uptake at concentrations of 75 mM and higher. As a whole these results suggest that ³H-D-glucose transport by hepatopancreatic BBMV occurs by a carrier system that is able to use both Na⁺ and K⁺ as drivers, is enhanced by membrane potential, is relatively refractory to phloridzin, and is only inhibited by itself, D-fructose, and D-mannose. These properties are similar to those exhibited by the mammalian SLC5A9/SGLT4 transporter

  13. Heat shock 70 protein interaction with Turnip mosaic virus RNA-dependent RNA polymerase within virus-induced membrane vesicles

    SciTech Connect

    Dufresne, Philippe J.; Thivierge, Karine; Cotton, Sophie; Beauchemin, Chantal; Ide, Christine; Ubalijoro, Eliane; Laliberte, Jean-Francois Fortin, Marc G.

    2008-04-25

    Tandem affinity purification was used in Arabidopsis thaliana to identify cellular interactors of Turnip mosaic virus (TuMV) RNA-dependent RNA polymerase (RdRp). The heat shock cognate 70-3 (Hsc70-3) and poly(A)-binding (PABP) host proteins were recovered and shown to interact with the RdRp in vitro. As previously shown for PABP, Hsc70-3 was redistributed to nuclear and membranous fractions in infected plants and both RdRp interactors were co-immunoprecipitated from a membrane-enriched extract using RdRp-specific antibodies. Fluorescently tagged RdRp and Hsc70-3 localized to the cytoplasm and the nucleus when expressed alone or in combination in Nicotiana benthamiana. However, they were redistributed to large perinuclear ER-derived vesicles when co-expressed with the membrane binding 6K-VPg-Pro protein of TuMV. The association of Hsc70-3 with the RdRp could possibly take place in membrane-derived replication complexes. Thus, Hsc70-3 and PABP2 are potentially integral components of the replicase complex and could have important roles to play in the regulation of potyviral RdRp functions.

  14. Cholesterol and F-actin are required for clustering of recycling synaptic vesicle proteins in the presynaptic plasma membrane.

    PubMed

    Dason, Jeffrey S; Smith, Alex J; Marin, Leo; Charlton, Milton P

    2014-02-15

    Synaptic vesicles (SVs) and their proteins must be recycled for sustained synaptic transmission. We tested the hypothesis that SV cholesterol is required for proper sorting of SV proteins during recycling in live presynaptic terminals. We used the reversible block of endocytosis in the Drosophila temperature-sensitive dynamin mutant shibire-ts1 to trap exocytosed SV proteins, and then examined the effect of experimental treatments on the distribution of these proteins within the presynaptic plasma membrane by confocal microscopy. SV proteins synaptotagmin, vglut and csp were clustered following SV trapping in control experiments but dispersed in samples treated with the cholesterol chelator methyl-β-cyclodextrin to extract SV cholesterol. There was accumulation of phosphatidylinositol (4,5)-bisphosphate (PIP2) in presynaptic terminals following SV trapping and this was reduced following SV cholesterol extraction. Reduced PIP2 accumulation was associated with disrupted accumulation of actin in presynaptic terminals. Similar to vesicular cholesterol extraction, disruption of actin by latrunculin A after SV proteins had been trapped on the plasma membrane resulted in the dispersal of SV proteins and prevented recovery of synaptic transmission due to impaired endocytosis following relief of the endocytic block. Our results demonstrate that vesicular cholesterol is required for aggregation of exocytosed SV proteins in the presynaptic plasma membrane and are consistent with a mechanism involving regulation of PIP2 accumulation and local actin polymerization by cholesterol. Thus, alteration of membrane or SV lipids may affect the ability of synapses to undergo sustained synaptic transmission by compromising the recycling of SV proteins.

  15. Endothelial Cell-Surface Gp60 Activates Vesicle Formation and Trafficking via Gi-Coupled Src Kinase Signaling Pathway

    PubMed Central

    Minshall, Richard D.; Tiruppathi, Chinnaswamy; Vogel, Stephen M.; Niles, Walter D.; Gilchrist, Annette; Hamm, Heidi E.; Malik, Asrar B.

    2000-01-01

    We tested the hypothesis that the albumin-docking protein gp60, which is localized in caveolae, couples to the heterotrimeric GTP binding protein Gi, and thereby activates plasmalemmal vesicle formation and the directed migration of vesicles in endothelial cells (ECs). We used the water-soluble styryl pyridinium dye N-(3-triethylaminopropyl)-4-(p-dibutylaminostyryl) pyridinium dibromide (FM 1-43) to quantify vesicle trafficking by confocal and digital fluorescence microscopy. FM 1-43 and fluorescently labeled anti-gp60 antibody (Ab) were colocalized in endocytic vesicles within 5 min of gp60 activation. Vesicles migrated to the basolateral surface where they released FM 1-43, the fluid phase styryl probe. FM 1-43 fluorescence disappeared from the basolateral EC surface without the loss of anti-gp60 Ab fluorescence. Activation of cell-surface gp60 by cross-linking (using anti-gp60 Ab and secondary Ab) in EC grown on microporous filters increased transendothelial 125I-albumin permeability without altering liquid permeability (hydraulic conductivity), thus, indicating the dissociation of hydraulic conductivity from the albumin permeability pathway. The findings that the sterol-binding agent, filipin, prevented gp60-activated vesicle formation and that caveolin-1 and gp60 were colocalized in vesicles suggest the caveolar origin of endocytic vesicles. Pertussis toxin pretreatment and expression of the dominant negative construct encoding an 11–amino acid Gαi carboxyl-terminal peptide inhibited endothelial 125I-albumin endocytosis and vesicle formation induced by gp60 activation. Expression of dominant negative Src (dn-Src) and overexpression of wild-type caveolin-1 also prevented gp60-activated endocytosis. Caveolin-1 overexpression resulted in the sequestration of Gαi with the caveolin-1, whereas dn-Src inhibited Gαi binding to caveolin-1. Thus, vesicle formation induced by gp60 and migration of vesicles to the basolateral membrane requires the interaction of gp60

  16. Incorporation of VSV-G produces fusogenic plasma membrane vesicles capable of efficient transfer of bioactive macromolecules and mitochondria.

    PubMed

    Lin, Hao-Peng; Zheng, De-Jin; Li, Yun-Pan; Wang, Na; Chen, Shao-Jun; Fu, Yu-Cai; Xu, Wen-Can; Wei, Chi-Ju

    2016-06-01

    The objective of this study was to determine if plasma membrane vesicles (PMVs) could be exploited for efficient transfer of macro-biomolecules and mitochondria. PMVs were derived from mechanical extrusion, and made fusogenic (fPMVs) by incorporating the glycoprotein G of vesicular stomatitis virus (VSV-G). Confocal microscopy examination revealed that cytoplasmic proteins and mitochondria were enclosed in PMVs as evidenced by tracing with cytoplasmically localized and mitochondria-targeted EGFP, respectively. However, no fluorescence signal was detected in PMVs from cells whose nucleus was labeled with an EGFP-tagged histone H2B. Consistently, qRT-PCR measurement showed that mRNA, miRNA and mitochondrial DNA decreased slightly; while nuclear DNA was not measureable. Further, Western blot analysis revealed that cytoplasmic and membrane-bound proteins fell inconspicuously while nuclear proteins were barely detecsle. In addition, fPMVs carrying cytoplamic DsRed proteins transduced about ~40 % of recipient cells. The transfer of protein was further confirmed by using the inducible Cre/loxP system. Mitochondria transfer was found in about 20 % recipient cells after incubation with fPMVs for 5 h. To verify the functionalities of transferred mitochondria, mitochodria-deficient HeLa cells (Rho0) were generated and cultivated with fPMVs. Cell enumeration demonstrated that adding fPMVs into culture media stimulated Rho0 cell growth by 100 % as compared to the control. Lastly, MitoTracker and JC-1 staining showed that transferred mitochondria maintained normal shape and membrane potential in Rho0 cells. This study established a time-saving and efficient approach to delivering proteins and mitochondria by using fPMVs, which would be helpful for finding a cure to mitochondria-associated diseases. Graphical abstract Schematic of the delivery of macro-biomolecules and organelles by fPMVs. VSV-G-expressing cells were extruded through a 3 μm polycarbonate membrane filter to

  17. Xanthomonas campestris pv. vesicatoria Secretes Proteases and Xylanases via the Xps Type II Secretion System and Outer Membrane Vesicles

    PubMed Central

    Solé, Magali; Scheibner, Felix; Hoffmeister, Anne-Katrin; Hartmann, Nadine; Hause, Gerd; Rother, Annekatrin; Jordan, Michael; Lautier, Martine; Arlat, Matthieu

    2015-01-01

    ABSTRACT Many plant-pathogenic bacteria utilize type II secretion (T2S) systems to secrete degradative enzymes into the extracellular milieu. T2S substrates presumably mediate the degradation of plant cell wall components during the host-pathogen interaction and thus promote bacterial virulence. Previously, the Xps-T2S system from Xanthomonas campestris pv. vesicatoria was shown to contribute to extracellular protease activity and the secretion of a virulence-associated xylanase. The identities and functions of additional T2S substrates from X. campestris pv. vesicatoria, however, are still unknown. In the present study, the analysis of 25 candidate proteins from X. campestris pv. vesicatoria led to the identification of two type II secreted predicted xylanases, a putative protease and a lipase which was previously identified as a virulence factor of X. campestris pv. vesicatoria. Studies with mutant strains revealed that the identified xylanases and the protease contribute to virulence and in planta growth of X. campestris pv. vesicatoria. When analyzed in the related pathogen X. campestris pv. campestris, several T2S substrates from X. campestris pv. vesicatoria were secreted independently of the T2S systems, presumably because of differences in the T2S substrate specificities of the two pathogens. Furthermore, in X. campestris pv. vesicatoria T2S mutants, secretion of T2S substrates was not completely absent, suggesting the contribution of additional transport systems to protein secretion. In line with this hypothesis, T2S substrates were detected in outer membrane vesicles, which were frequently observed for X. campestris pv. vesicatoria. We, therefore, propose that extracellular virulence-associated enzymes from X. campestris pv. vesicatoria are targeted to the Xps-T2S system and to outer membrane vesicles. IMPORTANCE The virulence of plant-pathogenic bacteria often depends on TS2 systems, which secrete degradative enzymes into the extracellular milieu. T2S

  18. S-Layered Aneurinibacillus and Bacillus spp. Are Susceptible to the Lytic Action of Pseudomonas aeruginosa Membrane Vesicles

    PubMed Central

    Kadurugamuwa, J. L.; Mayer, A.; Messner, P.; Sára, M.; Sleytr, U. B.; Beveridge, T. J.

    1998-01-01

    When S-layered strains of Bacillus stearothermophilus and Aneurinibacillus thermoaerophilus, possessing S-layers of different lattice type and lattice constant as well as S-(glyco)protein chemistry, and isogenic S-layerless variants were subjected to membrane vesicles (MVs) from P. aeruginosa during plaque assays on plates or CFU measurements on cell suspensions, all bacterial types lysed. Electron microscopy of negative stains, thin sections, and immunogold-labelled MV preparations revealed that the vesicles adhered to all bacterial surfaces, broke open, and digested the underlying peptidoglycan-containing cell wall of all cell types. Reassembled S-layer did not appear to be affected by MVs, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the S-(glyco)proteins remained intact. meso-Diaminopimelic acid, as a peptidoglycan breakdown product, was found in all culture supernatants after MV attack. These results suggest that even though MVs are much larger than the channels which penetrate these proteinaceous arrays, S-layers on gram-positive bacteria do not form a defensive barrier against the lytic action of MVs. The primary mode of attack is by the liberation from the MVs of a peptidoglycan hydrolase, which penetrates through the S-layer to digest the underlying peptidoglycan-containing cell wall. The S-layer is not affected by MV protease. PMID:9573179

  19. KIF3C and KIF3A Form a Novel Neuronal Heteromeric Kinesin That Associates with Membrane Vesicles

    PubMed Central

    Muresan, Virgil; Abramson, Tatiana; Lyass, Asya; Winter, Dirk; Porro, Elena; Hong, Filbert; Chamberlin, Nancy L.; Schnapp, Bruce J.

    1998-01-01

    We have cloned from rat brain the cDNA encoding an 89,828-Da kinesin-related polypeptide KIF3C that is enriched in brain, retina, and lung. Immunocytochemistry of hippocampal neurons in culture shows that KIF3C is localized to cell bodies, dendrites, and, in lesser amounts, to axons. In subcellular fractionation experiments, KIF3C cofractionates with a distinct population of membrane vesicles. Native KIF3C binds to microtubules in a kinesin-like, nucleotide-dependent manner. KIF3C is most similar to mouse KIF3B and KIF3A, two closely related kinesins that are normally present as a heteromer. In sucrose density gradients, KIF3C sediments at two distinct densities, suggesting that it may be part of two different multimolecular complexes. Immunoprecipitation experiments show that KIF3C is in part associated with KIF3A, but not with KIF3B. Unlike KIF3B, a significant portion of KIF3C is not associated with KIF3A. Consistent with these biochemical properties, the distribution of KIF3C in the CNS has both similarities and differences compared with KIF3A and KIF3B. These results suggest that KIF3C is a vesicle-associated motor that functions both independently and in association with KIF3A. PMID:9487132

  20. Voltage-morphology coupling in biomimetic membranes: dynamics of giant vesicles in applied electric fields.

    PubMed

    Vlahovska, Petia M

    2015-10-01

    An electric potential difference across the plasma membrane is common to all living cells and is essential to physiological functions such as the generation of action potentials for cell-to-cell communication. While the basics of cell electrical activity are well established (e.g. the Hodgkin-Huxley model of the action potential), the reciprocal coupling of voltage and membrane deformation has received limited attention. In recent years, studies of biomimetic membranes in externally applied electric fields have revealed a plethora of intriguing dynamics (formation of edges, pearling, and phase separation) that challenge the current understanding of membrane electromechanics. PMID:26314545

  1. Cation-induced aggregation of membrane vesicles isolated from vascular smooth muscle

    SciTech Connect

    Kwan, C.Y.

    1986-12-01

    Cations stimulated aortic muscle membrane aggregation with increasing potency according to their effective charge, e.g., K+ less than Mg2+ less than La3+, and the stimulation is reciprocally related to the apparent affinity for these cations. Divalent metal ion-induced membrane aggregation showed a dependence on the ionic radius, being optimal for Cd2+. Polyvalent cation-induced membrane aggregation was reversibly suppressed by high ionic strength as well as by metal ion chelators, irreversibly inhibited by the cross-linking agent glutaraldehyde, and enhanced by increasing concentrations of ethanol and increased temperature of the medium. When the pH is lowered below 6.0, membrane aggregation progressively increased with a concomitant decrease in cation-induced aggregation. The patterns of aggregation of microsomal membranes and further purified plasma membranes were almost identical whereas the aggregation of the heterogeneous mitochondrial membrane-enriched fraction was distinctly different in the initial rate of aggregation, its pH dependence, and metal ion concentration dependence. Our results indicate that cation-induced membrane aggregation can also be used to isolate a plasma membrane-enriched fraction from vascular smooth muscle.

  2. Quantitative proteomic analysis of the cell envelopes and native membrane vesicles derived from gram-negative bacteria.

    PubMed

    Zielke, Ryszard A; Gafken, Philip R; Sikora, Aleksandra E

    2014-08-01

    Proteins localized to the cell envelope and naturally released membrane vesicles (MVs) play diverse functions in physiology and pathogenesis of Gram-negative bacteria. Study of these proteome fractions is essential for better understanding the basic physiological processes, development of vaccines, and identification of potential drug targets. This unit presents gel-free quantitative proteomic methods for comprehensive proteomic profiling of the cell envelopes and MVs. The procedure starts with the precipitation of the isolated proteome fractions to remove any potential compounds that may interfere with downstream experimental steps. Subsequently, the proteins are reduced, alkylated, and subjected to trypsin digestion. The trypsinized peptides are labeled using isobaric tagging for relative and absolute quantification (iTRAQ), and analyzed samples are pooled and subjected to rigorous prefractionations by strong cation exchange (SCX) and reversed-phase (RP) liquid chromatography (LC). Finally, the tandem mass spectrometry (MS/MS) fragmentation enables peptides identification and quantification.

  3. A novel mechanism of generating extracellular vesicles during apoptosis via a beads-on-a-string membrane structure.

    PubMed

    Atkin-Smith, Georgia K; Tixeira, Rochelle; Paone, Stephanie; Mathivanan, Suresh; Collins, Christine; Liem, Michael; Goodall, Katharine J; Ravichandran, Kodi S; Hulett, Mark D; Poon, Ivan K H

    2015-06-15

    Disassembly of apoptotic cells into smaller fragments (a form of extracellular vesicle called apoptotic bodies) can facilitate removal of apoptotic debris and intercellular communication. However, the mechanism underpinning this process is unclear. While observing monocytes undergoing apoptosis by time-lapse microscopy, we discovered a new type of membrane protrusion that resembles a 'beads-on-a-string' structure. Strikingly, the 'beads' are frequently sheared off the 'string' to form apoptotic bodies. Generation of apoptotic bodies via this mechanism can facilitate a sorting process and results in the exclusion of nuclear contents from apoptotic bodies. Mechanistically, generation of 'beads-on-a-string' protrusion is controlled by the level of actomyosin contraction and apoptopodia formation. Furthermore, in an unbiased drug screen, we identified the ability of sertraline (an antidepressant) to block the formation of 'beads-on-a-string' protrusions and apoptotic bodies. These data uncover a new mechanism of apoptotic body formation in monocytes and also compounds that can modulate this process.

  4. Kinetics of D-glucose and L-leucine transport into sheep and pig intestinal brush border membrane vesicles.

    PubMed

    Wolffram, S; Eggenberger, E; Scharrer, E

    1986-01-01

    The kinetic parameters (Vmax, Kt) of Na+-dependent D-glucose transport into brush border membrane vesicles (BBMV) from sheep and pig jejunum were determined. Due to the fermentation of ingested carbohydrates in the rumen the small intestine of ruminants (sheep) has to absorb much less glucose than the small intestine of monogastric omnivores (pigs) or herbivores. Kinetic analysis of the concentration dependence of D-glucose transport revealed a ten-fold smaller Vmax value combined with a five times lower Kt value in sheep BBMV compared with pig BBMV. The Vmax value for L-leucine transport did not differ between the two species investigated, whereas the Kt value in the sheep exceeded that in the pig. It is concluded from these results that the mechanism for Na+-dependent D-glucose transport in ruminants is adapted to the small amounts of carbohydrates reaching the small intestine.

  5. Adenosine triphosphate-dependent copper transport in isolated rat liver plasma membranes.

    PubMed Central

    Dijkstra, M; In 't Veld, G; van den Berg, G J; Müller, M; Kuipers, F; Vonk, R J

    1995-01-01

    The process of hepatobiliary copper (Cu) secretion is still poorly understood: Cu secretion as a complex with glutathione and transport via a lysosomal pathway have been proposed. The recent cloning and sequencing of the gene for Wilson disease indicates that Cu transport in liver cells may be mediated by a Cu transporting P-type ATPase. Biochemical evidence for ATP-dependent Cu transport in mammalian systems, however, has not been reported so far. We have investigated Cu transport in rat liver plasma membrane vesicles enriched in canalicular or basolateral membranes in the presence and absence of ATP (4 mM) and an ATP-regenerating system. The presence of ATP clearly stimulated uptake of radiolabeled Cu (64Cu, 10 microM) into canalicular plasma membrane vesicles and, to a lesser extent, also into basolateral plasma membrane vesicles. ATP-dependent Cu transport was dose-dependently inhibited by the P-type ATPase inhibitor vanadate, and showed saturation kinetics with an estimated Km of 8.6 microM and a Vmax of 6.9 nmol/min/mg protein. ATP-stimulated Cu uptake was similar in canalicular membrane vesicles of normal Wistar rats and those of mutant GY rats, expressing a congenital defect in the activity of the ATP-dependent canalicular glutathione-conjugate transporter (cMOAT). These studies demonstrate the presence of an ATP-dependent Cu transporting system in isolated plasma membrane fractions of rat liver distinct from cMOAT. PMID:7814642

  6. ΔpH-Dependent Amino Acid Transport into Plasma Membrane Vesicles Isolated from Sugar Beet (Beta vulgaris L.) Leaves

    PubMed Central

    Li, Zhen-Chang; Bush, Daniel R.

    1991-01-01

    Proton-coupled aliphatic, neutral amino acid transport was investigated in plasma membrane vesicles isolated from sugar beet (Beta vulgaris L., cv Great Western) leaves. Two neutral amino acid symport systems were resolved based on inter-amino acid transport competition and on large variations in the specific activity of each porter in different species. Competitive inhibition was observed for transport competition between alanine, methionine, glutamine, and leucine (the alanine group) and between isoleucine, valine, and threonine (the isoleucine group). The apparent Km and Ki values were similar for transport competition among amino acids within the alanine group. In contrast, the kinetics of transport competition between these two groups of amino acids did not fit a simple competitive model. Furthermore, members of the isoleucine group were weak transport antagonists of the alanine group. These results are consistent with two independent neutral amino acid porters. In support of that conclusion, the ratio of the specific activity of alanine transport versus isoleucine transport varied from two- to 13-fold in plasma membrane vesicles isolated from different plant species. This ratio would be expected to remain relatively stable if these amino acids were moving through a single transport system and, indeed, the ratio of alanine to glutamine transport varied less than twofold. Analysis of the predicted structure of the aliphatic, neutral amino acids in solution shows that isoleucine, valine, and threonine contain a branched methyl or hydroxyl group at the β-carbon position that places a dense electron cloud close to the α-amino group. This does not occur for the unbranched amino acids or those that branch further away, e.g. leucine. We hypothesize that this structural feature of isoleucine, valine, and threonine results in unfavorable steric interactions with the alanine transport system that limits their flux through this porter. Hydrophobicity and hydrated

  7. Acellular approaches for regenerative medicine: on the verge of clinical trials with extracellular membrane vesicles?

    PubMed

    Fuster-Matanzo, Almudena; Gessler, Florian; Leonardi, Tommaso; Iraci, Nunzio; Pluchino, Stefano

    2015-01-01

    Extracellular vesicles (EVs) are a heterogeneous population of naturally occurring secreted small vesicles, with distinct biophysical properties and different functions both in physiology and under pathological conditions. In recent years, a number of studies have demonstrated that EVs might hold remarkable potential in regenerative medicine by acting as therapeutically promising nanodrugs. Understanding their final impact on the biology of specific target cells as well as clarification of their overall therapeutic impact remains a matter of intense debate. Here we review the key principles of EVs in physiological and pathological conditions with a specific highlight on the most recently described mechanisms regulating some of the EV-mediated effects. First, we describe the current debates and the upcoming research on EVs as potential novel therapeutics in regenerative medicine, either as unmodified agents or as functionalized small carriers for targeted drug delivery. Moreover, we address a number of safety aspects and regulatory limitations related to the novel nature of EV-mediated therapeutic applications. Despite the emerging possibilities of EV treatments, these issues need to be overcome in order to allow their safe and successful application in future explorative clinical studies. PMID:26631254

  8. Effects of different Brush Border Membrane Vesicle isolation protocols on proteomic analysis of Cry1Ac binding proteins from the midgut of Helicoverpa armigera

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The brush border membrane vesicles (BBMV) isolated from midgut cells of insect have been widely used for studying of the binding receptors and action mode of Cry proteins produced by Bacillus thuringiensis (Bt). There are several methods for isolating insect BBMV used in one-dimension electrophoresi...

  9. Size-selective permeation of water-soluble polymers through the bilayer membrane of cyclodextrin vesicles investigated by PFG-NMR.

    PubMed

    Himmelein, Sabine; Sporenberg, Nora; Schönhoff, Monika; Ravoo, Bart Jan

    2014-04-15

    Cyclodextrin vesicles (CDVs) consist of a bilayer of amphiphilic cyclodextrins (CDs). CDVs exhibit CD cavities at their surface that are able to recognize and bind hydrophobic guest molecules via size-selective inclusion. In this study, the permeability of α- and β-CDVs is investigated by pulsed field gradient-stimulated echo (PFG-STE) nuclear magnetic resonance. Diffusion experiments with water and two types of water-soluble polymers, polyethylene glycol (PEG) and polypropylene glycol (PPG), revealed three main factors that influence the exchange rate and permeability of CDVs. First, the length of the hydrophobic chain of the CD amphiphile plays a crucial role. Reasonably, vesicles consisting of amphiphiles with a longer aliphatic chain are less permeable since both membrane thickness and melting temperature T(m) increase. Second, the exchange rate through the bilayer membrane depends on the molecular weight of the polymer and decreases with increasing weight of the polymer. Most interestingly, a size-selective distinction of permeation due to the embedded CDs in the bilayer membrane was found. The mechanism of permeation is shown to occur through the CD cavity, such that depending on the size of the cavity, permeation of polymers with different cross-sectional diameters takes place. Whereas PPG permeates through the membrane of β-CD vesicles, it does not permeate α-CD vesicles. PMID:24650278

  10. Membrane vesicles from multidrug-resistant human carcinoma cells contain a specific 150,000-170,000 dalton protein detected by photoaffinity labeling

    SciTech Connect

    Cornwell, M.M.; Safa, A.R.; Felsted, R.L.; Gottesman, M.M.; Pastan, I.

    1986-05-01

    The authors have selected multidrug-resistant human KB carcinoma cells in high levels of colchicine (KB-C4) or vinblastine (KB-V1) which are cross-resistant to many other structurally unrelated chemotheraputic agents. To determine the mechanism of reduced drug accumulation, they measured /sup 3/H-vinblastine (/sup 3/H-VBL) association with membrane vesicles made from parental drug sensitive, drug-resistant and revertant cells. Membrane vesicles from highly multidrug resistant cells exhibited increased specific and saturable binding of vinblastine, (Kd = 1 ..mu..M) that was temperature dependent and trypsin sensitive. To identify the molecules which bind vinblastine, membrane vesicles were exposed to two photo-activatable analogs of vinblastine, (N-P-(azido-3,5,-(/sup 3/H)-benzoyl)-N'-..beta..-aminoethylvindisine (/sup 3/H-NAB) and N-P-(azido-3-(/sup 125/I)-solicyl)-N'-..beta..-aminoethylvindesine (/sup 125/I-NASV). The specific labeling of a 150,000-170,000 dalton protein in membrane vesicles from multidrug-resistant KB-C4 and KB-V1 cells was found. /sup 125/I-NASV labeling was inhibited by vinblastine, vincrinstine and verapamil but not by colchicine or dexamethasone. The 150,000-170,000 dalton protein may have an important role in the multidrug resistance phenotype.

  11. CLASP2-dependent microtubule capture at the neuromuscular junction membrane requires LL5β and actin for focal delivery of acetylcholine receptor vesicles

    PubMed Central

    Basu, Sreya; Sladecek, Stefan; Martinez de la Peña y Valenzuela, Isabel; Akaaboune, Mohammed; Smal, Ihor; Martin, Katrin; Galjart, Niels; Brenner, Hans Rudolf

    2015-01-01

    A hallmark of the neuromuscular junction (NMJ) is the high density of acetylcholine receptors (AChRs) in the postsynaptic muscle membrane. The postsynaptic apparatus of the NMJ is organized by agrin secreted from motor neurons. The mechanisms that underlie the focal delivery of AChRs to the adult NMJ are not yet understood in detail. We previously showed that microtubule (MT) capture by the plus end–tracking protein CLASP2 regulates AChR density at agrin-induced AChR clusters in cultured myotubes via PI3 kinase acting through GSK3β. Here we show that knockdown of the CLASP2-interaction partner LL5β by RNAi and forced expression of a CLASP2 fragment blocking the CLASP2/LL5β interaction inhibit microtubule capture. The same treatments impair focal vesicle delivery to the clusters. Consistent with these findings, knockdown of LL5β at the NMJ in vivo reduces the density and insertion of AChRs into the postsynaptic membrane. MT capture and focal vesicle delivery to agrin-induced AChR clusters are also inhibited by microtubule- and actin-depolymerizing drugs, invoking both cytoskeletal systems in MT capture and in the fusion of AChR vesicles with the cluster membrane. Combined our data identify a transport system, organized by agrin through PI3 kinase, GSK3β, CLASP2, and LL5β, for precise delivery of AChR vesicles from the subsynaptic nuclei to the overlying synaptic membrane. PMID:25589673

  12. Interaction of n-octyl β,D-glucopyranoside with giant magnetic-fluid-loaded phosphatidylcholine vesicles: direct visualization of membrane curvature fluctuations as a function of surfactant partitioning between water and lipid bilayer.

    PubMed

    Ménager, Christine; Guemghar, Dihya; Cabuil, Valérie; Lesieur, Sylviane

    2010-10-01

    The present study deals with the morphological modifications of giant dioleoyl phosphatidylcholine vesicles (DOPC GUVs) induced by the nonionic surfactant n-octyl β,D-glucopyranoside at sublytic levels, i.e., in the first steps of the vesicle-to-micelle transition process, when surfactant inserts into the vesicle bilayer without disruption. Experimental conditions were perfected to exactly control the surfactant bilayer composition of the vesicles, in line with former work focused on the mechanical properties of the membrane of magnetic-fluid-loaded DOPC GUVs submitted to a magnetic field. The purpose here was to systematically examine, in the absence of any external mechanical constraint, the dynamics of giant vesicle shape and membrane deformations as a function of surfactant partitioning between the aqueous phase and the lipid membrane, beforehand established by turbidity measurements from small unilamellar vesicles. PMID:20825201

  13. Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle (GUV) Membranes.

    PubMed

    Olety, Balaji; Veatch, Sarah L; Ono, Akira

    2016-01-01

    The structural protein of HIV-1, Pr55(Gag) (or Gag), binds to the plasma membrane in cells during the virus assembly process. Membrane binding of Gag is an essential step for virus particle formation, since a defect in Gag membrane binding results in severe impairment of viral particle production. To gain mechanistic details of Gag-lipid membrane interactions, in vitro methods based on NMR, protein footprinting, surface plasmon resonance, liposome flotation centrifugation, or fluorescence lipid bead binding have been developed thus far. However, each of these in vitro methods has its limitations. To overcome some of these limitations and provide a complementary approach to the previously established methods, we developed an in vitro assay in which interactions between HIV-1 Gag and lipid membranes take place in a "cell-like" environment. In this assay, Gag binding to lipid membranes is visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique. Here we describe the background and the protocols to obtain myristoylated full-length Gag proteins and GUV membranes necessary for the assay and to detect Gag-GUV binding by microscopy. PMID:27500610

  14. Linking microfilaments to intracellular membranes: the actin-binding and vesicle-associated protein comitin exhibits a mannose-specific lectin activity.

    PubMed Central

    Jung, E; Fucini, P; Stewart, M; Noegel, A A; Schleicher, M

    1996-01-01

    Comitin is a 24 kDa actin-binding protein from Dictyostelium discoideum that is located primarily on Golgi and vesicle membranes. We have probed the molecular basis of comitin's interaction with both actin and membranes using a series of truncation mutants obtained by expressing the appropriate cDNA in Escherichia coli. Comitin dimerizes in solution; its principle actin-binding activity is located between residues 90 and 135. The N-terminal 135 'core' residues of comitin contain a 3-fold sequence repeat that is homologous to several monocotyledon lectins and which retains key residues that determine these lectins' three-dimensional structure and mannose binding. These repeats of comitin appear to mediate its interaction with mannose residues in glycoproteins or glycolipids on the cytoplasmic surface of membrane vesicles from D.discoideum, and comitin can be released from membranes with mannose. Our data indicate that comitin binds to vesicle membranes via mannose residues and, by way of its interaction with actin, links these membranes to the cytoskeleton. Images PMID:8635456

  15. Critical particle sizes for the engulfment of nanoparticles by membranes and vesicles with bilayer asymmetry.

    PubMed

    Agudo-Canalejo, Jaime; Lipowsky, Reinhard

    2015-01-01

    The adhesion and engulfment of nanoparticles by biomembranes is essential for many processes such as biomedical imaging, drug delivery, nanotoxicity, and viral infection. Many studies have shown that both surface chemistry, which determines the adhesive strength of the membrane-particle interactions, and particle size represent key parameters for these processes. Here, we show that the asymmetry between the two leaflets of a bilayer membrane provides another key parameter for the engulfment of nanoparticles. The asymmetric membrane prefers to curve in a certain manner as quantitatively described by its spontaneous curvature. We derive two general relationships between particle size, adhesive strength, and spontaneous curvature that determine the instabilities of (i) the nonadhering or free state and (ii) the completely engulfed state of the particle. For model membranes such as lipid or polymer bilayers with a uniform composition, the two relationships lead to two critical particle sizes that determine four distinct engulfment regimes, both for the endocytic and for the exocytic engulfment process. For strong adhesion, the critical particle sizes are on the order of 10 nm, while they are on the order of 1000 nm for weak or ultraweak adhesion. Our theoretical results are therefore accessible to both experimental studies and computer simulations of model membranes. In order to address the more complex process of receptor-mediated endocytosis, we take the adhesion-induced segregation of membrane components into account and consider bound and unbound membrane segments that differ in their spontaneous curvatures. To model protein coats as formed during clathrin-dependent endocytosis, we focus on the case in which the bound membrane segments have a large spontaneous curvature compared to the unbound ones. We derive explicit expressions for the engulfment rate and the uptake of nanoparticles, which both depend on the particle size in a nonmonotonic manner, and provide a

  16. In vitro approach to the mechanics of lipid membrane area regulation: vesicle absorption and tube formation

    NASA Astrophysics Data System (ADS)

    Staykova, Margarita; Holmes, Douglas; Read, Clarke; Stone, Howard A.

    2011-03-01

    We have designed an experimental approach that allows us to study the response of supported lipid bilayers to cycles of biaxial expansion and compression. We observed that the bilayer effectively adjusts its area during dilatational or compressive strains in order to reduce its tension. For example, if there is a sufficient lipid reservoir in the form of attached vesicles, then a lipid bilayer may accommodate strains tens of times larger than the critical strain for rupture by expanding its area. Additionally, upon compression the bilayer reduces its area by expelling lipid tubes out of its plane. These observations offer new insights into how cells regulate their surface area in response to various mechanical stimuli, i.e. during physiological volume changes, locomotion, cyclic expansion and compression of the uro- and the alveolar- epithelium, etc.

  17. Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules.

    PubMed

    Heijnen, H F; Schiel, A E; Fijnheer, R; Geuze, H J; Sixma, J J

    1999-12-01

    Platelet activation leads to secretion of granule contents and to the formation of microvesicles by shedding of membranes from the cell surface. Recently, we have described small internal vesicles in multivesicular bodies (MVBs) and alpha-granules, and suggested that these vesicles are secreted during platelet activation, analogous to the secretion of vesicles termed exosomes by other cell types. In the present study we report that two different types of membrane vesicles are released after stimulation of platelets with thrombin receptor agonist peptide SFLLRN (TRAP) or alpha-thrombin: microvesicles of 100 nm to 1 microm, and exosomes measuring 40 to 100 nm in diameter, similar in size as the internal vesicles in MVBs and alpha-granules. Microvesicles could be detected by flow cytometry but not the exosomes, probably because of the small size of the latter. Western blot analysis showed that isolated exosomes were selectively enriched in the tetraspan protein CD63. Whole-mount immuno-electron microscopy (IEM) confirmed this observation. Membrane proteins such as the integrin chains alpha(IIb)-beta(3) and beta(1), GPIbalpha, and P-selectin were predominantly present on the microvesicles. IEM of platelet aggregates showed CD63(+) internal vesicles in fusion profiles of MVBs, and in the extracellular space between platelet extensions. Annexin-V binding was mainly restricted to the microvesicles and to a low extent to exosomes. Binding of factor X and prothrombin was observed to the microvesicles but not to exosomes. These observations and the selective presence of CD63 suggest that released platelet exosomes may have an extracellular function other than the procoagulant activity, attributed to platelet microvesicles.

  18. Ca2+ Transport in Membrane Vesicles from Pinto Bean Leaves and Its Alteration after Ozone Exposure 1

    PubMed Central

    Castillo, Federico J.; Heath, Robert L.

    1990-01-01

    The influence of ozone on Ca2+ transport in plant membranes from pinto bean (Phaseolus vulgaris L. var Pinto) leaves was investigated in vitro by means of a filtration method using purified vesicles. Two transport mechanisms located at the plasma membrane are involved in a response to ozone: (a) passive Ca2+ influx into the cell and (b) active Ca2+ efflux driven by an ATP-dependent system, which has two components: a primary Ca2+ transport directly linked to ATP which is partially activated by calmodulin and a H+/Ca2+ antiport coupled to activity of a H+-ATPase. The passive Ca2+ permeability is increased by ozone. A triangular pulse of ozone stimulates a higher influx of Ca2+ than does a square wave, even though the total dose was the same (0.6 microliter per liter × hour). Leaves exposed to a square wave did not exhibit visible injury and were still able to recover from oxidant stress by activation of calmodulin-dependent Ca2+ extrusion mechanisms. On the other hand, leaves exposed to a triangular wave of ozone, exhibit visible injury and lost the ability of extruding Ca2+ out of the cell. PMID:16667780

  19. Vesicle-associated membrane protein (VAMP) cleavage by a new metalloprotease from the Brazilian scorpion Tityus serrulatus.

    PubMed

    Fletcher, Paul L; Fletcher, Maryann D; Weninger, Keith; Anderson, Trevor E; Martin, Brian M

    2010-03-01

    We present evidence that venom from the Brazilian scorpion Tityus serrulatus and a purified fraction selectively cleave essential SNARE proteins within exocrine pancreatic tissue. Western blotting for vesicle-associated membrane protein type v-SNARE proteins (or synaptobrevins) reveals characteristic alterations to venom-treated excised pancreatic lobules in vitro. Immunocytochemistry by electron microscopy confirms both the SNARE identity as VAMP2 and the proteolysis of VAMP2 as a marked decrease in secondary antibody-conjugated colloidal gold particles that are predominantly associated with mature zymogen granules. Studies with recombinant SNARE proteins were used to determine the specific cleavage site in VAMP2 and the susceptibility of VAMP8 (endobrevin). The VAMP2 cleavage site is between the transmembrane anchor and the SNARE motif that assembles into the ternary SNARE complex. Inclusion of divalent chelating agents (EDTA) with fraction nu, an otherwise active purified component from venom, eliminates SNARE proteolysis, suggesting the active protein is a metalloprotease. The unique cleavages of VAMP2 and VAMP8 may be linked to pancreatitis that develops following scorpion envenomation as both of these v-SNARE proteins are associated with zymogen granule membranes in pancreatic acinar cells. We have isolated antarease, a metalloprotease from fraction nu that cleaves VAMP2, and report its amino acid sequence.

  20. Vesicle-associated Membrane Protein (VAMP) Cleavage by a New Metalloprotease from the Brazilian Scorpion Tityus serrulatus*

    PubMed Central

    Fletcher, Paul L.; Fletcher, Maryann D.; Weninger, Keith; Anderson, Trevor E.; Martin, Brian M.

    2010-01-01

    We present evidence that venom from the Brazilian scorpion Tityus serrulatus and a purified fraction selectively cleave essential SNARE proteins within exocrine pancreatic tissue. Western blotting for vesicle-associated membrane protein type v-SNARE proteins (or synaptobrevins) reveals characteristic alterations to venom-treated excised pancreatic lobules in vitro. Immunocytochemistry by electron microscopy confirms both the SNARE identity as VAMP2 and the proteolysis of VAMP2 as a marked decrease in secondary antibody-conjugated colloidal gold particles that are predominantly associated with mature zymogen granules. Studies with recombinant SNARE proteins were used to determine the specific cleavage site in VAMP2 and the susceptibility of VAMP8 (endobrevin). The VAMP2 cleavage site is between the transmembrane anchor and the SNARE motif that assembles into the ternary SNARE complex. Inclusion of divalent chelating agents (EDTA) with fraction ν, an otherwise active purified component from venom, eliminates SNARE proteolysis, suggesting the active protein is a metalloprotease. The unique cleavages of VAMP2 and VAMP8 may be linked to pancreatitis that develops following scorpion envenomation as both of these v-SNARE proteins are associated with zymogen granule membranes in pancreatic acinar cells. We have isolated antarease, a metalloprotease from fraction ν that cleaves VAMP2, and report its amino acid sequence. PMID:20026600

  1. Mycobacterial Membrane Vesicles Administered Systemically in Mice Induce a Protective Immune Response to Surface Compartments of Mycobacterium tuberculosis

    PubMed Central

    Carreño, Leandro J.; Batista-Gonzalez, Ana; Baena, Andres; Venkataswamy, Manjunatha M.; Xu, Jiayong; Yu, Xiaobo; Wallstrom, Garrick; Magee, D. Mitchell; LaBaer, Joshua; Achkar, Jacqueline M.; Jacobs, William R.; Chan, John; Porcelli, Steven A.; Casadevall, Arturo

    2014-01-01

    ABSTRACT Pathogenic and nonpathogenic species of bacteria and fungi release membrane vesicles (MV), containing proteins, polysaccharides, and lipids, into the extracellular milieu. Previously, we demonstrated that several mycobacterial species, including bacillus Calmette-Guerin (BCG) and Mycobacterium tuberculosis, release MV containing lipids and proteins that subvert host immune response in a Toll-like receptor 2 (TLR2)-dependent manner (R. Prados-Rosales et al., J. Clin. Invest. 121:1471–1483, 2011, doi:10.1172/JCI44261). In this work, we analyzed the vaccine potential of MV in a mouse model and compared the effects of immunization with MV to those of standard BCG vaccination. Immunization with MV from BCG or M. tuberculosis elicited a mixed humoral and cellular response directed to both membrane and cell wall components, such as lipoproteins. However, only vaccination with M. tuberculosis MV was able to protect as well as live BCG immunization. M. tuberculosis MV boosted BCG vaccine efficacy. In summary, MV are highly immunogenic without adjuvants and elicit immune responses comparable to those achieved with BCG in protection against M. tuberculosis. PMID:25271291

  2. Synaptobrevin/vesicle-associated membrane protein (VAMP) of Aplysia californica: structure and proteolysis by tetanus toxin and botulinal neurotoxins type D and F.

    PubMed Central

    Yamasaki, S; Hu, Y; Binz, T; Kalkuhl, A; Kurazono, H; Tamura, T; Jahn, R; Kandel, E; Niemann, H

    1994-01-01

    Synaptobrevin/vesicle-associated membrane protein (VAMP) and syntaxin are potential vesicle donor and target membrane receptors of a docking complex that requires N-ethylmaleimide-sensitive factor (NSF) and soluble NSF-attachment proteins as soluble factors for vesicle fusion with target membranes. Members of this docking complex are the target of clostridial neurotoxins that act as zinc-dependent proteases. Molecular cloning of the Aplysia californica synaptobrevin cDNA revealed a 180-residue polypeptide (M(r), 19,745) with a central transmembrane region and an atypically large C-terminal intravesicular domain. This polypeptide integrates into membranes at both the co- and posttranslational level, as shown by modification of an artificially introduced N-glycosylation site. The soluble and membrane-anchored forms of synaptobrevin are cleaved by the light chains of the botulinal toxins type D and F and by tetanus toxin involving the peptide bonds Lys49-Ile50, Gln48-Lys49, and Gln66-Phe67, respectively. The active center of teh tetanus toxin light chain was identified by site-specific mutagenesis. His233, His237, Glu234, and Glu270/271 are essential to this proteolytic activity. Modification of histidine residues resulted in loss of zinc binding, whereas a replacement of Glu234 only slightly reduced the zinc content. Images PMID:8197120

  3. Cdc42 and Actin Control Polarized Expression of TI-VAMP Vesicles to Neuronal Growth Cones and Their Fusion with the Plasma MembraneV⃞

    PubMed Central

    Alberts, Philipp; Rudge, Rachel; Irinopoulou, Theano; Danglot, Lydia; Gauthier-Rouvière, Cécile; Galli, Thierry

    2006-01-01

    Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP)-mediated fusion of intracellular vesicles with the plasma membrane is crucial for neurite outgrowth, a pathway not requiring synaptobrevin-dependent exocytosis. Yet, it is not known how the TI-VAMP membrane trafficking pathway is regulated or how it is coordinated with cytoskeletal dynamics within the growth cone that guide neurite outgrowth. Here, we demonstrate that TI-VAMP, but not synaptobrevin 2, concentrates in the peripheral, F-actin-rich region of the growth cones of hippocampal neurons in primary culture. Its accumulation correlates with and depends upon the presence of F-actin. Moreover, acute stimulation of actin remodeling by homophilic activation of the adhesion molecule L1 induces a site-directed, actin-dependent recruitment of the TI-VAMP compartment. Expression of a dominant-positive mutant of Cdc42, a key regulator of cell polarity, stimulates formation of F-actin- and TI-VAMP-rich filopodia outside the growth cone. Furthermore, we report that Cdc42 activates exocytosis of pHLuorin tagged TI-VAMP in an actin-dependent manner. Collectively, our data suggest that Cdc42 and regulated assembly of the F-actin network control the accumulation and exocytosis of TI-VAMP-containing membrane vesicles in growth cones to coordinate membrane trafficking and actin remodeling during neurite outgrowth. PMID:16381811

  4. Cdc42 and actin control polarized expression of TI-VAMP vesicles to neuronal growth cones and their fusion with the plasma membrane.

    PubMed

    Alberts, Philipp; Rudge, Rachel; Irinopoulou, Theano; Danglot, Lydia; Gauthier-Rouvière, Cécile; Galli, Thierry

    2006-03-01

    Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP)-mediated fusion of intracellular vesicles with the plasma membrane is crucial for neurite outgrowth, a pathway not requiring synaptobrevin-dependent exocytosis. Yet, it is not known how the TI-VAMP membrane trafficking pathway is regulated or how it is coordinated with cytoskeletal dynamics within the growth cone that guide neurite outgrowth. Here, we demonstrate that TI-VAMP, but not synaptobrevin 2, concentrates in the peripheral, F-actin-rich region of the growth cones of hippocampal neurons in primary culture. Its accumulation correlates with and depends upon the presence of F-actin. Moreover, acute stimulation of actin remodeling by homophilic activation of the adhesion molecule L1 induces a site-directed, actin-dependent recruitment of the TI-VAMP compartment. Expression of a dominant-positive mutant of Cdc42, a key regulator of cell polarity, stimulates formation of F-actin- and TI-VAMP-rich filopodia outside the growth cone. Furthermore, we report that Cdc42 activates exocytosis of pHLuorin tagged TI-VAMP in an actin-dependent manner. Collectively, our data suggest that Cdc42 and regulated assembly of the F-actin network control the accumulation and exocytosis of TI-VAMP-containing membrane vesicles in growth cones to coordinate membrane trafficking and actin remodeling during neurite outgrowth.

  5. Delivery of membrane proteins into small and giant unilamellar vesicles by charge-mediated fusion.

    PubMed

    Biner, Olivier; Schick, Thomas; Müller, Yannic; von Ballmoos, Christoph

    2016-07-01

    One of the current challenges in synthetic biology is the production of stable membrane mimetic systems and the insertion of components in these systems. Here, we employ fusion of oppositely charged liposomes to deliver separately reconstituted membrane proteins into a common lipid bilayer. After a systematic evaluation of different lipid compositions by lipid mixing and size distribution analysis, suitable conditions were further investigated for proteoliposome fusion. With this technique, we functionally coreconstituted bo3 oxidase and ATP synthase from Escherichia coli into unilamellar liposomes ranging from 100 nm to 50 μm in size. The presented method is a simple and versatile tool for oriented membrane protein reconstitution to produce biomimetic systems with increased complexity. PMID:27264202

  6. Delivery of analgesic peptides to the brain by nano-sized bolaamphiphilic vesicles made of monolayer membranes.

    PubMed

    Popov, Mary; Abu Hammad, Ibrahim; Bachar, Tzach; Grinberg, Sarina; Linder, Charles; Stepensky, David; Heldman, Eliahu

    2013-11-01

    Inefficient drug delivery to the brain is a major obstacle for pharmacological management of brain diseases. We investigated the ability of bolavesicles - monolayer membrane vesicles self-assembled from synthetic bolaamphiphiles that contain two hydrophilic head groups at each end of a hydrophobic alkyl chain - to permeate the blood-brain barrier and to deliver the encapsulated materials into the brain. Cationic vesicles with encapsulated kyotorphin and leu-enkephalin (analgesic peptides) were prepared from the bolalipids GLH-19 and GLH-20 and studied for their analgesic effects in vivo in experimental mice. The objectives were to determine: (a) whether bolavesicles can efficiently encapsulate analgesic peptides, (b) whether bolavesicles can deliver these peptides to the brain in quantities sufficient for substantial analgesic effect, and to identify the bolavesicle formulation/s that provides the highest analgetic efficiency. The results indicate that the investigated bolavesicles can deliver analgesic peptides across the blood-brain barrier and release them in the brain in quantities sufficient to elicit efficient and prolonged analgesic activity. The analgesic effect is enhanced by using bolavesicles made from a mixture the bolas GLH-19 (that contains non-hydrolyzable acetylcholine head group) and GLH-20 (that contains hydrolysable acetylcholine head group) and by incorporating chitosan pendants into the formulation. The release of the encapsulated materials (the analgesic peptides kyotorphin and leu-enkephalin) appears to be dependent on the choline esterase (ChE) activity in the brain vs. other organs and tissues. Pretreatment of experimental animals with pyridostigmine (the BBB-impermeable ChE inhibitor) enhances the analgesic effects of the studied formulations. The developed formulations and the approach for their controlled decapsulation can serve as a useful modality for brain delivery of therapeutically-active compounds. PMID:23791683

  7. Permeation of bacterial cells, permeation of cytoplasmic and artificial membrane vesicles, and channel formation on lipid bilayers by peptide antibiotic AS-48.

    PubMed Central

    Gálvez, A; Maqueda, M; Martínez-Bueno, M; Valdivia, E

    1991-01-01

    Peptide AS-48 induces ion permeation, which is accompanied by the collapse of the cytoplasmic membrane potential, in sensitive bacteria. Active transport by cytoplasmic membrane vesicles is also impaired by AS-48. At low concentrations, this peptide also causes permeability of liposomes to low-molecular-weight compounds without a requirement for a membrane potential. Higher antibiotic concentrations induce severe disorganization, which is visualized under electron microscopy as aggregation and formation of multilamellar structures. Electrical measurements suggest that AS-48 can form channels in lipid bilayers. Images PMID:1702784

  8. Effects of Lipid Composition and Solution Conditions on the Mechanical Properties of Membrane Vesicles

    PubMed Central

    Kato, Nobuhiko; Ishijima, Akihiko; Inaba, Takehiko; Nomura, Fumimasa; Takeda, Shuichi; Takiguchi, Kingo

    2015-01-01

    The mechanical properties of cell-sized giant unilamellar liposomes were studied by manipulating polystyrene beads encapsulated within the liposomes using double-beam laser tweezers. Mechanical forces were applied to the liposomes from within by moving the beads away from each other, which caused the liposomes to elongate. Subsequently, a tubular membrane projection was generated in the tip at either end of the liposome, or the bead moved out from the laser trap. The force required for liposome transformation reached maximum strength just before formation of the projection or the moving out of the bead. By employing this manipulation system, we investigated the effects of membrane lipid compositions and environment solutions on the mechanical properties. With increasing content of acidic phospholipids, such as phosphatidylglycerol or phosphatidic acid, a larger strength of force was required for the liposome transformation. Liposomes prepared with a synthetic dimyristoylphosphatidylcholine, which has uniform hydrocarbon chains, were transformed easily compared with liposomes prepared using natural phosphatidylcholine. Surprisingly, bovine serum albumin or fetuin (soluble proteins that do not bind to membranes) decreased liposomal membrane rigidity, whereas the same concentration of sucrose showed no particular effect. These results show that the mechanical properties of liposomes depend on their lipid composition and environment. PMID:25611306

  9. Mouse Norovirus Replication Is Associated with Virus-Induced Vesicle Clusters Originating from Membranes Derived from the Secretory Pathway▿ †

    PubMed Central

    Hyde, Jennifer L.; Sosnovtsev, Stanislav V.; Green, Kim Y.; Wobus, Christiane; Virgin, Herbert W.; Mackenzie, Jason M.

    2009-01-01

    Human noroviruses (family Caliciviridae) are the leading cause of nonbacterial gastroenteritis worldwide. Despite the prevalence of these viruses within the community, the study of human norovirus has largely been hindered due to the inability to cultivate the viruses ex vivo and the lack of a small-animal model. In 2003, the discovery of a novel murine norovirus (MNV-1) and the identification of the tropism of MNV-1 for cells of a mononuclear origin led to the establishment of the first norovirus tissue culture system. Like other positive-sense RNA viruses, MNV-1 replication is associated with host membranes, which undergo significant rearrangement during infection. We characterize here the subcellular localization of the MNV-1 open reading frame 1 proteins and viral double-stranded RNA (dsRNA). Over the course of infection, dsRNA and the MNV-1 RNA-dependent RNA polymerase (NS7) were observed to proliferate from punctate foci located in the perinuclear region. All of the MNV-1 open reading frame 1 proteins were observed to colocalize with dsRNA during the course of infection. The MNV-1 replication complex was immunolocalized to virus-induced vesicle clusters formed in the cytoplasm of infected cells. Both dsRNA and MNV-1 NS7 were observed to localize to the limiting membrane of the individual clusters by cryo-immunoelectron microscopy. We show that the MNV-1 replication complex initially associates with membranes derived from the endoplasmic reticulum, trans-Golgi apparatus, and endosomes. In addition, we show that MNV-1 replication is insensitive to the fungal metabolite brefeldin A and consistently does not appear to recruit coatomer protein complex I (COPI) or COPII component proteins during replication. These data provide preliminary insights into key aspects of replication of MNV-1, which will potentially further our understanding of the pathogenesis of noroviruses and aid in the identification of potential targets for drug development. PMID:19587041

  10. Engraftment of plasma membrane vesicles into liposomes: A new method for designing of liposome-based vaccines

    PubMed Central

    Samiei, Afshin; Tamadon, Ali Mohammad; Samani, Soliman Mohammadi; Manolios, Nicholas; Sarvestani, Eskandar Kamali

    2014-01-01

    Objective(s): One of the major challenges in the field of vaccine design is choosing immunogenic antigens which can induce a proper immune response against complex targets like malignant cells or recondite diseases caused by protozoan parasites such as leishmaniasis. The aim of this study was to find a way to construct artificial liposome-based cells containing fragments of target's cell membrane. This structure not only mimics the real biological properties of proteins in the cell membrane of target cells, but also may induce the required immune responses, which culminate in eradication of target cells. Materials and Methods: Five different techniques have been investigated to engraft the plasma membrane's vesicles (PMVs) derived from a characterized Leishmania parasite into liposomes. The most efficient method was tested again on the PMVs derived from well-known breast cancer cell line SK-BR-3. The percentage of engraftment was determined by two-color flowcytometry after staining the engrafted dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine DiI-labeled liposomes with FITC-labeled PMVs. Results: Among the investigated techniques, freeze-drying method with 91±2% and 90±3% of engraftment for Leishmania and SK-BR-3 derived PMVs, respectively, showed superiority over the other methods. In addition, after 9 weeks storage in refrigerator, freeze-dried fused particles kept their original size (660±350 nm) and fusion efficiency (94±3%). Conclusion: Among five different engraftment techniques, freeze-drying is preferred over the other methods due to its simplicity, more fusion efficiency and stability of produced particles during storage. PMID:25729546

  11. Binding Analyses of Bacillus thuringiensis Cry δ-Endotoxins Using Brush Border Membrane Vesicles of Ostrinia nubilalis

    PubMed Central

    Hua, Gang; Masson, Luke; Jurat-Fuentes, Juan Luis; Schwab, George; Adang, Michael J.

    2001-01-01

    Transgenic corn expressing the Bacillus thuringiensis Cry1Ab gene is highly insecticidal to Ostrinia nubilalis (European corn borer) larvae. We ascertained whether Cry1F, Cry9C, or Cry9E recognizes the Cry1Ab binding site on the O. nubilalis brush border by three approaches. An optical biosensor technology based on surface plasmon resonance measured binding of brush border membrane vesicles (BBMV) injected over a surface of immobilized Cry toxin. Preincubation with Cry1Ab reduced BBMV binding to immobilized Cry1Ab, whereas preincubation with Cry1F, Cry9C, or Cry9E did not inhibit BBMV binding. BBMV binding to a Cry1F-coated surface was reduced when vesicles were preincubated in Cry1F or Cry1Ab but not Cry9C or Cry9E. A radioligand approach measured 125I-Cry1Ab toxin binding to BBMV in the presence of homologous (Cry1Ab) and heterologous (Cry1Ac, Cry1F, Cry9C, or Cry9E) toxins. Unlabeled Cry1Ac effectively competed for 125I-Cry1Ab binding in a manner comparable to Cry1Ab itself. Unlabeled Cry9C and Cry9E toxins did not inhibit 125I-Cry1Ab binding to BBMV. Cry1F inhibited 125I-Cry1Ab binding at concentrations greater than 500 nM. Cry1F had low-level affinity for the Cry1Ab binding site. Ligand blot analysis identified Cry1Ab, Cry1Ac, and Cry1F binding proteins in BBMV. The major Cry1Ab signals on ligand blots were at 145 kDa and 154 kDa, but a strong signal was present at 220 kDa and a weak signal was present at 167 kDa. Cry1Ac and Cry1F binding proteins were detected at 220 and 154 kDa. Anti-Manduca sexta aminopeptidase serum recognized proteins of 145, 154, and 167 kDa, and anti-cadherin serum recognized the 220 kDa protein. We speculate that isoforms of aminopeptidase and cadherin in the brush border membrane serve as Cry1Ab, Cry1Ac, and Cry1F binding proteins. PMID:11157257