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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. 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.

  3. 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.

  4. 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

  5. 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.

  6. Calcium transport in canine renal basolateral membrane vesicles. Effects of parathyroid hormone.

    PubMed Central

    Scoble, J E; Mills, S; Hruska, K A

    1985-01-01

    The effects of parathyroid hormone were studied on Ca2+ fluxes in canine renal proximal tubular basolateral membrane vesicles (BLMV). Efflux of Ca2+ from preloaded BLMV was found to be stimulated by an external Na+ gradient, and this was inhibited by the Na+ ionophore, monensin, and enhanced by intravesicular negative electrical potentials, which indicated electrogenic Na+/Ca2+ exchange activity. There was a Na+ gradient independent Ca2+ flux, but membrane binding of Ca2+ was excluded from contributing to the Na+ gradient-dependent efflux. The Na+ gradient-dependent flux of Ca2+ was very rapid, and even 2- and 5-s points may not fully represent absolute initial rates. It was saturable with respect to the interaction of Ca2+ and Na+ with an apparent (5 s) Km for Na+-dependent Ca2+ uptake of 10 microM, and an apparent (5 s) Vmax of 0.33 nmol/mg protein per 5 s. The Na+ concentration that yielded half maximal Ca2+ efflux (2 s) was 11 mM, and the Hill coefficient was two or greater. Both Na+ gradient dependent and independent Ca2+ efflux were decreased in BLMV prepared from kidneys of thyroparathyroidectomized (TPTX) dogs, and both were stimulated by parathyroid hormone (PTH) infusion to TPTX dogs. BLMV from TPTX dogs exhibited significantly reduced maximal stimulation of Na+ gradient-dependent Ca2+ uptake with an apparent (5 s) Vmax of 0.23 nmol/mg protein per 5 s, but the apparent Km was 8 microM, which was unchanged from normal. The Na+ gradient independent Ca2+ uptake was also reduced in BLMV from TPTX dogs compared with normal. Thus, PTH stimulated both Na+/Ca2+ exchange activity and Na+ independent Ca2+ flux. In vivo, the latter could result in an elevation of cytosolic Ca2+ by PTH, and this might contribute to the observed decrease in solute transport in the proximal tubule. Images PMID:3988932

  7. Hydroxyl/bile acid exchange. A new mechanism for the uphill transport of cholate by basolateral liver plasma membrane vesicles.

    PubMed

    Blitzer, B L; Terzakis, C; Scott, K A

    1986-09-15

    In order to characterize the driving forces for the concentrative uptake of unconjugated bile acids by the hepatocyte, the effects of pH gradients on the uptake of [3H]cholate by rat basolateral liver plasma membrane vesicles were studied. In the presence of an outwardly directed hydroxyl gradient (pH 6.0 outside and pH 7.5 inside the vesicle), cholate uptake was markedly stimulated and the bile acid was transiently accumulated at a concentration 1.5- to 2-fold higher than at equilibrium ("overshoot"). In the absence of a pH gradient (pH 6.0 or 7.5 both inside and outside the vesicle), uptake was relatively slower and no overshoot was seen. Reductions in the magnitude of the transmembrane pH gradient were associated with slower initial uptake rates and smaller overshoots. Cholate uptake under pH gradient conditions was inhibited by furosemide and bumetanide but not by 4, 4'-diisothiocyano-2,2'-disulfonic stilbene (SITS), 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (DIDS), or probenecid. In the absence of a pH gradient, an inside-positive valinomycin-induced K+ diffusion potential caused a slight increase in cholate uptake which was insensitive to furosemide. Moreover, in the presence of an outwardly directed hydroxyl gradient, uphill cholate transport was observed even under voltage clamped conditions. These findings suggest that pH gradient-driven cholate uptake was not due to associated electrical potentials. Despite an identical pKa to that of cholate, an outwardly directed hydroxyl gradient did not drive uphill transport of three other unconjugated bile acids (deoxycholate, chenodeoxycholate, ursodeoxycholate), suggesting that a non-ionic diffusion mechanism cannot account for uphill cholate transport. In canalicular vesicles, although cholate uptake was relatively faster in the presence of a pH gradient than in the absence of a gradient, peak uptake was only slightly above that found at equilibrium under voltage clamped conditions. These findings

  8. Glucose and fructose uptake by Limulus polyphemus hepatopancreatic brush border and basolateral membrane vesicles: evidence for Na+-dependent sugar transport activity.

    PubMed

    Sterling, Kenneth M; Ahearn, Gregory A

    2011-05-01

    [(3)H]-fructose and [(3)H]-glucose transport activities were determined in brush border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) from Limulus polyphemus (horseshoe crab) hepatopancreas. Glucose transport was equilibrative in the absence of sodium and sodium dependent in the presence of sodium in BBMV, suggesting GLUT-like and SGLT-like transport activity. Glucose transport by BLMV was equilibrative and sodium independent. Fructose uptake by BBMV and BLMV was equilibrative in the absence of sodium and sodium dependent in the presence of sodium. Western blot analysis using a rabbit anti-mouse SGLT-1 polyclonal antibody indicated the presence of a cross-reacting horseshoe crab BBMV protein of similar molecular weight to the mammalian SGLT1. Sequence alignment of the mouse SGLT-4 and SGLT1 with a translated, horseshoe crab-expressed sequence tag also indicated significant identity between species. Fructose and glucose uptake in the absence and presence of sodium by hepatopancreas BBMV and BLMV indicated the presence of sodium-dependent transport activity for each sugar that may result from the presence of transporters similar to those described for other species. PMID:21184084

  9. Use of fluorescence-activated vesicle sorting for isolation of Naked2-associated, basolaterally targeted exocytic vesicles for proteomics analysis.

    PubMed

    Cao, Zheng; Li, Cunxi; Higginbotham, James N; Franklin, Jeffrey L; Tabb, David L; Graves-Deal, Ramona; Hill, Salisha; Cheek, Kristin; Jerome, W Gray; Lapierre, Lynne A; Goldenring, James R; Ham, Amy-Joan L; Coffey, Robert J

    2008-09-01

    By interacting with the cytoplasmic tail of a Golgi-processed form of transforming growth factor-alpha (TGFalpha), Naked2 coats TGFalpha-containing exocytic vesicles and directs them to the basolateral corner of polarized epithelial cells where the vesicles dock and fuse in a Naked2 myristoylation-dependent manner. These TGFalpha-containing Naked2-associated vesicles are not directed to the subapical Sec6/8 exocyst complex as has been reported for other basolateral cargo, and thus they appear to represent a distinct set of basolaterally targeted vesicles. To identify constituents of these vesicles, we exploited our finding that myristoylation-deficient Naked2 G2A vesicles are unable to fuse at the plasma membrane. Isolation of a population of myristoylation-deficient, green fluorescent protein-tagged G2A Naked2-associated vesicles was achieved by biochemical enrichment followed by flow cytometric fluorescence-activated vesicle sorting. The protein content of these plasma membrane de-enriched, flow-sorted fluorescent G2A Naked2 vesicles was determined by LC/LC-MS/MS analysis. Three independent isolations were performed, and 389 proteins were found in all three sets of G2A Naked2 vesicles. Rab10 and myosin IIA were identified as core machinery, and Na(+)/K(+)-ATPase alpha1 was identified as an additional cargo within these vesicles. As an initial validation step, we confirmed their presence and that of three additional proteins tested (annexin A1, annexin A2, and IQGAP1) in wild-type Naked2 vesicles. To our knowledge, this is the first large scale protein characterization of a population of basolaterally targeted exocytic vesicles and supports the use of fluorescence-activated vesicle sorting as a useful tool for isolation of cellular organelles for comprehensive proteomics analysis. PMID:18504258

  10. Characterization of the basolateral membrane conductance of Necturus urinary bladder.

    PubMed

    Demarest, J R; Finn, A L

    1987-04-01

    Necturus urinary bladders stripped of serosal muscle and connective tissue were impaled through their basolateral membranes with microelectrodes in experiments that permitted rapid changes in the ion composition of the serosal solution. The transepithelial electrical properties exhibited a marked seasonal variation that could be attributed to variations in the conductance of the shunt pathway, apical membrane selectivity, and basolateral Na+ transport. In contrast, the passive electrical properties of the basolateral membrane remained constant throughout the year. The apparent transference numbers (Ti) of the basolateral membrane for K+ and Cl- were determined from the effect on the basolateral membrane equivalent electromotive force of a sudden increase in the serosal K+ concentration from 2.5 to 50 mM/liter or a decrease in the Cl- concentration from 101 to 10 mM/liter. TK and TCl were 0.71 +/- 0.05 and 0.04 +/- 0.01, respectively. The basolateral K+ conductance could be blocked by Ba2+ (0.5 mM), Cs+ (10 mM), or Rb+ (10 mM), but was unaffected by 3,4-diaminopyridine (100 microM), decamethonium (100 microM), or tetraethylammonium (10 mM). We conclude that a highly selective K+ conductance dominates the electrical properties of the basolateral membrane and that this conductance is different from those found in nerve and muscle membranes. PMID:2438371

  11. 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

  12. Active urea transport and an unusual basolateral membrane composition in the gills of a marine elasmobranch.

    PubMed

    Fines, G A; Ballantyne, J S; Wright, P A

    2001-01-01

    In elasmobranch fishes, urea occurs at high concentrations (350-600 mM) in the body fluids and tissues, where it plays an important role in osmoregulation. Retention of urea by the gill against this huge blood-to-water diffusion gradient requires specialized adaptations to the epithelial cell membranes. Experiments were performed to determine the mechanisms and structural features that facilitate urea retention by the gill of the spiny dogfish Squalus acanthias. Analysis of urea uptake by gill basolateral membrane vesicles revealed the presence of a phloretin-sensitive (half inhibition 0.09 mM), sodium-coupled, secondary active urea transporter (Michaelis constant = 10.1 mM, maximal velocity = 0.34 micromol. h(-1). mg protein(-1)). We propose that this system actively transports urea out of the gill epithelial cells back into the blood against the urea concentration gradient. Lipid analyses of the basolateral membrane revealed high levels of cholesterol contributing to the highest reported cholesterol-to-phospholipid molar ratio (3.68). This unique combination of active urea transport and modification of the phospholipid bilayer membrane is responsible for decreasing the gill permeability to urea and facilitating urea retention by the gill of Squalus acanthias. PMID:11124129

  13. 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

  14. Differences in neutral amino acid and glucose transport between brush border and basolateral plasma membrane of intestinal epithelial cells.

    PubMed

    Hopfer, U; Sigrist-Nelson, K; Ammann, E; Murer, H

    1976-12-01

    A comparison of L-valine and D-glucose transport was carried out with vesicles of plasma membrane isolated either from the luminal (brush border) or from the contra-luminal (basolateral) region of small intestinal epithelial cells. The existence of transport systems for both non-electrolytes was demonstrated by stereospecificity and saturability of uptake, as well as tracer coupling. Transport of L-valine and D-glucose differs markedly in the two types of plasma membrane with respect to stimulation by Na+. The presence of Na+ stimulated initial L-valine and D-glucose uptake in brush border, but not in basolateral membrane. Moreover, an electro-chemical Na+ gradient, oriented with the lower potential on the inside, supported accumulation of the non-electrolytes above medium concentration only in the brush border membrane. L-Valine and D-glucose transport also were saturated at lower concentrations in brush border (10-20 mM) than in basolateral plasma membranes (30-50 mM). A third difference between the two membranes was found in the effectiveness of known inhibitors of D-glucose transport. In brush border membranes phlorizin was more potent than phloretin and 2', 3', 4'-trihydroxy-4-methoxy chalcone and cytochalasin B did not inhibit at all. In contrast, with the basolateral plasma membranes the order of potency was changed to phloretin = 2',3',4'-trihydroxy-4-methoxy chalcone greater than cytochalasin B greater than phlorizin. These results indicate the presence of different types of transport systems for monosaccharides and neutral amino acids in the luminal and contra-luminal region of the plasma membrane. Active transepithelial transport can be explained on the basis of the different properties of the non-electrolyte transport systems in the two cellular regions and an electro-chemical Na+ gradient that is dependent on cellular metabolism. PMID:137908

  15. Membrane tensiometer for heavy giant vesicles

    NASA Astrophysics Data System (ADS)

    Puech, P.-H.; Brochard-Wyart, F.

    2004-10-01

    One key parameter of giant-vesicles adhesion is their membrane tension, σ. A theoretically simple but delicate way to impose (and measure) it is to use micropipette manipulation techniques. But usually, the vesicles are free and their tension is unknown, until an adhesion patch grows. σ can be deduced from the detailed profile of the membrane close to the substrate, but this method is limited to very low tensions. We present here a rather simple way to estimate the membrane tension of heavy vesicles, which sediment close to a surface, by observing by RIM the size of the flat region of the vesicle. As an application, we follow the slow flattening of vesicles, when the surrounding sugar solution is evaporating, and their light-induced tensioning.

  16. 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

  17. 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. PMID:26465512

  18. Effects of ADH on the apical and basolateral membranes of toad urinary bladder epithelial cells.

    PubMed

    Donaldson, P J; Leader, J P

    1993-11-01

    Short-circuited urinary bladders from Bufo marinus were supported on their apical surface by an agar mounting method and impaled with microelectrodes via their basolateral membrane. This arrangement provided stable and long-lasting impalements of epithelial cells and yielded reliable membrane potentials and voltage divider ratios (Ra/Rb), where Ra and Rb are apical and basolateral membrane resistances respectively. The membrane potential under short-circuit conditions (Vsc) was -51.4 +/- 2.2 mV (n = 59), while under open-circuit conditions apical membrane potential (Va) and basolateral membrane potential (Vb) were -31.0 +/- 2.4 and 59.5 +/- 2.4 mV, respectively. This yields a "well-shaped" potential profile across the toad urinary bladder, where Va is inversely related to the rate of transport, Isc. Antidiuretic hormone (ADH) produced a hyperpolarisation of Vsc and Vb but had no significant effect on Va. In addition, Ra/Rb was significantly increased by ADH (4.6 +/- 0.5 to 10.2 +/- 3.6). Calculation of individual membrane resistances following the addition of amiloride showed that ADH produced a parallel decrease in Ra and Rb membrane resistance, with the observed increase in Ra/Rb being due to a greater percentage decrease in Rb than in Ra. The ability of ADH to effect parallel changes in apical and basolateral membrane conductance helps to maintain a constant cellular volume despite an increase in transepithelial transport. PMID:8309781

  19. 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.

  20. Bolaamphiphiles Promote Phospholipid Translocation Across Vesicle Membranes

    PubMed Central

    Forbes, Christopher C.; DiVittorio, Kristy M.; Smith, Bradley D.

    2008-01-01

    A series of membrane-spanning bolaamphiphiles (molecules with two hydrophilic end-groups connected by a hydrophobic linker) were prepared by a modular synthetic method and evaluated for their abilities to affect the dynamics of a surrounding bilayer membrane. The goal was to determine if the bolaamphiphiles promote the translocation of phospholipids across vesicle membranes. The bolaamphiphiles were incorporated at low levels (up to 5 mol%) in vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Inward translocation assays were performed using fluorescent, NBD-labeled phospholipid probes with phosphocholine (PC) or phosphoglycerol (PG) head-groups. The membrane-spanning bolaamphiphiles promote the translocation of both phospholipid probes in the order PG > PC, while shorter bolaamphiphiles (structures that must adopt a U-shape and keep both end-groups in the same leaflet of the membrane), and regular amphiphiles with one hydrophilic end-group, are inactive. These results are an exception to the rule-of-thumb that membrane-spanning bolaamphiphiles are inherently membrane stabilizing molecules that inhibit all types of membrane transport. PMID:16834395

  1. The N-terminal basolateral targeting signal unlikely acts alone in the differential trafficking of membrane transporters in MDCK cells.

    PubMed

    Kuo, Shiu-Ming; Wang, Li-Yuan; Yu, Siyuan; Campbell, Christine E; Valiyaparambil, Sujith A; Rance, Mark; Blumenthal, Kenneth M

    2013-07-30

    We have shown previously, using confocal imaging and transport assays, that the N-terminus of sodium-dependent vitamin C transporter 2 (SVCT2) can redirect apical SVCT1 to the basolateral membrane. Here, the SVCT model was used to further characterize the basolateral targeting peptide signal. Both the length (31 amino acids) and sequence accuracy of the N-terminus of SVCT2 were found to be important in basolateral targeting activity, suggesting a structural requirement. However, the N-terminal basolateral targeting sequence did not appear to act alone, based on analyses of heterologous chimeras. Although diverse N-terminal basolateral targeting signals from multipass membrane proteins can all redirect apical protein from the same gene family to the basolateral membrane, none of the N-terminal basolateral targeting signals can redirect the transmembrane and C-terminal regions from a different gene family. Instead, the presence of these heterologous N-terminal basolateral targeting signals affected the trafficking of otherwise apical protein, causing their accumulation in a stable tubulin-like non-actin structure. Nontargeting N-terminal sequences had no effect. Similar protein retention was observed previously and in this study when the C-terminus of apical or basolateral protein was mutated. These results suggest that the N- and C-termini interact, directly or indirectly, within each gene family for basolateral targeting. Circular dichroism and two-dimensional nuclear magnetic resonance analyses both found a lack of regular secondary structure in the conserved N-terminus of SVCT2, consistent with the presence of partner(s) in the targeting unit. Our finding, a departure from the prevailing single-peptide motif model, is consistent with the evolution of basolateral transporters from the corresponding apical genes. The interaction among the N-terminus, its partner(s), and the cellular basolateral targeting machinery needs to be further elucidated. PMID:23837633

  2. 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.

  3. Expression of two membrane fusion proteins, synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein, in choroid plexus epithelium.

    PubMed

    Chung, I; Burkart, A; Szmydynger-Chodobska, J; Dodd, K A; Trimble, W S; Miller, K V; Shim, M; Chodobski, A

    2003-01-01

    In addition to being the major site of cerebrospinal fluid formation, the choroid plexus epithelium emerges as an important source of polypeptides in the brain. Physiologically regulated release of some polypeptides synthesized by the choroid plexus has been shown. The molecular mechanisms underlying this polypeptide secretion have not been characterized, however. In the present study, synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein, two membrane fusion proteins playing a critical role in exocytosis in neurons and endocrine cells, were found to be expressed in the choroid plexus epithelium. It was also shown that in choroidal epithelium, synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein stably interact. Two members of the vesicle-associated membrane protein family, vesicle-associated membrane protein-1 and vesicle-associated membrane protein-2, were expressed in the rat choroid plexus at the messenger RNA and protein level. However, their newly discovered isoforms, vesicle-associated membrane protein-1b and vesicle-associated membrane protein-2b, produced by alternative RNA splicing, were not detected in choroidal tissue. Immunohistochemistry demonstrated that vesicle-associated membrane protein is confined to the cytoplasm of choroidal epithelium, whereas synaptosome-associated protein of 25 kDa is associated with plasma membranes, albeit with a varied cellular distribution among species studied. Specifically, in the rat choroid plexus, synaptosome-associated protein of 25 kDa was localized to the basolateral membrane domain of choroidal epithelium and was expressed in small groups of cells. In comparison, in ovine and human choroidal tissues, apical staining for synaptosome-associated protein of 25 kDa was found in the majority of epithelial cells. These species-related differences in cellular synaptosome-associated protein of 25 kDa distribution suggested that the synaptosome-associated protein of

  4. Evidence for basolateral but not apical membrane localization of outwardly rectifying depolarization-induced Cl(-) channel in airway epithelia.

    PubMed

    Hwang, T H; Lee, H J; Lee, N K; Choi, Y C

    2000-08-01

    The rat primary cultured-airway monolayer had been an excellent model for deciphering the ion channel after nystatin permeabilization of its basolateral or apical membrane (Hwang et al., 1996). After apical membrane permeabilization of rat primary cultured-airway monolayer, 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS)-sensitive outwardly rectifying depolarization-induced Cl(-) (BORDIC) currents were observed across the basolateral membrane in symmetrical NMG-Cl solution in this study. No significant Cl(-) current induced by the application of voltage clamping was observed across the apical membrane in symmetrical NMG-Cl solution after basolateral membrane permeabilization. The halide permeability sequence for BORDIC current was Br(-) = I(-) > Cl(-). BORDIC current was not affected by basolaterally applied bumetanide (0.5 mm). Basolateral DIDS (0.2 mm) but not apical DIDS inhibited CFTR mediated short-circuit current (I(sc)) in an intact monolayer of rat airway epithelia, a T84 human colonal epithelial cell line, and a Calu-3 human airway epithelial cell line. This is the first report showing that depolarization induced Cl(-) current is present on the basolateral membrane of airway epithelia. PMID:10931973

  5. 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

  6. Bacterial outer membrane vesicles and vaccine applications.

    PubMed

    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

  7. Identification of a vesicle-associated membrane protein (VAMP)-like membrane protein in zymogen granules of the rat exocrine pancreas.

    PubMed

    Braun, J E; Fritz, B A; Wong, S M; Lowe, A W

    1994-02-18

    Zymogen granules of the exocrine pancreas are the secretory organelles responsible for the regulated secretion of digestive enzymes. Several proteins are associated with or are integral components of the lipid bilayer that forms the zymogen granule membrane. These proteins likely represent important components in the regulated secretion of digestive enzymes. VAMPs (vesicle-associated membrane proteins)/synaptobrevins are a family of 18-kDa integral membrane proteins originally characterized in synaptic vesicles. Polyclonal antisera raised against either a VAMP/glutathione S-transferase (GST) fusion protein or rat brain synaptic vesicles, detected an 18-kDa immunoreactive protein in zymogen granule membranes that co-migrates electrophorectically with rat brain synaptic vesicle VAMP. Rat brain synaptic vesicle VAMP was detected by both antisera. Botulinum-B toxin treatment of zymogen granule membranes did not result in cleavage of zymogen granule membrane VAMP, indicating that exocrine pancreatic VAMP is either VAMP1 or a novel VAMP-isoform. Immunofluorescent studies demonstrated that exocrine pancreatic VAMP localized with GP2, a zymogen granule membrane protein, to the apical region of pancreatic acinar cells. No significant labeling was observed in basolateral regions of pancreatic acinar cells. These results establish the presence of a VAMP protein in the zymogen granule of the rat pancreas and suggest that VAMPs have a role in exocrine secretion. PMID:8106518

  8. Transmembrane flux and receptor desensitization measured with membrane vesicles. Homogeneity of vesicles investigated by computer simulation.

    PubMed Central

    Cash, D J; Langer, R M; Subbarao, K; Bradbury, J R

    1988-01-01

    The use of membrane vesicles to make quantitative studies of transmembrane transport and exchange processes involves an assumption of homogeneity of the membrane vesicles. In studies of 86Rb+ exchange mediated by acetylcholine receptor from the electric organ of Electrophorus electricus and of 36Cl- exchange mediated by GABA receptor from rat brain, measurements of ion exchange and receptor desensitization precisely followed first order kinetics in support of this assumption. In other measurements a biphasic decay of receptor activity was seen. To elucidate the molecular properties of receptors from such measurements it is important to appreciate what the requirements of vesicle monodispersity are for meaningful results and what the effect of vesicle heterogeneity would be. The experiments were simulated with single vesicle populations with variable defined size distributions as well as with mixtures of different populations of vesicles. The properties of the receptors and their density in the membrane could be varied. Different receptors could be present on the same or different membrane vesicles. The simulated measurements were not very sensitive to size dispersity. A very broad size distribution of a single vesicle population was necessary to give rise to detectable deviations from first order kinetics or errors in the determined kinetic constants. Errors could become significant with mixtures of different vesicle populations, where the dispersity in initial ion exchange rate constant, proportional to the receptor concentration per internal volume, became large. In this case the apparent rate of receptor desensitization would diverge in opposite directions from the input value when measured by two different methods, suggesting an experimental test for such kinetic heterogeneity. A biphasic decrease of receptor activity could not be attributed to vesicle heterogeneity and must be due to desensitization processes with different rates. Significant errors would not

  9. Redox-Reactive Membrane Vesicles produced by Shewanella

    SciTech Connect

    Gorby, Yuri A.; McLean, Jeffrey S.; Korenevsky, Anton A.; Rosso, Kevin M.; El-Naggar, Mohamed Y.; Beveridge, Terrance J.

    2008-06-01

    Dissimilatory iron reducing bacteria produce and release membrane vesicles with diameters ranging from 50 to 250 nm. The vesicles, which arise from the outer membrane of these Gram-negative bacteria, lack DNA but contain proteins that catalyze the reduction of ferric iron and other multivalent heavy metals and radionuclides. This enzymatic process results in the formation of nano-size biogenic mineral assemblages that resemble nanofossils. 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 paleontological indicators of early life on earth and as biosignatures of like on other planets is recognized.

  10. Vesicle-associated membrane protein 7 is expressed in intestinal ER.

    PubMed

    Siddiqi, Shadab A; Mahan, James; Siddiqi, Shahzad; Gorelick, Fred S; Mansbach, Charles M

    2006-03-01

    Intestinal dietary triacylglycerol absorption is a multi-step process. Triacylglycerol exit from the endoplasmic reticulum (ER) is the rate-limiting step in the progress of the lipid from its apical absorption to its basolateral membrane export. Triacylglycerol is transported from the ER to the cis Golgi in a specialized vesicle, the pre-chylomicron transport vesicle (PCTV). The vesicle-associated membrane protein 7 (VAMP7) was found to be more concentrated on PCTVs compared with ER membranes. VAMP7 has been previously identified associated with post-Golgi sites in eukaryotes. To examine the potential role of VAMP7 in PCTV trafficking, antibodies were generated that identified a 25 kDa band consistent with VAMP7 but did not crossreact with VAMP1,2. VAMP7 was concentrated on intestinal ER by immunofluorescence microscopy. Immunoelectron microscopy showed that the ER proteins Sar1 and rBet1 were present on PCTVs and colocalized with VAMP7. Iodixanol gradient centrifugation showed VAMP7 to be isodense with ER and endosomes. Although VAMP7 localized to intestinal ER, it was not present in the ER of liver and kidney. Anti-VAMP7 antibodies reduced the transfer of triacylglycerol, but not newly synthesized proteins, from the ER to the Golgi by 85%. We conclude that VAMP7 is enriched in intestinal ER and that it plays a functional role in the delivery of triacylglycerol from the ER to the Golgi. PMID:16495485

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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

  16. Electro-hydrodynamic effects on lipid membranes in giant vesicles

    NASA Astrophysics Data System (ADS)

    Staykova, Margarita; Yamamoto, Tetsuya; Lipowsky, Reinhard; Dimova, Rumiana

    2009-11-01

    Electric fields are widely applied for cell manipulation in numerous micron-scale systems. Here, we show for the first time that alternating electric fields may cause pronounced flows in the membrane of giant lipid vesicles as well as in the surrounding fluid media.^ The lipid vesicles are not only biomimetic model for the cell membrane but also have many potential biotechnological applications, e.g. as drug-delivery systems and micro-reactors. The reported effects should be considered in electric micro-manipulation procedures on cells and vesicles. They might be useful for applications in microfluidic technologies, for lipid mixing, trapping and displacement, as will be demonstrated. We also believe that our method for visualization of the lipid flows by fluorescently labeled intra-membrane domains will be helpful for studies on membrane behavior in vesicles subjected to shear or mechanical stresses.

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

    PubMed

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

    1998-01-01

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

  18. Aminosilane/oleic acid vesicles as model membranes of protocells.

    PubMed

    Douliez, Jean-Paul; Zhendre, Vanessa; Grélard, Axelle; Dufourc, Erick J

    2014-12-16

    Oleic acid vesicles represent good models of membrane protocells that could have existed in prebiotic times. Here, we report the formation, growth polymorphism, and dynamics of oleic acid spherical vesicles (1-10 μm), stable elongated vesicles (>50 μm length; 1-3 μm diameter), and chains of vesicles (pearl necklaces, >50 μm length; 1-3 μm diameter) in the presence of aminopropyl triethoxysilane and guanidine hydrochloride. These vesicles exhibit a remarkable behavior with temperature: spherical vesicles only are observed when keeping the sample at 4 °C for 2 h, and self-aggregated spherical vesicles occur upon freezing/unfreezing (-20/20 °C) samples. Rather homogeneous elongated vesicles are reformed upon heating samples at 80 °C. The phenomenon is reversible through cycles of freezing/heating or cooling/heating of the same sample. Deuterium NMR evidences a chain packing rigidity similar to that of phospholipid bilayers in cellular biomembranes. We expect these bilayered vesicles to be surrounded by a layer of aminosilane oligomers, offering a variant model for membrane protocells. PMID:25420203

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

    PubMed

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

    1988-06-01

    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. PMID:3380805

  20. Functional Advantages Conferred by Extracellular Prokaryotic Membrane Vesicles

    PubMed Central

    Manning, Andrew J.; Kuehn, Meta J.

    2015-01-01

    The absence of subcellular organelles is a characteristic typically used to distinguish prokaryotic from eukaryotic cells. But recent discoveries do not support this dogma. Over the past 50 years, researchers have begun to appreciate and characterize Gram-negative bacterial outer membrane derived vesicles and Gram-positive and archaeal membrane vesicles. These extracellular, membrane-bound organelles can perform a variety of functions, including binding and delivery of DNA, transport of virulence factors, protection of the cell from outer membrane targeting antimicrobials, and ridding the cell of toxic envelope proteins. Here we review the contributions of these extracellular organelles to prokaryotic physiology and compare these with the contributions of the bacterial interior membrane bound organelles responsible for harvesting light energy and for generating magnetic crystals of heavy metals. Understanding the roles of these multifunctional extracellular vesicle organelles as microbial tools will help us to better realize the diverse interactions that occur in our polymicrobial world. PMID:23615201

  1. Na sup + uptake into colonic enterocyte membrane vesicles

    SciTech Connect

    Bridges, R.J.; Garty, H.; Benos, D.J.; Rummel, W. Weizmann Institute of Science, Rehovot Univ. of Alabama, Birmingham )

    1988-04-01

    Na{sup +} uptake was studied in colonic enterocyte membrane vesicles prepared from normal and dexamethasone-treated rats. Vesicles from rats treated with dexamethasone demonstrated a fivefold greater {sup 22}Na{sup +} uptake compared with vesicles from normal rats. Most of the tracer uptake in membranes derived from treated rats occurred through a conductive, amiloride-blockable pathway located in vesicles with low native K{sup +} permeability and high Cl{sup {minus}} permeability. Kinetic analysis of the amiloride inhibition curve revealed the presence of two amiloride-blockable pathways, one with a high affinity accounting for 85% of the uptake, and one with a low affinity accounting for only 12% of the uptake. Only the low-affinity pathway was detected with vesicles from normal rats. The high sensitivity to amiloride, the dependence on dexamethasone pretreatment, and the relative permeabilities to K{sup +} and Cl{sup {minus}} indicate that most of the {sup 22}Na{sup +} uptake in membranes derived from treated rats is through a Na{sup +}-specific channel located in apical membrane vesicles. Preincubation of the isolated cells from dexamethasone-treated rats at 37{degree}C in Ca{sup 2+}-free solutions before homogenization and membrane vesicle purification caused a 5- to 10-fold increase in amiloride-blockable {sup 22}Na{sup +} uptake compared with vesicles derived from cells maintained at 0{degree}C. The addition of Ca{sup 2+}, but not of Mg{sup 2+}, to the incubation solution markedly reduced this temperature-dependent enhancement in {sup 22}Na{sup +} uptake. These results suggest that Na{sup +} transport in colonic enterocytes from dexamethasone-treated rats is regulated by a Ca{sup 2+}-dependent, temperature-sensitive process which causes a sustained change in the apical membrane.

  2. Virulence and Immunomodulatory Roles of Bacterial Outer Membrane Vesicles

    PubMed Central

    Ellis, Terri N.; Kuehn, Meta J.

    2010-01-01

    Summary: Outer membrane (OM) vesicles are ubiquitously produced by Gram-negative bacteria during all stages of bacterial growth. OM vesicles are naturally secreted by both pathogenic and nonpathogenic bacteria. Strong experimental evidence exists to categorize OM vesicle production as a type of Gram-negative bacterial virulence factor. A growing body of data demonstrates an association of active virulence factors and toxins with vesicles, suggesting that they play a role in pathogenesis. One of the most popular and best-studied pathogenic functions for membrane vesicles is to serve as natural vehicles for the intercellular transport of virulence factors and other materials directly into host cells. The production of OM vesicles has been identified as an independent bacterial stress response pathway that is activated when bacteria encounter environmental stress, such as what might be experienced during the colonization of host tissues. Their detection in infected human tissues reinforces this theory. Various other virulence factors are also associated with OM vesicles, including adhesins and degradative enzymes. As a result, OM vesicles are heavily laden with pathogen-associated molecular patterns (PAMPs), virulence factors, and other OM components that can impact the course of infection by having toxigenic effects or by the activation of the innate immune response. However, infected hosts can also benefit from OM vesicle production by stimulating their ability to mount an effective defense. Vesicles display antigens and can elicit potent inflammatory and immune responses. In sum, OM vesicles are likely to play a significant role in the virulence of Gram-negative bacterial pathogens. PMID:20197500

  3. 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. PMID:25568329

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

    PubMed Central

    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

    2015-01-01

    Summary 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. PMID:26687357

  5. 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. PMID:25534673

  6. Quinidine-sensitive K+ channels in the basolateral membrane of embryonic coprodeum epithelium: regulation by aldosterone and thyroxine.

    PubMed

    Illek, B; Fischer, H; Clauss, W

    1993-01-01

    Basolateral K+ channels and their regulation during aldosterone- and thyroxine-stimulated Na+ transport were studied in the lower intestinal epithelium (coprodeum) of embryonic chicken in vitro. Isolated tissues of the coprodeum were mounted in Ussing chambers and investigated under voltage-clamped conditions. Simultaneous stimulation with aldosterone (1 mumol.l-1) and thyroxine (1 mumol.l-1) raised short-circuit current after a 1- to 2-h latent period. Maximal values were reached after 6-7 h of hormonal treatment, at which time transepithelial Na+ absorption was more than tripled (77 +/- 11 microA.cm-2) compared to control (24 +/- 8 microA.cm-2). K+ currents across the basolateral membrane were investigated after permeabilizing the apical membrane with the pore-forming antibiotic amphotericin B and application of a mucosal-to-serosal K+ gradient. This K+ current could be dose dependently depressed by the K+ channel blocker quinidine. Fluctuation analysis of the short-circuit current revealed a spontaneous and a blocker-induced Lorentzian noise component in the power density spectra. The Lorentzian corner frequencies increased linearly with the applied blocker concentration. This enabled the calculation of single K+ channel current and K+ channel density. Single K+ channel current was not affected by stimulation, whereas the number of quinidine-sensitive K+ channels in the basolateral membrane increased from 11 to 26.10(6).cm-2 in parallel to the hormonal stimulation transepithelial Na+ transport. This suggests that the basolateral membrane is a physiological target during synergistic aldosterone and thyroxine regulation of transepithelial Na+ transport for maintaining intracellular K+ homeostasis. PMID:8151014

  7. 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

  8. 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

  9. 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. PMID:26244517

  10. 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

  11. 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.

  12. Contribution of bacterial outer membrane vesicles to innate bacterial defense

    PubMed Central

    2011-01-01

    Background Outer membrane vesicles (OMVs) are constitutively produced by Gram-negative bacteria throughout growth and have proposed roles in virulence, inflammation, and the response to envelope stress. Here we investigate outer membrane vesiculation as a bacterial mechanism for immediate short-term protection against outer membrane acting stressors. Antimicrobial peptides as well as bacteriophage were used to examine the effectiveness of OMV protection. Results We found that a hyper-vesiculating mutant of Escherichia coli survived treatment by antimicrobial peptides (AMPs) polymyxin B and colistin better than the wild-type. Supplementation of E. coli cultures with purified outer membrane vesicles provided substantial protection against AMPs, and AMPs significantly induced vesiculation. Vesicle-mediated protection and induction of vesiculation were also observed for a human pathogen, enterotoxigenic E. coli (ETEC), challenged with polymyxin B. When ETEC with was incubated with low concentrations of vesicles concomitant with polymyxin B treatment, bacterial survival increased immediately, and the culture gained resistance to polymyxin B. By contrast, high levels of vesicles also provided immediate protection but prevented acquisition of resistance. Co-incubation of T4 bacteriophage and OMVs showed fast, irreversible binding. The efficiency of T4 infection was significantly reduced by the formation of complexes with the OMVs. Conclusions These data reveal a role for OMVs in contributing to innate bacterial defense by adsorption of antimicrobial peptides and bacteriophage. Given the increase in vesiculation in response to the antimicrobial peptides, and loss in efficiency of infection with the T4-OMV complex, we conclude that OMV production may be an important factor in neutralizing environmental agents that target the outer membrane of Gram-negative bacteria. PMID:22133164

  13. Insertion of isolated insulin receptors into placental membrane vesicles.

    PubMed Central

    Christiansen, K; Carlsen, J

    1992-01-01

    Purified human insulin receptors were inserted into placental plasma-membrane vesicles by fusion of membranes with receptor-lysophosphatidylcholine micelles. Scatchard analysis of insulin binding showed that about 10-15% of the added receptors became inserted into the membrane. The receptor number could be increased about 3-fold, corresponding to approx. 5 pmol of receptor/mg of membrane protein. The receptors became firmly bound to the membrane, as they could not be removed by extensive wash. The insertion of exogenous receptors could be demonstrated by immunoblotting. The inserted insulin receptor had the same insulin-binding affinity as the isolated receptor and the endogenous receptor of the membrane. Insulin binding in the presence or absence of Triton X-100 revealed that more than 80% of the exogenous receptors had a right-side-out orientation. Function of the inserted receptors, as observed by insulin-stimulated autophosphorylation, could be demonstrated. About 80% of the added lysophospholipid, corresponding to approx. 160 nmol of lysophospholipid/mg of membrane protein, became integrated into the membrane and was partly metabolized to phospholipid and to non-esterified fatty acid. The method of insertion of isolated insulin receptors using the natural detergent, lysophospholipid, may be a method for insertion of receptors into intact cells, where the lysophospholipid, as in the plasma-membrane vesicles, will be acylated to phospholipid. Images Fig. 3. Fig. 4. PMID:1736892

  14. Role of basolateral cell membranes in organic solute reabsorption in rabbit kidneys.

    PubMed

    Foulkes, E C

    1987-06-01

    The present work explores the contributions of basolateral carrier systems in tubular reabsorption of organic solutes. Reabsorption of sugars and amino acids, as previously shown, can be represented by a three-compartment linear model that predicts that 1) if basolateral transport contributes to sugar reabsorption, alpha-methylglucoside reabsorption compared with that of glucose should be characterized by a longer transepithelial transit time (TET) and a correspondingly increased cellular transport pool (S), and 2) saturation of basolateral amino acid carriers, or presence of competing amino acids or other basolateral transport inhibitors, should prolong TET of a test amino acid, and increase S above the expected value. Both predictions were fully confirmed. Heavy metal intoxication not only inhibits transport of amino acids at the brush border, but also prolongs their TET and increases the size of S for a given reabsorbed load. Basolateral extrusion of amino acids is more sensitive to metals than is uptake across the brush border. Although basolateral carriers accelerate return of reabsorbed solute to blood, their contribution to reabsorption does not seem to be mandatory. PMID:3591952

  15. Giant vesicles functionally expressing membrane receptors for an insect pheromone.

    PubMed

    Hamada, Satoshi; Tabuchi, Masashi; Toyota, Taro; Sakurai, Takeshi; Hosoi, Tomohiro; Nomoto, Tomonori; Nakatani, Kei; Fujinami, Masanori; Kanzaki, Ryohei

    2014-03-18

    To date, biochemical approaches to membrane receptors have been limited to the following methods: knockout or overexpression of membrane receptors by gene introduction and genome engineering or extraction of membrane receptor-surfactant complexes from innate cells and their introduction into model biomembranes. Here, we describe the development of a third method involving gene expression using cell-free in situ protein synthesis inside model biomembrane capsules. We verified this method by synthesizing olfactory receptors from the silkmoth Bombyx mori inside giant vesicles and found that they were excited in the presence of their ligand the Bombyx mori sex pheromone. PMID:24509495

  16. 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

  17. Preparation of right-side-out, acetylcholine receptor enriched intact vesicles from Torpedo californica electroplaque membranes.

    PubMed

    Hartig, P R; Raftery, M A

    1979-04-01

    Intact vesicles enriched in acetylcholine receptor from Torpedo californica electroplaque membranes can be separated from collapsed or leaky vesicles and membrane sheets on sucrose density gradients. alpha-Bungarotoxin binding in intact vesicles reveals that approximately 95% of the acetylcholine receptor containing vesicles are formed outside-out (with the synaptic membrane face exposed on the vesicle exterior). The binding data also indicated that only 5% or less of the sites for alpha-bungarotoxin binding to synaptic membranes are located on the interior, cytoplasmic face. Intact vesicles are stable to gentle pelleting and resuspension but are easily osmotically shocked. The vesicles are impermeable to sucrose and Ficoll, but glycerol readily transverses to membrane barrier. Intact vesicles provide a sealed, oriented membrane preparation for studies of vectorial acetylcholine receptor mediated processes. PMID:427105

  18. 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. PMID:25909915

  19. A mini Cl- channel sensitive to external pH in the basolateral membrane of guinea-pig parietal cells.

    PubMed Central

    Kajita, H; Morishima, S; Shirakata, Y; Kotera, T; Ueda, S; Okuma, M; Okada, Y

    1995-01-01

    1. Voltage-independent whole-cell Cl- currents were recorded from both single, isolated parietal cells and parietal cells within gastric glands obtained from the fundus of guinea-pig stomach. 2. The Cl- currents were rapidly suppressed by a Cl- channel blocker, NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate), added to the (basolateral) bathing solution in a concentration-dependent manner with a half-maximal inhibition concentration of 12 microM. 3. The selectivity sequence among anions was I- > Br- > Cl- > F-, corresponding to Eisenman's sequence I. 4. The Cl- currents were independent of cytosolic Ca2+, cyclic AMP, cyclic GMP, GTP-gamma-S and cell volume, and were not affected by application of acid secretagogues, omeprazol, arachidonic acid or prostaglandin E2. 5. Reduction of pH in the (basolateral) bathing solution immediately inhibited the Cl- current with a pK (-log of KD) of 6.3, whereas changes in intracellular pH had no effect. 6. The single-channel conductance was estimated to be 0.46-0.6 pS by variance noise analysis during inhibition of whole-cell Cl- currents by NPPB or acidic pH. 7. It is concluded that pH-sensitive 'mini' Cl- channels, with a sub-picosiemens unitary conductance, exist in the basolateral membrane of guinea-pig parietal cells. PMID:8568665

  20. Lipid Bilayer Membrane-Triggered Presynaptic Vesicle Assembly

    PubMed Central

    2009-01-01

    The formation of functional synapses on artificial substrates is a very important step in the development of engineered in vitro neural networks. Spherical supported bilayer lipid membranes (SS-BLMs) are used here as a novel substrate to demonstrate presynaptic vesicle accumulation at an in vitro synaptic junction. Confocal fluorescence microscopy, cryo-transmission electron microscopy (cryo-TEM), and fluorescence recovery after photobleaching (FRAP) experiments have been used to characterize the SS-BLMs. Conventional immunocytochemistry combined with confocal fluorescence microscopy was used to observe the formation of presynaptic vesicles at the neuron−SS-BLM contacts. These results indicate that lipid phases may play a role in the observed phenomenon, in addition to the chemical and electrostatic interactions between the neurons and SS-BLMs. The biocompatibility of lipid bilayers along with their membrane tunability makes the suggested approach a useful “toolkit” for many neuroengineering applications including artificial synapse formation and synaptogenesis in vivo. PMID:22778819

  1. 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

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

    PubMed

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

    1983-10-01

    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

  3. 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

  4. 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.

  5. KCNJ10 (Kir4.1) is expressed in the basolateral membrane of the cortical thick ascending limb.

    PubMed

    Zhang, Chengbiao; Wang, Lijun; Su, Xiao-Tong; Lin, Dao-Hong; Wang, Wen-Hui

    2015-06-01

    The aim of the present study is to examine the role of Kcnj10 (Kir.4.1) in contributing to the basolateral K conductance in the cortical thick ascending limb (cTAL) using Kcnj10(+/+) wild-type (WT) and Kcnj10(-/-) knockout (KO) mice. The patch-clamp experiments detected a 40- and an 80-pS K channel in the basolateral membrane of the cTAL. Moreover, the probability of finding the 40-pS K was significantly higher in the late part of the cTAL close to the distal convoluted tubule than those in the initial part. Immunostaining showed that Kcnj10 staining was detected in the basolateral membrane of the cTAL but the expression was not uniformly distributed. The disruption of Kcnj10 completely eliminated the 40-pS K channel but not the 80-pS K channel, suggesting the role of Kcnj10 in forming the 40-pS K channel of the cTAL. Also, the disruption of Kcnj10 increased the probability of finding the 80-pS K channel in the cTAL, especially in the late part of the cTAL. Because the channel open probability of the 80-pS K channel in KO was similar to those of WT mice, the increase in the 80-pS K channel may be achieved by increasing K channel number. The whole cell recording further showed that K reversal potential measured with 5 mM K in the bath and 140 mM K in the pipette was the same in the WT and KO mice. Moreover, Western blot and immunostaining showed that the disruption of Kcnj10 did not affect the expression of Na-K-Cl cotransporter 2 (NKCC2). We conclude that Kir.4.1 is expressed in the basolateral membrane of cTAL and that the disruption of Kir.4.1 has no significant effect on the membrane potential of the cTAL and NKCC2 expression. PMID:25834074

  6. 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.

  7. 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

  8. 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

  9. Binding of E. coli heat-stable enterotoxin to rat intestinal brush borders and to basolateral membranes

    SciTech Connect

    Guarino, A.; Cohen, M.B.; Overmann, G.; Thompson, M.R.; Giannella, R.A.

    1987-09-01

    We studied the binding of E. coli heat-stable enterotoxin (STa) to rat brush borders (BB) and to basolateral membranes (BLM) using a biologically active monoiodinated radioligand (( /sup 125/I)STa) and highly enriched BB and BLM preparations free of other significant organelle contamination. Binding of (/sup 125/I)STa to BB was specific; time-, temperature-, and pH-dependent; saturable; and partially reversible. Nonlabeled toxin competitively inhibited the binding of radioligand to BB in a dose-related manner. Scatchard analysis revealed a single class of receptors with an apparent affinity constant of 8.7 +/- 1.5 X 10(8) l/mol. Binding was not affected by amino acids, sugars, and lectins. Proteolytic enzymes significantly decreased binding, although several did so by modifying the radioligand. Trypsin inhibited binding without modifying the radioligand thus supporting the proteinaceous nature of the receptor. Since the enrichment in binding activity in the BB over the homogenate was significantly lower than the enrichment in sucrase activity, we concluded that binding activity is probably associated with other membranous domains, but direct examination revealed no binding activity on basolateral membranes.

  10. Characterization of Water Channels in Wheat Root Membrane Vesicles.

    PubMed Central

    Niemietz, C. M.; Tyerman, S. D.

    1997-01-01

    The functional significance of water channels in wheat (Triticum aestivum L.) root membranes was assessed using light scattering to measure vesicle shrinking in response to osmotic gradients rapidly imposed in a stopped flow apparatus. Vesicles were obtained from both a plasma membrane fraction and a plasma membrane-depleted endomembrane fraction including tonoplast vesicles. Osmotic water permeability (Pos) in the endomembrane fraction was high (Pos= 86.0 [mu]m s-1) with a low activation energy (EA= 23.32 kJ mol-1 [plus or minus] 3.88 SE), and was inhibited by mercurials (K1= 40 [mu]M HgCl2, where K1 is the inhibition constant for half-maximal inhibition), suggesting participation of water channels. A high ratio of osmotic to diffusional permeability (Pd) (using D2O as a tracer, Pos/Pd = 7 [plus or minus] 0.5 SE) also supported this view. For the endomembrane fraction there was a marked decrease in Pos with increasing osmotic gradient that was not observed in the plasma membrane fraction. Osmotic water permeability in the plasma membrane fraction was lower (Pos= 12.5 [mu]m s-1) with a high activation energy (EA= 48.07 kJ mol-1 [plus or minus] 3.63 SE) and no mercury inhibition. Nevertheless, Pos/Pd was found to be substantially higher than one (Pos= 3 [plus or minus] 0.2 SE), indicating that water channels mediated water flow in this fraction, too. Possible distortion of the Pos/Pd value by unstirred layer effects was shown to be unlikely. PMID:12223824

  11. Use of inside-out chloroplast thylakoid membrane vesicles for studying electron transport and membrane structure

    SciTech Connect

    Atta-Asafo-Adjei, E.

    1987-01-01

    Inside-out and right-side-out thylakoid vesicles were isolated from spinach chloroplasts by aqueous-polymer two-phase partitioning following mechanical fragmentation of thylakoid membranes by Yeda press treatment. Externally added plastocyanin stimulated the whole-chain and PSI electron transport rates in the inside-out thylakoid vesicles by about 500 and 350%, respectively, compared to about 50% stimulation for both assays in the fraction enriched in right-side-out vesicles. The electron transport between PSII and PSI in inside-out thylakoid vesicles appears to be interrupted due to plastocyanin release from the thylakoids by the Yeda press treatment, but it was restored by externally added plastocyanin. Acetic anhydride chemical modification and uncoupler-induced proton release from dark-adapted membranes are probes for detecting the sequested proton domains in thylakoid membranes. Both assays were used to find out if inside-out membranes retain metastable, localized proton binding domains. Treatment of dark-maintained inside-out thylakoid membrane vesicles with ({sup 3}H)acetic anhydride showed no uncoupler-induced increase in acetylation of the 33, 24, and 18 kDa polypeptides of the oxygen-evolving-complex, indicating complete loss of the implicated proton domains in these polypeptides. The various steps in the inside-out preparation were studied to discern which steps(s) leads to the loss of the metastable domain proton pool.

  12. 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

  13. 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...

  14. 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...

  15. 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

  16. 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. PMID:26684926

  17. Identification of Outer Membrane Vesicles Derived from Orientia tsutsugamushi

    PubMed Central

    Lee, Sun-Myoung; Kwon, Hea Yoon; Im, Jae-Hyong; Baek, Ji Hyeon; Kang, Jae-Seung

    2015-01-01

    Orientia tsutsugamushi, a causative pathogen of Scrub typhus, is a gram-negative intracellular bacterium. Outer membrane vesicles (OMVs) are produced from the membrane of bacteria and play many roles related to the survival of the pathogen. However, there have been no reports confirming whether O. tsutsugamushi indeed produce OMVs. O. tsutsugamushi boryong was cultured in ECV-304 cells for the purification of OMVs. Western blot analysis and immunoenrichment using anti-O. tsutsugamushi monoclonal antibody and electron microscopy were employed for identification and characterization of OMVs. We confirm the presence of OMVs derived from O. tsutsugamushi, and also found that those OMVs contain a major surface antigen of 56-kDa protein and variant immunogenic antigens. PMID:26130947

  18. 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

  19. Ciprofloxacin encapsulation into giant unilamellar vesicles: membrane binding and release.

    PubMed

    Kaszás, Nóra; Bozó, Tamás; Budai, Marianna; Gróf, Pál

    2013-02-01

    This study aimed at investigating some respects of binding and interaction between water-soluble drugs and liposomal carrier systems depending on their size and lamellarity. As model substance, ciprofloxacin hydrochloride (CPFX) was incorporated into giant unilamellar vesicles (GUVs) to study their CPFX encapsulation/binding capacity. To characterize molecular interactions of various CPFX microspecies with lipid bilayer, zeta potential and electron paramagnetic resonance (EPR) spectroscopy measurements were performed. The increase of the zeta potential at pH 5.4 but no change at pH 7.2 was interpreted in terms of the CPFX microspecies' distribution at the two pH values. EPR observations showed an increased fluidity because of CPFX binding to GUVs. We worked out and applied a three-compartment dialysis model to separately determine the rate of drug diffusion through the liposomal membrane. Equilibrium dialysis showed (a) different permeation of CPFX through the membranes of GUVs and multilamellar vesicles (MLVs), with characteristic half-lives of 54.4 and 18.1 h, respectively; and (b) increased retention of CPFX in case of GUVs with released amounts of 70% compared with about 97% in case of MLVs. Our results may provide further details for efficient design of liposomal formulations incorporating water-soluble drugs. PMID:23233199

  20. Continuous Microfluidic Fabrication of Synthetic Asymmetric Vesicles for Membrane Biology Studies

    NASA Astrophysics Data System (ADS)

    Lu, Li; Schertzer, Jeffrey; Chiarot, Paul

    2015-11-01

    Membrane vesicles are spherical structures comprised of a single lipid bilayer enclosing an aqueous lumen. In nature, vesicles carry out many important functions in both eukaryotic and prokaryotic organisms. When preparing vesicles artificially, it is difficult to simultaneously control vesicle membrane asymmetry, size, unilamellarity, throughput, and monodispersity. Membrane asymmetry, where each leaflet of the lipid bilayer consists of a different lipid distribution, is of particular importance as it is a feature of nearly all natural membranes. In this study, we report on a novel microfluidic strategy to build monodisperse asymmetric vesicles with customized membrane composition, size, and luminal content at high-throughput. The microfluidic device consists of a triangular post region and two flow-focusing regions. The major steps of the vesicle fabrication process include: (1) assembly of the inner-leaflet, (2) continuous flow separation - replacing the inner-leaflet-lipid with the outer-leaflet-lipid, (3) assembly of the outer-leaflet, and (4) extraction of the intermediate oil layer. Membrane asymmetry and unilamellarity are confirmed using a fluorescence quenching assay and a membrane protein insertion assay, respectively. Our vesicle fabrication method can yield membrane asymmetries as high as 95%, which is maintained at a high-degree for over 30 hours. In addition, over 80% of the vesicles remain stable for at least 6 weeks. The effect of bilayer composition on the mechanical properties of the membrane and the role of small molecules on membrane architecture will be investigated.

  1. 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

  2. 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

  3. Parameters affecting the fusion of unilamellar phospholipid vesicles with planar bilayer membranes.

    PubMed

    Cohen, F S; Akabas, M H; Zimmerberg, J; Finkelstein, A

    1984-03-01

    It was previously shown (Cohen, F. S., J. Zimmerberg, and A. Finkelstein, 1980, J. Gen. Physiol., 75:251-270) that multilamellar phospholipid vesicles can fuse with decane-containing phospholipid bilayer membranes. An essential requirement for fusion was an osmotic gradient across the planar membrane, with the vesicle-containing (cis) side hyperosmotic with respect to the opposite (trans) side. We now report that unilamellar vesicles will fuse with "hydrocarbon-free" membranes subject to these same osmotic conditions. Thus the same conditions that apply to fusion of multilamellar vesicles with planar bilayer membranes also apply to fusion of unilamellar vesicles with these membranes, and hydrocarbon is not required for the fusion process. If the vesicles and/or planar membrane contain negatively charged lipids, divalent cation (approximately 15 mM Ca++) is required in the cis compartment (in addition to the osmotic gradient across the membrane) to obtain substantial fusion rates. On the other hand, vesicles made from uncharged lipids readily fuse with planar phosphatidylethanolamine planar membranes in the near absence of divalent cation with just an osmotic gradient. Vesicles fuse much more readily with phosphatidylethanolamine-containing than with phosphatidylcholine-containing planar membranes. Although hydrocarbon (decane) is not required in the planar membrane for fusion, it does affect the rate of fusion and causes the fusion process to be dependent on stirring in the cis compartment. PMID:6699081

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. Detection of outer membrane vesicles in Synechocystis PCC 6803.

    PubMed

    Pardo, Yehudah A; Florez, Catalina; Baker, Kristopher M; Schertzer, Jeffrey W; Mahler, Gretchen J

    2015-10-01

    It has been well established that many species of Gram-negative bacteria release nanoscale outer membrane vesicles (OMVs) during normal growth. Furthermore, the roles of these structures in heterotrophic bacteria have been extensively characterized. However, little is known about the existence or function of OMVs in photoautotrophs. In the present study, we report for the first time the production of OMVs by the model photosynthetic organism Synechocystis sp. PCC 6803, a species of biotechnological importance. We detected extracellular proteins and lipids in cell-free supernatants derived from Synechocystis culture, yet the cytoplasmic and thylakoid membrane markers NADH oxidase and chlorophyll were absent. This indicated that the extracellular proteins and lipids derived from the outer membrane, and not from cell lysis. Furthermore, we identified spherical structures within the expected size range of OMVs in Synechocystis culture using scanning electron microscopy. Taken together, these results suggest that the repertoire of Gram-negative bacteria that are known to produce OMVs may be expanded to include Synechocystis PCC6803. Because of the considerable genetic characterization of Synechocystis in particular, our discovery has the potential to support novel biotechnological applications as well. PMID:26363014

  10. Purification of outer membrane vesicles from Pseudomonas aeruginosa and their activation of an IL-8 response

    PubMed Central

    Bauman, Susanne J.; Kuehn, Meta J.

    2012-01-01

    Considerable lung injury results from the inflammatory response to Pseudomonas aeruginosa infections in patients with cystic fibrosis (CF). The P. aeruginosa laboratory strain PAO1, an environmental isolate, and isolates from CF patients were cultured in vitro and outer membrane vesicles from those cultures were quantitated, purified, and characterized. Vesicles were produced throughout the growth phases of the culture and vesicle yield was strain-independent. Strain-dependent differences in the protein composition of vesicles were quantitated and identified. The aminopeptidase PaAP (PA2939) was highly enriched in vesicles from CF isolates. Vesicles from all strains elicited IL-8 secretion by lung epithelial cells. These results suggest that P. aeruginosa colonizing the CF lung may produce vesicles with a particular composition and that the vesicles could contribute to inflammation. PMID:16807039

  11. Interaction of chloride and bicarbonate transport across the basolateral membrane of rabbit proximal straight tubule. Evidence for sodium coupled chloride/bicarbonate exchange.

    PubMed Central

    Sasaki, S; Yoshiyama, N

    1988-01-01

    The existence of chloride/bicarbonate exchange across the basolateral membrane and its physiologic significance were examined in rabbit proximal tubules. S2 segments of the proximal straight tubule were perfused in vitro and changes in intracellular pH (pHi) and chloride activity (aCli) were monitored by double-barreled microelectrodes. Total peritubular chloride replacement with gluconate increased pHi by 0.8, and this change was inhibited by a pretreatment with an anion transport inhibitor, SITS. Peritubular bicarbonate reduction increased aCli, and most of this increase was lost when ambient sodium was totally removed. The reduction rates of pHi induced by a peritubular bicarbonate reduction or sodium removal were attenuated by 20% by withdrawal of ambient chloride. SITS application to the bath in the control condition quickly increased pHi, but did not change aCli. However, the aCli slightly decreased in response to SITS when the basolateral bicarbonate efflux was increased by reducing peritubular bicarbonate concentration. It is concluded that sodium coupled chloride/bicarbonate exchange is present in parallel with sodium-bicarbonate cotransport in the basolateral membrane of the rabbit proximal tubule, and it contributes to the basolateral bicarbonate and chloride transport. PMID:2450891

  12. Immunogenicity of Pasteurella multocida and Mannheimia haemolytica outer membrane vesicles

    PubMed Central

    Roier, Sandro; Fenninger, Judith C.; Leitner, Deborah R.; Rechberger, Gerald N.; Reidl, Joachim; Schild, Stefan

    2013-01-01

    Pasteurella multocida is able to cause disease in humans and in a wide range of animal hosts, including fowl cholera in birds, atrophic rhinitis in pigs, and snuffles in rabbits. Together with Mannheimia haemolytica, P. multocida also represents a major bacterial causative agent of bovine respiratory disease (BRD), which is one of the most important causes for economic losses for the cattle backgrounding and feedlot industry. Commercially available vaccines only partially prevent infections caused by P. multocida and M. haemolytica. Thus, this study characterized the immunogenicity of P. multocida and M. haemolytica outer membrane vesicles (OMVs) upon intranasal immunization of BALB/c mice. Enzyme-linked immunosorbent assays (ELISA) revealed that OMVs derived from P. multocida or M. haemolytica are able to induce robust humoral and mucosal immune responses against the respective donor strain. In addition, also significant cross-immunogenic potential was observed for both OMV types. Colonization studies showed that a potential protective immune response against P. multocida is not only achieved by immunization with P. multocida OMVs, but also by immunization with OMVs derived from M. haemolytica. Immunoblot and immunoprecipitation analyses demonstrated that M. haemolytica OMVs induce a more complex immune response compared to P. multocida OMVs. The outer membrane proteins OmpA, OmpH, and P6 were identified as the three major immunogenic proteins of P. multocida OMVs. Amongst others, the serotype 1-specific antigen, an uncharacterized outer membrane protein, as well as the outer membrane proteins P2 and OmpA were found to be the most important antigens of M. haemolytica OMVs. These findings are useful for the future development of broad-spectrum OMV based vaccines against BRD and other infections caused by P. multocida or M. haemolytica. PMID:23731905

  13. 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

  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. Complexin-1 Enhances the On-Rate of Vesicle Docking via Simultaneous SNARE and Membrane Interactions

    PubMed Central

    2013-01-01

    In synaptic terminals, complexin is thought to have inhibitory and activating roles for spontaneous “mini” release and evoked synchronized neurotransmitter release, respectively. We used single vesicle–vesicle microscopy imaging to study the effect of complexin-1 on the on-rate of docking between vesicles that mimic synaptic vesicles and the plasma membrane. We found that complexin-1 enhances the on-rate of docking of synaptic vesicle mimics containing full-length synaptobrevin-2 and full-length synaptotagmin-1 to plasma membrane-mimicking vesicles containing full-length syntaxin-1A and SNAP-25A. This effect requires the C-terminal domain of complexin-1, which binds to the membrane, the presence of PS in the membrane, and the core region of complexin-1, which binds to the SNARE complex. PMID:24083833

  16. 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

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

    PubMed Central

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

    1996-01-01

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

  18. Membrane carbonic anhydrase (IV) and ciliary epithelium. Carbonic anhydrase activity is present in the basolateral membranes of the non-pigmented ciliary epithelium of rabbit eyes.

    PubMed

    Matsui, H; Murakami, M; Wynns, G C; Conroy, C W; Mead, A; Maren, T H; Sears, M L

    1996-04-01

    Carbonic anhydrase inhibitors (CAIs) lower intraocular pressure by reducing aqueous flow. It has been thought that this pharmacologic reduction of aqueous flow is mediated by the ciliary epithelium, but it is not known whether this cellular action is effected by inhibition of the membranal (CA IV) and/or cytosolic (CA II) carbonic anhydrases of the ciliary epithelium. The isolated ciliary epithelial bilayer maintains its anatomic and functional polarity and generates a transepithelial potential difference (TEP) in an Ussing type chamber. Depletion of HCO3-, accomplished either with an HCO3(-)-free solution bathing the epithelial bilayer, or, with addition of freely permeant CAIs to HCO3(-)-containing media, (from either the PE or NPE side of the bilayer) depolarizes the preparation. Addition of CAIs to an HCO3(-)-depleted preparation has no further effect, indicating the specific action of the CAIs. The CAI, 2-p-NH2 benzenesulfonamido-1,3,4,-thiadiazole-5-SO2NH2, linked to polybutadiene maleic acid yields an impermeant polymer of 20000 Da with no loss of activity. At 45 microM this impermeant polymer caused a 60% increase in the SCC, seen only when the compound was applied to the NPE side of the bilayer. This latter result indicates an effect from inhibition of CA IV in the basolateral membranes of the NPE. Thus there are probably two different cellular actions of CAIs upon the ciliary epithelium to reduce aqueous inflow, cytoplasmic and membranal. The action of NPE basolateral membranal CA IV is probably linked to the chloride/bicarbonate exchanger. PMID:8795459

  19. Membrane stress causes inhibition of water channels in brush border membrane vesicles from kidney proximal tubule.

    PubMed

    Soveral, G; Macey, R I; Moura, T F

    1997-08-01

    Brush border membrane vesicles (BBMV) from rabbit kidney proximal tubule cells, prepared with different internal solute concentrations (cellobiose buffer 13, 18 or 85 mosM) developed an hydrostatic pressure difference across the membrane of 18.7 mosM, that causes a membrane tension close to 5 x 10(-5) N cm-1. When subjected to several hypertonic osmotic shocks an initial delay of osmotic shrinkage (a lag time), corresponding to a very small change in initial volume was apparent. This initial osmotic response, which is significantly retarded, was correlated with the initial period of elevated membrane tension, suggesting that the water permeability coefficient is inhibited by membrane stress. We speculate that this inhibition may serve to regulate cell volume in the proximal tubule. PMID:9468597

  20. 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

  1. 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

  2. Vesicles

    MedlinePlus

    ... pox Contact dermatitis (may be caused by poison ivy) Herpes simplex (cold sores, genital herpes ) Herpes zoster ( ... for certain conditions that cause vesicles, including poison ivy and cold sores.

  3. 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

  4. Bestrophin, the product of the Best vitelliform macular dystrophy gene (VMD2), localizes to the basolateral plasma membrane of the retinal pigment epithelium

    PubMed Central

    Marmorstein, Alan D.; Marmorstein, Lihua Y.; Rayborn, Mary; Wang, Xinxing; Hollyfield, Joe G.; Petrukhin, Konstantin

    2000-01-01

    Best vitelliform macular dystrophy is a dominantly inherited, early onset, macular degenerative disease that exhibits some histopathologic similarities to age-related macular degeneration. Although the vitelliform lesion is common in the fundus of individuals with Best disease, diagnosis is based on a reduced ratio of the light peak to dark trough in the electrooculogram. Recently, the VMD2 gene on chromosome 11q13, encoding the protein bestrophin, was identified. The function of bestrophin is unknown. To facilitate studies of bestrophin, we produced both rabbit polyclonal and mouse monoclonal antibodies that proved useful for Western blotting, immunoprecipitation, and immunocytochemistry. To characterize bestrophin, we initially probed the retinal pigment epithelium (RPE)-derived cell lines ARPE-19, D407, and RPE-J. All of the cell lines expressed bestrophin mRNA by reverse transcription-PCR, but not on Western blots. Bestrophin in human RPE partitioned in the detergent phase during Triton X-114 extraction and could be modified by biotin in intact cells, indicative of a plasma membrane localization. Immunocytochemical staining of macaque and porcine eyes indicated that bestrophin is localized at the basolateral plasma membrane of RPE cells. When expressed in RPE-J cells by adenovirus-mediated gene transfer, bestrophin again was determined by confocal microscopy and cell surface biotinylation to be a basolateral plasma membrane protein. The basolateral plasma membrane localization of bestrophin suggests the possibility that bestrophin plays a role in generating the altered electrooculogram of individuals with Best disease. PMID:11050159

  5. Bestrophin, the product of the Best vitelliform macular dystrophy gene (VMD2), localizes to the basolateral plasma membrane of the retinal pigment epithelium.

    PubMed

    Marmorstein, A D; Marmorstein, L Y; Rayborn, M; Wang, X; Hollyfield, J G; Petrukhin, K

    2000-11-01

    Best vitelliform macular dystrophy is a dominantly inherited, early onset, macular degenerative disease that exhibits some histopathologic similarities to age-related macular degeneration. Although the vitelliform lesion is common in the fundus of individuals with Best disease, diagnosis is based on a reduced ratio of the light peak to dark trough in the electrooculogram. Recently, the VMD2 gene on chromosome 11q13, encoding the protein bestrophin, was identified. The function of bestrophin is unknown. To facilitate studies of bestrophin, we produced both rabbit polyclonal and mouse monoclonal antibodies that proved useful for Western blotting, immunoprecipitation, and immunocytochemistry. To characterize bestrophin, we initially probed the retinal pigment epithelium (RPE)-derived cell lines ARPE-19, D407, and RPE-J. All of the cell lines expressed bestrophin mRNA by reverse transcription-PCR, but not on Western blots. Bestrophin in human RPE partitioned in the detergent phase during Triton X-114 extraction and could be modified by biotin in intact cells, indicative of a plasma membrane localization. Immunocytochemical staining of macaque and porcine eyes indicated that bestrophin is localized at the basolateral plasma membrane of RPE cells. When expressed in RPE-J cells by adenovirus-mediated gene transfer, bestrophin again was determined by confocal microscopy and cell surface biotinylation to be a basolateral plasma membrane protein. The basolateral plasma membrane localization of bestrophin suggests the possibility that bestrophin plays a role in generating the altered electrooculogram of individuals with Best disease. PMID:11050159

  6. 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.

  7. Cell-cell communication via extracellular membrane vesicles and its role in the immune response.

    PubMed

    Hwang, Inkyu

    2013-08-01

    The host immune response involves a variety of cell types, including specialized immune and non-immune cells. The delicate coordination among these cells via close communication is central for the proper operation of immune system. Cell-cell communication is mediated by a complex network that includes soluble factors such as cytokines, chemokines, and metabolites exported from cells, as well as membrane-bound receptors and their ligands. Cell-cell communication is also mediated by membrane vesicles (e.g., exosomes, ectosomes), which are either shed by distant cells or exchanged by cells that are making direct contact. Intercellular communication via extracellular membrane vesicles has drawn much attention recently, as they have been shown to carry various biomolecules that modulate the activities of recipient cells. In this review, I will discuss current views on cell-cell communication via extra-cellular membrane vesicles, especially shedded membrane vesicles, and their effects on the control of the immune system. PMID:23807045

  8. 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.

  9. Translocation of cationic amphipathic peptides across the membranes of pure phospholipid giant vesicles.

    PubMed

    Wheaten, Sterling A; Ablan, Francis D O; Spaller, B Logan; Trieu, Julie M; Almeida, Paulo F

    2013-11-01

    The ability of amphipathic polypeptides with substantial net positive charges to translocate across lipid membranes is a fundamental problem in physical biochemistry. These peptides should not passively cross the bilayer nonpolar region, but they do. Here we present a method to measure peptide translocation and test it on three representative membrane-active peptides. In samples of giant unilamellar vesicles (GUVs) prepared by electroformation, some GUVs enclose inner vesicles. When these GUVs are added to a peptide solution containing a membrane-impermeant fluorescent dye (carboxyfluorescein), the peptide permeabilizes the outer membrane, and dye enters the outer GUV, which then exhibits green fluorescence. The inner vesicles remain dark if the peptide does not cross the outer membrane. However, if the peptide translocates, it permeabilizes the inner vesicles as well, which then show fluorescence. We also measure translocation, simultaneously on the same GUV, by the appearance of fluorescently labeled peptides on the inner vesicle membranes. All three peptides examined are able to translocate, but to different extents. Peptides with smaller Gibbs energies of insertion into the membrane translocate more easily. Further, translocation and influx occur broadly over the same period, but with very different kinetics. Translocation across the outer membrane follows approximately an exponential rise, with a characteristic time of 10 min. Influx occurs more abruptly. In the outer vesicle, influx happens before most of the translocation. However, some peptides cross the membrane before any influx is observed. In the inner vesicles, influx occurs abruptly sometime during peptide translocation across the membrane of the outer vesicle. PMID:24152283

  10. 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

  11. 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.

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

    PubMed

    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

  13. Proteomic analysis of Vibrio cholerae outer membrane vesicles

    PubMed Central

    Altindis, Emrah; Fu, Yang; Mekalanos, John J.

    2014-01-01

    Outer membrane vesicles (OMVs) produced by Gram-negative bacteria provide an interesting research material for defining cell-envelope proteins without experimental cell disruption. OMVs are also promising immunogenic platforms and may play important roles in bacterial survival and pathogenesis. We used in-solution trypsin digestion coupled to mass spectrometry to identify 90 proteins present in OMVs of Vibrio cholerae when grown under conditions that activate the TCP pilus virulence regulatory protein (ToxT) virulence regulon. The ToxT expression profile and potential contribution to virulence of these proteins were assessed using ToxT and in vivo RNA-seq, Tn-seq, and cholera stool proteomic and other genome-wide data sets. Thirteen OMV-associated proteins appear to be essential for cell growth, and therefore may represent antibacterial drug targets. Another 12 nonessential OMV proteins, including DegP protease, were required for intestinal colonization in rabbits. Comparative proteomics of a degP mutant revealed the importance of DegP in the incorporation of nine proteins into OMVs, including ones involved in biofilm matrix formation and various substrates of the type II secretion system. Taken together, these results suggest that DegP plays an important role in determining the content of OMVs and also affects phenotypes such as intestinal colonization, proper function of the type II secretion system, and formation of biofilm matrix. PMID:24706774

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

    PubMed Central

    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. PMID:25840612

  15. Molecular characterisation of plasma membrane-derived vesicles.

    PubMed

    Antwi-Baffour, Samuel S

    2015-01-01

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

  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. 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

  19. 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

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

    PubMed

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

    2015-07-01

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

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

    SciTech Connect

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

    1987-05-01

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

  3. 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.

  4. 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

  5. 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

  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. Vesicular transport system in myotubes: ultrastructural study and signposting with vesicle-associated membrane proteins.

    PubMed

    Tajika, Yuki; Takahashi, Maiko; Khairani, Astrid Feinisa; Ueno, Hitoshi; Murakami, Tohru; Yorifuji, Hiroshi

    2014-04-01

    Myofibers have characteristic membrane compartments in their cytoplasm and sarcolemma, such as the sarcoplasmic reticulum, T-tubules, neuromuscular junction, and myotendinous junction. Little is known about the vesicular transport that is believed to mediate the development of these membrane compartments. We determined the locations of organelles in differentiating myotubes. Electron microscopic observation of a whole myotube revealed the arrangement of Golgi apparatus, rough endoplasmic reticulum, autolysosomes, mitochondria, and smooth endoplasmic reticulum from the perinuclear region toward the end of myotubes and the existence of a large number of vesicles near the ends of myotubes. Vesicles in myotubes were further characterized using immunofluorescence microscopy to analyze expression and localization of vesicle-associated membrane proteins (VAMPs). VAMPs are a family of seven proteins that regulate post-Golgi vesicular transport via the fusion of vesicles to the target membranes. Myotubes express five VAMPs in total. Vesicles with VAMP2, VAMP3, or VAMP5 were found near the ends of the myotubes. Some of these vesicles are also positive for caveolin-3, suggesting their participation in the development of T-tubules. Our morphological analyses revealed the characteristic arrangement of organelles in myotubes and the existence of transport vesicles near the ends of the myotubes. PMID:24263617

  8. 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

  9. Outer membrane vesicles displaying engineered glycotopes elicit protective antibodies.

    PubMed

    Chen, Linxiao; Valentine, Jenny L; Huang, Chung-Jr; Endicott, Christine E; Moeller, Tyler D; Rasmussen, Jed A; Fletcher, Joshua R; Boll, Joseph M; Rosenthal, Joseph A; Dobruchowska, Justyna; Wang, Zhirui; Heiss, Christian; Azadi, Parastoo; Putnam, David; Trent, M Stephen; Jones, Bradley D; DeLisa, Matthew P

    2016-06-28

    The O-antigen polysaccharide (O-PS) component of lipopolysaccharides on the surface of gram-negative bacteria is both a virulence factor and a B-cell antigen. Antibodies elicited by O-PS often confer protection against infection; therefore, O-PS glycoconjugate vaccines have proven useful against a number of different pathogenic bacteria. However, conventional methods for natural extraction or chemical synthesis of O-PS are technically demanding, inefficient, and expensive. Here, we describe an alternative methodology for producing glycoconjugate vaccines whereby recombinant O-PS biosynthesis is coordinated with vesiculation in laboratory strains of Escherichia coli to yield glycosylated outer membrane vesicles (glycOMVs) decorated with pathogen-mimetic glycotopes. Using this approach, glycOMVs corresponding to eight different pathogenic bacteria were generated. For example, expression of a 17-kb O-PS gene cluster from the highly virulent Francisella tularensis subsp. tularensis (type A) strain Schu S4 in hypervesiculating E. coli cells yielded glycOMVs that displayed F. tularensis O-PS. Immunization of BALB/c mice with glycOMVs elicited significant titers of O-PS-specific serum IgG antibodies as well as vaginal and bronchoalveolar IgA antibodies. Importantly, glycOMVs significantly prolonged survival upon subsequent challenge with F. tularensis Schu S4 and provided complete protection against challenge with two different F. tularensis subsp. holarctica (type B) live vaccine strains, thereby demonstrating the vaccine potential of glycOMVs. Given the ease with which recombinant glycotopes can be expressed on OMVs, the strategy described here could be readily adapted for developing vaccines against many other bacterial pathogens. PMID:27274048

  10. v-SNARE cellubrevin is required for basolateral sorting of AP-1B–dependent cargo in polarized epithelial cells

    PubMed Central

    Fields, Ian C.; Shteyn, Elina; Pypaert, Marc; Proux-Gillardeaux, Véronique; Kang, Richard S.; Galli, Thierry; Fölsch, Heike

    2007-01-01

    The epithelial cell–specific adaptor complex AP-1B is crucial for correct delivery of many transmembrane proteins from recycling endosomes to the basolateral plasma membrane. Subsequently, membrane fusion is dependent on the formation of complexes between SNARE proteins located at the target membrane and on transport vesicles. Although the t-SNARE syntaxin 4 has been localized to the basolateral membrane, the v-SNARE operative in the AP-1B pathway remained unknown. We show that the ubiquitously expressed v-SNARE cellubrevin localizes to the basolateral membrane and to recycling endosomes, where it colocalizes with AP-1B. Furthermore, we demonstrate that cellubrevin coimmunoprecipitates preferentially with syntaxin 4, implicating this v-SNARE in basolateral fusion events. Cleavage of cellubrevin with tetanus neurotoxin (TeNT) results in scattering of AP-1B localization and missorting of AP-1B–dependent cargos, such as transferrin receptor and a truncated low-density lipoprotein receptor, LDLR-CT27. These data suggest that cellubrevin and AP-1B cooperate in basolateral membrane trafficking. PMID:17485489

  11. Leda-1/Pianp is targeted to the basolateral plasma membrane by a distinct intracellular juxtamembrane region and modulates barrier properties and E-Cadherin processing.

    PubMed

    Evdokimov, Konstantin; Biswas, Siladitta; Schledzewski, Kai; Winkler, Manuel; Gorzelanny, Christian; Schneider, Stefan W; Goerdt, Sergij; Géraud, Cyrill

    2016-07-01

    Leda-1/Pianp is a type-I transmembrane protein which is sorted to the basolateral membrane domain of polarized epithelial cells. Here, we investigated trafficking mechanisms and functions of Leda-1/Pianp in MDCK and MCF-7 cells. Basolateral sorting and posttranslational modifications depended on the intracellular juxtamembrane region. Functionally, Leda-1/Pianp increased the transepithelial electrical resistance generated by a polarized cell sheet. Furthermore, resistance to junctional destabilization by tumor cells was enhanced by Leda-1/Pianp indicating increased stability and tightness of intercellular junctions. While Claudin 1 and 4 expression and activities of small GTPases were not affected, γ-Secretase-mediated cleavage of E-Cadherin was attenuated by Leda-1/Pianp. Regulation of proteolytic processing is thus a molecular mechanism by which Leda-1/Pianp can affect junctional integrity and function. PMID:27216462

  12. Insulin and IGF-1 activate Kir4.1/5.1 channels in cortical collecting duct principal cells to control basolateral membrane voltage.

    PubMed

    Zaika, Oleg; Palygin, Oleg; Tomilin, Viktor; Mamenko, Mykola; Staruschenko, Alexander; Pochynyuk, Oleh

    2016-02-15

    Potassium Kir4.1/5.1 channels are abundantly expressed at the basolateral membrane of principal cells in the cortical collecting duct (CCD), where they are thought to modulate transport rates by controlling transepithelial voltage. Insulin and insulin-like growth factor-1 (IGF-1) stimulate apically localized epithelial sodium channels (ENaC) to augment sodium reabsorption in the CCD. However, little is known about their actions on potassium channels localized at the basolateral membrane. In this study, we implemented patch-clamp analysis in freshly isolated murine CCD to assess the effect of these hormones on Kir4.1/5.1 at both single channel and cellular levels. We demonstrated that K(+)-selective conductance via Kir4.1/5.1 is the major contributor to the macroscopic current recorded from the basolateral side in principal cells. Acute treatment with 10 μM amiloride (ENaC blocker), 100 nM tertiapin-Q (TPNQ; ROMK inhibitor), and 100 μM ouabain (Na(+)-K(+)-ATPase blocker) failed to produce a measurable effect on the macroscopic current. In contrast, Kir4.1 inhibitor nortriptyline (100 μM), but not fluoxetine (100 μM), virtually abolished whole cell K(+)-selective conductance. Insulin (100 nM) markedly increased the open probability of Kir4.1/5.1 and nortriptyline-sensitive whole cell current, leading to significant hyperpolarization of the basolateral membrane. Inhibition of the phosphatidylinositol 3-kinase cascade with LY294002 (20 μM) abolished action of insulin on Kir4.1/5.1. IGF-1 had similar stimulatory actions on Kir4.1/5.1-mediated conductance only when applied at a higher (500 nM) concentration and was ineffective at 100 nM. We concluded that both insulin and, to a lesser extent, IGF-1 activate Kir4.1/5.1 channel activity and open probability to hyperpolarize the basolateral membrane, thereby facilitating Na(+) reabsorption in the CCD. PMID:26632606

  13. Characterization of proteins in membrane vesicles from scrapie-infected hamster brain.

    PubMed

    Dees, C; German, T L; Wade, W F; Marsh, R F

    1985-04-01

    Previous studies have shown that the scrapie agent is highly membrane-associated. We examined the protein composition of gradient fractions enriched for large membrane vesicles prepared from scrapie-infected and uninfected hamster brain using various methods to extract membrane proteins. We also examined proteins in detergent-extracted membrane vesicles fractionated on CsCl gradients. No qualitative differences in protein composition were seen comparing scrapie-infected and uninfected samples by one-dimensional gel electrophoresis. Extraction of proteins from membrane vesicles by phenol, pyridine, perchloric acid or lithium diiodosalicylate also failed to reveal any unique proteins in scrapie-infected hamster brain. Attempts to solubilize hydrophobic proteins (proteolipids) from CsCl gradient fractions into organic solvents were unsuccessful. These findings indicate that any hydrophobic protein associated with the scrapie agent is not a proteolipid, and that the ability of solvents to reduce scrapie infectivity is not a result of extraction of a proteolipid. PMID:3920349

  14. Membrane curvature induced by Arf1-GTP is essential for vesicle formation

    PubMed Central

    Beck, Rainer; Sun, Zhe; Adolf, Frank; Rutz, Chistoph; Bassler, Jochen; Wild, Klemens; Sinning, Irmgard; Hurt, Ed; Brügger, Britta; Béthune, Julien; Wieland, Felix

    2008-01-01

    The GTPase Arf1 is considered as a molecular switch that regulates binding and release of coat proteins that polymerize on membranes to form transport vesicles. Here, we show that Arf1-GTP induces positive membrane curvature and find that the small GTPase can dimerize dependent on GTP. Investigating a possible link between Arf dimerization and curvature formation, we isolated an Arf1 mutant that cannot dimerize. Although it was capable of exerting the classical role of Arf1 as a coat receptor, it could not mediate the formation of COPI vesicles from Golgi-membranes and was lethal when expressed in yeast. Strikingly, this mutant was not able to deform membranes, suggesting that GTP-induced dimerization of Arf1 is a critical step inducing membrane curvature during the formation of coated vesicles. PMID:18689681

  15. Retardation of Abeta fibril formation by phospholipid vesicles depends on membrane phase behavior.

    PubMed

    Hellstrand, Erik; Sparr, Emma; Linse, Sara

    2010-05-19

    An increasing amount of evidence suggests that in several amyloid diseases, the fibril formation in vivo and the mechanism of toxicity both involve membrane interactions. We have studied Alzheimer's disease related amyloid beta peptide (Abeta). Recombinant Abeta(M1-40) and Abeta(M1-42) produced in Escherichia coli, allows us to carry out large scale kinetics assays with good statistics. The amyloid formation process is followed in means of thioflavin T fluorescence at relatively low (down to 380 nM) peptide concentration approaching the physiological range. The lipid membranes are introduced in the system as large and small unilamellar vesicles. The aggregation lagtime increases in the presence of lipid vesicles for all situations investigated and the phase behavior of the membrane in the vesicles has a large effect on the aggregation kinetics. By comparing vesicles with different membrane phase behavior we see that the solid gel phase dipalmitoylphosphatidylcholine bilayers cause the largest retardation of Abeta fibril formation. The membrane-induced retardation reaches saturation and is present when the vesicles are added during the lag time up to the nucleation point. No significant difference is detected in lag time when increasing amount of negative charge is incorporated into the membrane. PMID:20483329

  16. Heterogeneous nucleation of calcium oxalate crystals in the presence of membrane vesicles

    NASA Astrophysics Data System (ADS)

    Khan, Saeed R.; Whalen, Patrick O.; Glenton, Patricia A.

    1993-12-01

    Membrane-assisted crystallization of calcium oxalate was studied in vitro, using constant composition methodology. Rat renal tubular brush border membrane vesicles were incubated in supersaturated solution of calcium oxalate. Calcium and oxalate depletion started much earlier in the presence of the vesicles than in their absence; within 8, 32, or 258 min of the incubation of vesicles in calcium oxalate solutions of relative supersaturation of 12, 10 or 6 respectively. Thin plate-like crystals with jagged edges formed in association with the membrane vesicles. Since crystal nucleation in the presence of membrane vesicles started within 8 min at a relative supersaturation as low as 12, it will start significantly earlier in the urine of stone formers which is known to have higher relative supersaturation with respect to calcium oxalate. These results demonstrate that cellular membranes can efficiently induce nucleation of calcium oxalate crystals from a metastable solution in an vitro system. Similar membrane induced heterogeneous nucleation of calcium oxalate in vivo within the renal tubules is a distinct possibility.

  17. Rapid determination of internal volumes of membrane vesicles with electron spin resonance-stopped flow technique.

    PubMed

    Anzai, K; Higashi, K; Kirino, Y

    1988-01-13

    We have developed an electron spin resonance (ESR)-stopped flow technique and employed it for the simple and rapid determination of internal volumes of biomembrane vesicles and liposomes. A vesicle suspension containing a neutral and membrane-permeable spin label, 2,2,6,6-tetramethyl-4-oxopiperidine-1-oxyl (TEMPONE), was mixed in the stopped-flow apparatus with an isotonic solution of relatively impermeable line broadening agents, potassium tris(oxalato)chromate(III) or potassium ferricyanide, and an ESR spectrum was recorded. From the relative intensity of the sharp triplet signal due to TEMPONE in the aqueous space within vesicles, the determination of the internal aqueous volume was straightforward. Using this technique, it is possible to measure intravesicular volumes in 0.1 s. The internal volume of sonicated phospholipid vesicles was approximately 0.3 microliter/mg lipid. The light fraction of sarcoplasmic reticulum membrane vesicles isolated from rabbit skeletal muscle was estimated to have an internal volume of 2.2-2.6 microliter/mg protein in its resting state. Activation of Ca2+ pumps in the membrane upon addition of ATP and Ca2+ ions decreased the internal volume by about 10%. This finding supports the hypothesis that the Ca2+ pump is electrogenic and that the efflux of potassium ions compensates for the influx of positive charges. The present technique is widely applicable to the simple and rapid determination of the internal volumes of membrane vesicles. PMID:2825810

  18. 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.

  19. 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.

  20. 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.

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

    PubMed

    Noguchi, Hiroshi

    2015-12-28

    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. PMID:26723594

  2. The theory of semipermeable vesicles and membranes: An integral-equation approach. III. Vesicles with internal nonpermeating ions

    NASA Astrophysics Data System (ADS)

    Zhou, Yaoqi; Stell, George

    1989-09-01

    The system of a fluid in the presence of a spherical semipermeable vesicle (SPV) with the freely mobile nonpermeating species inside the vesicle is investigated via an integral-equation approach. This system can be used to model certain feature of a biological cell, permeable to simple ions, in which solute proteins inside the cell are unable to permeate its walls. As an illustrative example of the use of our integral equations, the analytical solution for density profiles in the mean-spherical approximation/Debye-Hückel approximation (MSA/DH) is obtained, where the MSA is used to obtain the density profiles near a membrane and the DH approximation to obtain the bulk pair correlation functions. A method which applies to nonmobile protein fixed inside a cell is also considered.

  3. Electron microscopy during release and purification of mesosomal vesicles and protoplast membranes from Staphylococcus aureus.

    PubMed

    Popkin, T J; Theodore, T S; Cole, R M

    1971-09-01

    The mesosomes of log-phase Staphylococcus aureus ATCC 6538P and Staphylococcus aureus phage-type 80/81, as seen in situ in ultrathin sections, were of the vesicular type. The constituent vesicles ranged from 35 to 50 nm in diameter when the glutaraldehyde-osmium-uranium-lead sequence of fixation and staining was used. During protoplasting in hypertonic buffer containing a muralytic enzyme, vesicles of the same size were extruded and required magnesium ion to maintain structural integrity. The vesicles, purified from the protoplasting supernatant medium by density gradient centrifugation, maintained size and configuration in a homogeneous preparation. Cytoplasmic membranes, produced by osmotic shock and nuclease treatment of protoplasts, were similarly concentrated in gradients. However, they were not free of membrane-associated ribosomes nor of mesosomal vesicles except when prepared in the absence of magnesium. PMID:4106221

  4. 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.

  5. 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

  6. Structural Basis for Membrane Binding and Remodeling by the Exomer Secretory Vesicle Cargo Adaptor

    PubMed Central

    Paczkowski, Jon E.; Fromme, J. Christopher

    2014-01-01

    Summary Cargo adaptor subunits of vesicle coat protein complexes sort transmembrane proteins to distinct membrane compartments in eukaryotic cells. The exomer complex is the only cargo adaptor known to sort proteins at the trans-Golgi network into secretory vesicles. Exomer function is regulated by the Arf1 GTPase, a master regulator of trafficking at the Golgi. We report the structure of exomer bound to two copies of Arf1. Exomer interacts with each Arf1 molecule via two surfaces; one is a non-canonical interface that regulates GTP hydrolysis. The structure uncovers an unexpected membrane-proximal hydrophobic element that exomer uses in cooperation with Arf1 to remodel membranes. Given the constrained motion of the exomer hinge region, we envision that exomer dynamically positions multiple membrane insertion elements to drive membrane fission. In contrast to other known cargo adaptors, exomer therefore couples two functions, cargo sorting and membrane fission, into a single complex. PMID:25203211

  7. 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.

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

    PubMed

    Richardson, P J; Luzio, J P

    1980-03-15

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

  9. 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

  10. A synaptic vesicle antigen is restricted to the junctional region of the presynaptic plasma membrane.

    PubMed Central

    Buckley, K M; Schweitzer, E S; Miljanich, G P; Clift-O'Grady, L; Kushner, P D; Reichardt, L F; Kelly, R B

    1983-01-01

    The plasma membrane of electric organ nerve terminals has two domains that can be distinguished by monoclonal antibodies. A library of 111 mouse monoclonal antibodies raised to nerve terminals from Torpedo californica contains 4 antibodies that bind specifically to the outside of intact synaptosomes. The distribution of the binding sites of these monoclonal antibodies on the outside of intact nerve terminals was examined by immunofluorescence and immunoelectron microscopy. The binding sites of 3 (tor23, 25, and 132) are distributed uniformly over nerve trunks and fine terminal branches. The binding site of the fourth (tor70) is restricted to synaptic junctional regions. This antibody, but not the other 3, recognizes a major component of synaptic vesicles, a proteoglycan associated with the inner surface of the vesicle membrane. The difference in the pattern of binding of these monoclonal antibodies suggests that the region of the plasma membrane containing active zones is antigenically distinguishable from other nerve terminal plasma membrane. We suggest that the antigen recognized by tor70 is externalized by exocytosis of synaptic vesicles while other plasma antigens take a different route to the surface. The unexpected observation that the vesicle antigen remains on the surface after exocytosis and is prevented from diffusion from the synaptic junctional region would be consistent with an interaction between the vesicle proteoglycan and elements of the synaptic cleft. Images PMID:6359167

  11. Formation of secretory vesicles in permeabilized cells: a salt extract from yeast membranes promotes budding of nascent secretory vesicles from the trans-Golgi network of endocrine cells.

    PubMed Central

    Ling, W L; Shields, D

    1996-01-01

    The mechanism of secretory-vesicle formation from the trans-Golgi network (TGN) of endocrine cells is poorly understood. To identify cytosolic activities that facilitate the formation and fission of nascent secretory vesicles, we treated permeabilized pituitary GH3 cells with high salt to remove endogenous budding factors. Using this cell preparation, secretory-vesicle budding from the TGN required addition of exogenous cytosol and energy. Mammalian cytosols (GH3 cells and bovine brain) promoted post-TGN vesicle formation. Most significantly, a salt extract of membranes from the yeast Saccharomyces cerevisiae, a cell lacking a regulated secretory pathway, stimulated secretory vesicle budding in the absence of mammalian cytosolic factors. These results demonstrate that the factors which promote secretory-vesicle release from the TGN are conserved between yeast and mammalian cells. PMID:8615761

  12. 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.

  13. Light-induced membrane potential and pH gradient in Halobacterium halobium envelope vesicles.

    PubMed

    Renthal, R; Lanyi, J K

    1976-05-18

    Illumination of envelope vesicles prepared from Halobacterium halobium cells causes translocation of protons from inside to outside, due to the light-induced cycling of bacteriorhodopsin. This process results in a pH gradient across the membranes, an electrical potential, and the movements of K+ and Na+. The electrical potential was estimated by following the fluorescence of a cyanine dye, 3,3'-dipentyloxadicarbocyanine. Illumination of H. halobium vesicles resulted in a rapid, reversible decrease of the dye fluorescence, by as much as 35%. This effect was not seen in nonvesicular patches of purple membrane. Observation of maximal fluorescence decreases upon ilumination of vesicles required an optimal dye/membrane protein ratio. The pH optimum for the lightinduced fluorescence decrease was 6.0. The decrease was linear with actinic light intensity up to about 4 X 10(5) ergs cn-2 s-1. Valinomycin, gramicidin, and triphenylmethylphosphonium ion all abolished the fluorescence changes. However, the light-induced pH change was enhanced by these agents. Conversely, buffered vesicles showed no pH change but gave the same or larger fluorescence changes. Thus, we have identified the fluorescence decrease with a light-induced membrane potential, inside negative. By using valinomycin-K+-induced membrane potentials, we calibrated the fluorescence decrease with calculated Nernst diffusion potentials. We found a linear dependence between potential and fluorescence decrease of 3 mV/%, up to 90 mV. When the envelope vesicles were illuminated, the total proton-motive force generated was dependent on the presence of Na+ and K+ and their concentration gradients across the membrane. In general, K+ appeared to be more permeable than Na+ and, thus, permitted development of greater pH gradients and lower electrical potentials. By calculating the total proton-motive force from the sum of the pH and potential terms, we found that the vesicles can produce proton-motive forces near--200 m

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

    SciTech Connect

    Rengasamy, A.; Feinberg, H.

    1988-02-15

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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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.

  1. Membrane Viscosity Determined from Shear-Driven Flow in Giant Vesicles

    NASA Astrophysics Data System (ADS)

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

    2013-07-01

    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.

  2. 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

  3. 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

  4. Staphylococcus aureus α-Toxin-Dependent Induction of Host Cell Death by Membrane-Derived Vesicles

    PubMed Central

    Thay, Bernard; Wai, Sun Nyunt; Oscarsson, Jan

    2013-01-01

    Staphylococcus aureus causes a wide spectrum of infections in humans, ranging from superficial cutaneous infections, infections in the circum-oral region, to life-threatening bacteremia. It was recently demonstrated that Gram-positive organisms such as S. aureus liberate membrane-derived vesicles (MVs), which analogously to outer membrane vesicles (OMVs) of Gram-negative bacteria can play a role in delivering virulence factors to host cells. In the present study we have shown that cholesterol-dependent fusion of S. aureus MVs with the plasma membrane represents a route for delivery of a key virulence factor, α-toxin (α-hemolysin; Hla) to human cells. Most S. aureus strains produce this 33-kDa pore-forming protein, which can lyse a wide range of human cells, and induce apoptosis in T-lymphocytes. Our results revealed a tight association of biologically active α-toxin with membrane-derived vesicles isolated from S. aureus strain 8325-4. Concomitantly, α-toxin contributed to HeLa cell cytotoxicity of MVs, and was the main vesicle-associated protein responsible for erythrocyte lysis. In contrast, MVs obtained from an isogenic hla mutant were significantly attenuated with regards to both causing lysis of erythrocytes and death of HeLa cells. This is to our knowledge the first recognition of an S. aureus MV-associated factor contributing to host cell cytotoxicity. PMID:23382935

  5. The Enolase of Borrelia burgdorferi Is a Plasminogen Receptor Released in Outer Membrane Vesicles

    PubMed Central

    Toledo, A.; Coleman, J. L.; Kuhlow, C. J.; Crowley, J. T.

    2012-01-01

    The agent of Lyme disease, Borrelia burgdorferi, has a number of outer membrane proteins that are differentially regulated during its life cycle. In addition to their physiological functions in the organism, these proteins also likely serve different functions in invasiveness and immune evasion. In borreliae, as well as in other bacteria, a number of membrane proteins have been implicated in binding plasminogen. The activation and transformation of plasminogen into its proteolytically active form, plasmin, enhances the ability of the bacteria to disseminate in the host. Outer membrane vesicles of B. burgdorferi contain enolase, a glycolytic-cycle enzyme that catalyzes 2-phosphoglycerate to form phosphoenolpyruvate, which is also a known plasminogen receptor in Gram-positive bacteria. The enolase was cloned, expressed, purified, and used to generate rabbit antienolase serum. The enolase binds plasminogen in a lysine-dependent manner but not through ionic interactions. Although it is present in the outer membrane, microscopy and proteinase K treatment showed that enolase does not appear to be exposed on the surface. However, enolase in the outer membrane vesicles is accessible to proteolytic degradation by proteinase K. Samples from experimentally and tick-infected mice and rabbits as well as from Lyme disease patients exhibit recognition of enolase in serologic assays. Thus, this immunogenic plasminogen receptor released in outer membrane vesicles could be responsible for external proteolysis in the pericellular environment and have roles in nutrition and in enhancing dissemination. PMID:22083700

  6. 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

  7. 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.

  8. Improved stability of rabbit and rat intestinal brush border membrane vesicles using phospholipase inhibitors.

    PubMed

    Maenz, D D; Chenu, C; Bellemare, F; Berteloot, A

    1991-11-01

    The initial rates of Na(+)-dependent D-aspartate and D-glucose uptakes were shown to decline from the time of resuspension of brush border membrane vesicles isolated from rabbit and rat jejunum by standard divalent cation precipitation procedures. The former were however more stable than the latter and followed quite closely the decrease in the intravesicular volume, thus suggesting that the loss of transport activity may involve both nonspecific opening of the vesicles and either direct or indirect specific inactivation of the transporters. Uptake rates for both substrates did tend to stabilize at 6-24 h from resuspension, however this final 'next day' uptake activity was too low to be of practical use in kinetic studies. Freezing aliquots of rabbit jejunal vesicles in liquid N2 until the time of assay resulted in complete stabilization of D-glucose uptake. A modified homogenate buffer designed to inhibit a broad spectrum of phospholipase activities resulted in a partial stabilization of glucose transport by rabbit jejunal vesicles with, on average, an over 6-fold enrichment in the 'next day' stable specific activity of uptake as compared to unfrozen vesicles. The modified homogenate buffer also improved the stability and the 'next day' specific activities of D-glucose uptake in rat jejunal brush border vesicles and D-aspartic acid uptake in rabbit jejunal vesicles. It also completely stabilized the intravesicular volume in the latter preparation. An evaluation of the kinetic parameters of Na(+)-dependent D-glucose transport in rabbit vesicles prepared from either the standard homogenate media and frozen in liquid N2 or the modified media and allowed to stabilize overnight, revealed a single transport system with a Km of 0.31-0.32 mM as the best model to fit the data. As such the modifications to the homogenate media do not appear to effect the functional properties of D-glucose transport in the membrane. While being less efficient in stabilizing the vesicles than

  9. 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

  10. 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

  11. Formation of Mitochondrial Outer Membrane Derived Protrusions and Vesicles in Arabidopsis thaliana.

    PubMed

    Yamashita, Akihiro; Fujimoto, Masaru; Katayama, Kenta; Yamaoka, Shohei; Tsutsumi, Nobuhiro; Arimura, Shin-Ichi

    2016-01-01

    Mitochondria are dynamic organelles that have inner and outer membranes. In plants, the inner membrane has been well studied but relatively little is known about the outer membrane. Here we report that Arabidopsis cells have mitochondrial outer membrane-derived structures, some of which protrude from the main body of mitochondria (mitochondrial outer-membrane protrusions; MOPs), while others form vesicle-like structures without a matrix marker. The latter vesicle-like structures are similar to some mammalian MDVs (mitochondrial-derived vesicles). Live imaging demonstrated that a plant MDV budded off from the tip of a MOP. MDVs were also observed in the drp3a drp3b double mutant, indicating that they could be formed without the mitochondrial fission factors DRP3A and DRP3B. Double staining studies showed that the MDVs were not peroxisomes, endosomes, Golgi apparatus or trans-Golgi network (TGN). The numbers of MDVs and MOPs increased in senescent leaves and after dark treatment. Together, these results suggest that MDVs and MOPs are related to leaf senescence. PMID:26752045

  12. Formation of Mitochondrial Outer Membrane Derived Protrusions and Vesicles in Arabidopsis thaliana

    PubMed Central

    Yamashita, Akihiro; Fujimoto, Masaru; Katayama, Kenta; Yamaoka, Shohei; Tsutsumi, Nobuhiro; Arimura, Shin-ichi

    2016-01-01

    Mitochondria are dynamic organelles that have inner and outer membranes. In plants, the inner membrane has been well studied but relatively little is known about the outer membrane. Here we report that Arabidopsis cells have mitochondrial outer membrane-derived structures, some of which protrude from the main body of mitochondria (mitochondrial outer-membrane protrusions; MOPs), while others form vesicle-like structures without a matrix marker. The latter vesicle-like structures are similar to some mammalian MDVs (mitochondrial-derived vesicles). Live imaging demonstrated that a plant MDV budded off from the tip of a MOP. MDVs were also observed in the drp3a drp3b double mutant, indicating that they could be formed without the mitochondrial fission factors DRP3A and DRP3B. Double staining studies showed that the MDVs were not peroxisomes, endosomes, Golgi apparatus or trans-Golgi network (TGN). The numbers of MDVs and MOPs increased in senescent leaves and after dark treatment. Together, these results suggest that MDVs and MOPs are related to leaf senescence. PMID:26752045

  13. 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

  14. Kinetics of ATP- and Na(+)-gradient driven Ca2+ transport in basolateral membranes from gills of freshwater- and seawater-adapted tilapia.

    PubMed

    Verbost, P M; Schoenmakers, T J; Flik, G; Wendelaar Bonga, S E

    1994-01-01

    Plasma membranes of the gills of freshwater- and seawater-adapted tilapia were analyzed for Ca(2+)-ATPase and Na+/Ca2+ exchange activity. The relative importance of ATP-driven and Na(+)-gradient-driven Ca2+ transport in Ca2+ extrusion was evaluated on the basis of kinetic analyses in vitro. The Na+/Ca2+ exchangers in branchial membranes from freshwater or seawater fish displayed similar kinetics. The ATP-driven Ca2+ pump, however, showed a somewhat lower affinity for Ca2+ in membranes isolated from seawater gills than in membranes from freshwater gills; no difference in Vmax was found. The activity of the exchanger was estimated to be 50% of that of the ATP-driven pump at prevailing cytosolic Ca2+ concentrations (10(-7) mol l-1). Opercular ionocyte densities and branchial Na+/K(+)-ATPase content were not significantly different in fish residing in fresh water or sea water. We conclude that the gills of tilapia living for prolonged periods in fresh water or sea water do not differ in the make-up of their basolateral membrane with regard to Ca(2+)-ATPase, Na+/Ca2+ exchange and Na+/K(+)-ATPase activity. Apparently, the densities of these carriers suffice for calcium and sodium homeostasis under these vastly different ambient conditions. PMID:7964377

  15. 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.

  16. 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

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

    PubMed

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

    2016-07-01

    Their biodegradable nature and ability to target cells make biological vesicles potential nanocarriers for bioactives delivery. In this work, the interaction between proteoliposomes enriched in aquaporins derived from broccoli plants and the glucosinolates was evaluated. The vesicles were stored at different temperatures and their integrity was studied. Determination of glucosinolates, showed that indolic glucosinolates were more sensitive to degradation in aqueous solution than aliphatic glucosinolates. Glucoraphanin was stabilized by leaf and root proteoliposomes at 25°C through their interaction with aquaporins. An extensive hydrogen bond network, including different aquaporin residues, and hydrophobic interactions, as a consequence of the interaction between the linear alkane chain of glucoraphanin and Glu31 and Leu34 protein residues, were established as the main stabilizing elements. Combined our results showed that plasma membrane vesicles from leaf and root tissues of broccoli plants may be considered as suitable carriers for glucosinolate which stabilization can be potentially attributed to aquaporins. PMID:27022872

  18. Tubular lipid membranes pulled from vesicles: Dependence of system equilibrium on lipid bilayer curvature

    NASA Astrophysics Data System (ADS)

    Golushko, I. Yu.; Rochal, S. B.

    2016-01-01

    Conditions of joint equilibrium and stability are derived for a spherical lipid vesicle and a tubular lipid membrane (TLM) pulled from this vesicle. The obtained equations establish relationships between the geometric and physical characteristics of the system and the external parameters, which have been found to be controllable in recent experiments. In particular, the proposed theory shows that, in addition to the pressure difference between internal and external regions of the system, the variable spontaneous average curvature of the lipid bilayer (forming the TLM) also influences the stability of the lipid tube. The conditions for stability of the cylindrical phase of TLMs after switching off the external force that initially formed the TLM from a vesicle are discussed. The loss of system stability under the action of a small axial force compressing the TLM is considered.

  19. 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.

  20. 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

  1. 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

  2. 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

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

    PubMed

    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

  4. 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

  5. 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

  6. A Bcl-xL-Drp1 complex regulates synaptic vesicle membrane dynamics during endocytosis

    PubMed Central

    Li, Hongmei; Alavian, Kambiz N.; Lazrove, Emma; Mehta, Nabil; Jones, Adrienne; Zhang, Ping; Licznerski, Pawel; Graham, Morven; Uo, Takuma; Guo, Junhua; Rahner, Christoph; Duman, Ronald S.; Morrison, Richard S.; Jonas, Elizabeth A.

    2013-01-01

    Following exocytosis, the rate of recovery of neurotransmitter release is determined by vesicle retrieval from the plasma membrane and by recruitment of vesicles from reserve pools within the synapse, the latter of which is dependent on mitochondrial ATP. The Bcl-2 family protein Bcl-xL, in addition to its role in cell death, regulates neurotransmitter release and recovery in part by increasing ATP availability from mitochondria. We now find, however, that, Bcl-xL directly regulates endocytotic vesicle retrieval in hippocampal neurons through protein/protein interaction with components of the clathrin complex. Our evidence suggests that, during synaptic stimulation, Bcl-xL translocates to clathrin-coated pits in a calmodulin-dependent manner and forms a complex of proteins with the GTPase Drp1, Mff and clathrin. Depletion of Drp1 produces misformed endocytotic vesicles. Mutagenesis studies suggest that formation of the Bcl-xL-Drp1 complex is necessary for the enhanced rate of vesicle endocytosis produced by Bcl-xL, thus providing a mechanism for presynaptic plasticity. PMID:23792689

  7. 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

  8. 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

  9. Membrane permeability of fructose-1,6-diphosphate in lipid vesicles and endothelial cells.

    PubMed

    Ehringer, W D; Niu, W; Chiang, B; Wang, O L; Gordon, L; Chien, S

    2000-07-01

    Fructose-1,6-diphosphate (FDP) is a glycolytic intermediate which has been used an intervention in various ischemic conditions for two decades. Yet whether FDP can enter the cell is under constant debate. In this study we examined membrane permeability of FDP in artificial membrane bilayers and in endothelial cells. To examine passive diffusion of FDP through the membrane bilayer, L-alpha-phosphatidylcholine from egg yolk (Egg PC) (10 mM) multi-lamellar vesicles were created containing different external concentrations of FDP (0, 0.5, 5 and 50 mM). The passive diffusion of FDP into the vesicles was followed spectrophotometrically. The results indicate that FDP diffuses through the membrane bilayer in a dose-dependent fashion. The movement of FDP through Egg PC membrane bilayers was confirmed by measuring the conversion of FDP to dihydroxyacetone-phosphate and the formation of hydrozone. FDP (0, 0.5, 5 or 50 mM) was encapsulated in Egg PC multilamellar vesicles and placed in a solution containing aldolase. In the 5 and 50 mM FDP groups there was a significant increase in dihydroxyacetone/hydrazone indicating that FDP crossed the membrane bilayer intact. We theorized that the passive diffusion of FDP might be due to disruption of the membrane bilayer. To examine this hypothesis, small unilamellar vesicles composed of Egg PC were created in the presence of 60 mM carboxyfluorescein, and the leakage of the sequestered dye was followed upon addition of various concentrations of FDP, fructose, fructose-6-phosphate, or fructose-1-phosphate (0, 5 or 50 mM). These results indicate that increasing concentrations of FDP increase the leakage rate of carboxyfluorescein. In contrast, no concentration of fructose, fructose-6-phosphate, or fructose-1-phosphate resulted in any significant increase in membrane permeability to carboxyfluorescein. To examine whether FDP could pass through cellular membranes, we examined the uptake of 14C-FDP by endothelial cells cultured under hypoxia

  10. 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

  11. The Xylella fastidiosa PD1063 Protein Is Secreted in Association with Outer Membrane Vesicles

    PubMed Central

    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

  12. 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...

  13. Binding of the vesicle docking protein p115 to the GTPase Rab1b regulates membrane recruitment of the COPI vesicle coat

    PubMed Central

    Guo, Yusong; Linstedt, Adam D

    2014-01-01

    Membrane recruitment of the COPI vesicle coat is fundamental to its function and contributes to compartment identity in the early secretory pathway. COPI recruitment is triggered by guanine nucleotide exchange activating the Arf1 GTPase, but the key exchange factor, GBF1, is a peripheral membrane component whose membrane association is dependent on another GTPase, Rab1. Inactive Rab GTPases are in a soluble complex with guanine nucleotide dissociation inhibitor (GDI) and activation of Rab GTPases by exchange factors can be enhanced by GDI dissociation factors (GDFs). In the present study, we investigated the vesicle docking protein p115 and it’s binding to the Rab1 isoform Rab1b. Inhibition of p115 expression induced dissociation of Rab1b from Golgi membranes. Rab1b bound the cc2 domain of p115 and p115 lacking this domain failed to recruit Rab1b. Further, p115 inhibition blocked association of the COPI coat with Golgi membranes and this was suppressed by constitutive activation of Rab1b. These findings show p115 enhancement of Rab1b activation leading to COPI recruitment suggesting a connection between the vesicle docking machinery and the vesicle coat complex during the establishment of post-ER compartment identity. PMID:25332841

  14. Measuring localization and diffusion coefficients of basolateral proteins in lateral versus basal membranes using functionalized substrates and kICS analysis.

    PubMed

    Marlar, Saw; Arnspang, Eva C; Pedersen, Gitte A; Koffman, Jennifer S; Nejsum, Lene N

    2014-10-01

    Micropatterning enabled semiquantitation of basolateral proteins in lateral and basal membranes of the same cell. Lateral diffusion coefficients of basolateral aquaporin-3 (AQP3-EGFP) and EGFP-AQP4 were extracted from "lateral" and "basal" membranes using identical live-cell imaging and k-space Image Correlation Spectroscopy (kICS). To simultaneously image proteins in "lateral" and "basal" membranes, micropatterning with the extracellular domain of E-cadherin and collagen, to mimic cell-cell and cell-extracellular matrix (ECM) adhesion, respectively, was used. In kidney collecting duct principal cells AQP3 localizes lateral and basal whereas AQP4 localizes mainly basal. On alternating stripes of E-cadherin and collagen, AQP3-EGFP was predominantly localized to "lateral" compared to "basal" membranes, whereas Orange-AQP4 was evenly distributed. Average diffusion coefficients were extracted via kICS analysis of rapid time-lapse sequences of AQP3-EGFP and EGFP-AQP4 on uniform substrates of either E-cadherin or collagen. AQP3-EGFP was measured to 0.022±0.010μm(2)/s on E-cadherin and 0.019±0.004μm(2)/s on collagen, whereas EGFP-AQP4 was measured to 0.044±0.009μm(2)/s on E-cadherin and 0.037±0.009μm(2)/s on collagen, thus, diffusion did not differ between substrates. Cholesterol depletion by methyl-beta-cyclodextrin (MBCD) reduced the AQP3-EGFP diffusion coefficient by 43% from 0.024±0.007μm(2)/s (water) to 0.014±0.003μm(2)/s (MBCD) (p<0.05) on collagen surfaces, and by 41% from 0.023±0.011μm(2)/s (water) to 0.014±0.005μm(2)/s (MBCD) (p<0.05) on E-cadherin surfaces. Thus, protein patterning enables the semiquantitation of protein distribution between the "lateral" and "basal" membranes as well as measurements of lateral diffusion coefficients. PMID:24950246

  15. Characterization of nucleic acids in membrane vesicles from scrapie-infected hamster brain.

    PubMed Central

    Dees, C; McMillan, B C; Wade, W F; German, T L; Marsh, R F

    1985-01-01

    This study reports the partial characterization of nucleic acids present in gradient fractions enriched for large membrane vesicles from scrapie-infected and uninfected hamster brains. Labeling of phenol-extracted nucleic acids at the 3' or 5' ends revealed abundant amounts of low-molecular-weight RNA and little or no DNA. These nucleic acids survived nuclease treatment of membrane vesicles but were sensitive to RNase after phenol extraction. Analysis of 5'-end-labeled nucleic acids by one- and two-dimensional gel electrophoresis revealed an RNA of ca. 100 bases in preparations from scrapie-infected hamster brain that could not be detected in uninfected brain. The possibility that this apparently unique small RNA may result from tissue damage or abnormal RNA processing or may be a component of the infectious complex is discussed. Images PMID:2409296

  16. Tissue-specific alternative RNA splicing of rat vesicle-associated membrane protein-1 (VAMP-1).

    PubMed

    Mandic, R; Trimble, W S; Lowe, A W

    1997-10-15

    The vesicle-associated membrane protein (VAMP) family is essential to vesicle-mediated protein transport. Three mammalian isoforms, VAMP-1, VAMP-2, and cellubrevin, play a role in protein transport to the plasma membrane. In this study, we describe a new rat VAMP-1 isoform produced by alternative pre-mRNA splicing. Only one VAMP-1 isoform dominates in each tissue. Analysis of the nucleotide sequence for the newly discovered isoform, VAMP-1b, reveals that its expression is determined by whether an intron is retained or removed. The predicted amino acid sequences for the VAMP-1 isoforms differ at the carboxy-terminal end of the protein. A similar process has been described for VAMPs in Drosophila melanogaster and suggests a conserved function for the carboxy-terminal domain that can be modulated. PMID:9358054

  17. Amino acid transport by membrane vesicles of an obligate anaerobic bacterium, Clostridium acetobutylicum.

    PubMed Central

    Driessen, A J; Ubbink-Kok, T; Konings, W N

    1988-01-01

    Membrane vesicles were isolated from the obligate anaerobic bacterium Clostridium acetobutylicum. Beef heart mitochondrial cytochrome c oxidase was inserted in these membrane vesicles by membrane fusion by using the freeze-thaw sonication technique (A. J. M. Driessen, W. de Vrij, and W. N. Konings, Proc. Natl. Acad. Sci. USA 82:7555-7559, 1985) to accommodate them with a functional proton motive force-generating system. With ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine-cytochrome c as the electron donor, a proton motive force (delta p) of -80 to -120 mV was generated in these fused membranes. This delta p drove the accumulation of leucine and lysine up to 40- and 100-fold, respectively. High transport activities were observed in fused membranes containing Escherichia coli lipids, whereas the transport activities in fused membranes containing mainly soybean lipids or phosphatidylcholine were low. It is suggested that branched-chain amino acids and lysine were taken up by separate systems. The effects of the ionophores nigericin and valinomycin indicated that lysine and leucine were translocated in symport with a proton. PMID:2828326

  18. Intra- and Interspecies Effects of Outer Membrane Vesicles from Stenotrophomonas maltophilia on β-Lactam Resistance.

    PubMed

    Devos, Simon; Stremersch, Stephan; Raemdonck, Koen; Braeckmans, Kevin; Devreese, Bart

    2016-04-01

    The treatment ofStenotrophomonas maltophiliainfection with β-lactam antibiotics leads to increased release of outer membrane vesicles (OMVs), which are packed with two chromosomally encoded β-lactamases. Here, we show that these β-lactamase-packed OMVs are capable of establishing extracellular β-lactam degradation. We also show that they dramatically increase the apparent MICs of imipenem and ticarcillin for the cohabituating speciesPseudomonas aeruginosaandBurkholderia cenocepacia. PMID:26787686

  19. Fluorescence quenching in β-cyclodextrin vesicles: membrane confinement and host-guest interactions.

    PubMed

    Schibilla, Frauke; Stegemann, Linda; Strassert, Cristian A; Rizzo, Fabio; Ravoo, Bart Jan

    2016-02-01

    Fluorescent β-cyclodextrin vesicles (β-CDV) that display host cavities available for host-guest interactions at the vesicle surface were prepared by incorporation of the hydrophobic spirobifluorene-based dye 1 into the membrane of unilamellar vesicles. Fluorescence quenching of dye 1 was observed in the presence of different quenchers. Methyl viologen 2 does not quench dye 1 because it does not bind to β-CDV. 4-Nitrophenol 3 and 4-nitrophenol covalently connected to adamantane 4 quench the fluorescence of dye 1 in neutral solution, but by different mechanisms according to lifetime measurements. The quenching efficiency of 3 is pH dependent due to the presence of the phenolate form. Competition experiments with excess host and guest showed that 3 is likely to diffuse in and out of the membrane, while 4 forms an inclusion complex with β-CDV leading to close contact and efficient quenching. Our findings confirm that this dynamic supramolecular system is a versatile model to investigate quenching and recognition processes in bilayer membranes. PMID:26777315

  20. 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

  1. Vesicle formation from the nuclear membrane is induced by coexpression of two conserved herpesvirus proteins

    PubMed Central

    Klupp, Barbara G.; Granzow, Harald; Fuchs, Walter; Keil, Günther M.; Finke, Stefan; Mettenleiter, Thomas C.

    2007-01-01

    Although the nuclear envelope is a dynamic structure that disassembles and reforms during mitosis, the formation of membranous vesicles derived from the nuclear envelope has not yet been described in noninfected cells. However, during herpesvirus maturation, intranuclear capsids initiate transit to the cytosol for final maturation by budding at the inner nuclear membrane. Two conserved herpesvirus proteins are required for this primary envelopment, designated in the alphaherpesviruses as pUL31 and pUL34. Here, we show that simultaneous expression of pUL31 and pUL34 of the alphaherpesvirus pseudorabies virus in stably transfected rabbit kidney cells resulted in the formation of vesicles in the perinuclear space that resemble primary envelopes without a nucleocapsid. They contain pUL31 and pUL34 as shown by immunolabeling and are derived from the nuclear envelope. Thus, coexpression of only two conserved herpesvirus proteins without any other viral factor is sufficient to induce the formation of vesicles from the nuclear membrane. This argues for the contribution of cellular factors in this process either recruited from their natural cytoplasmic location or not yet identified as components of the nuclear compartment. PMID:17426144

  2. Identification of the endoplasmic reticulum targeting signal in vesicle-associated membrane proteins.

    PubMed

    Kim, P K; Hollerbach, C; Trimble, W S; Leber, B; Andrews, D W

    1999-12-24

    The vesicle-associated membrane proteins (Vamp(s)) function as soluble N-ethylmaleimide-sensitive factor attachment receptor proteins in the intracellular trafficking of vesicles. The membrane attachment of Vamps requires a carboxyl-terminal hydrophobic sequence termed an insertion sequence. Unlike other insertion sequence-containing proteins, targeting of the highly homologous Vamp1 and Vamp2 to the endoplasmic reticulum requires ATP and a membrane-bound receptor. To determine if this mechanism of targeting to the endoplasmic reticulum extends to other Vamps, we compared the membrane binding of Vamp1 and Vamp2 with the distantly related Vamp8. Similar to the other Vamps, Vamp8 requires both ATP and a membrane component to target to the endoplasmic reticulum. Furthermore, binding curves for the three Vamps overlap, suggesting a common receptor-mediated process. We identified a minimal endoplasmic reticulum targeting domain that is both necessary and sufficient to confer receptor-mediated, ATP-dependent, binding of a heterologous protein to microsomes. Surprisingly, this conserved sequence includes four positively charged amino acids spaced along an amphipathic sequence, which unlike the carboxyl-terminal targeting sequence in mitochondrial Vamp isoforms, is amino-terminal to the insertion sequence. Because Vamps do not bind to phospholipid vesicles, it is likely that these residues mediate an interaction with a protein, rather than bind to acidic phospholipids. Therefore, we suggest that a bipartite motif is required for the specific targeting and integration of Vamps into the endoplasmic reticulum with receptor-mediated recognition of specifically configured positive residues leading to the insertion of the hydrophobic tail into the membrane. PMID:10601239

  3. 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. PMID:26751083

  4. 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

  5. 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

  6. Mixing Water, Transducing Energy, and Shaping Membranes: Autonomously Self-Regulating Giant Vesicles.

    PubMed

    Ho, James C S; Rangamani, Padmini; Liedberg, Bo; Parikh, Atul N

    2016-03-01

    Giant lipid vesicles are topologically closed compartments bounded by semipermeable flexible shells, which isolate femto- to picoliter quantities of the aqueous core from the surrounding bulk. Although water equilibrates readily across vesicular walls (10(-2)-10(-3) cm(3) cm(-2) s(-1)), the passive permeation of solutes is strongly hindered. Furthermore, because of their large volume compressibility (∼10(9)-10(10) N m(-2)) and area expansion (10(2)-10(3) mN m(-1)) moduli, coupled with low bending rigidities (10(-19) N m), vesicular shells bend readily but resist volume compression and tolerate only a limited area expansion (∼5%). Consequently, vesicles experiencing solute concentration gradients dissipate the available chemical energy through the osmotic movement of water, producing dramatic shape transformations driven by surface-area-volume changes and sustained by the incompressibility of water and the flexible membrane interface. Upon immersion in a hypertonic bath, an increased surface-area-volume ratio promotes large-scale morphological remodeling, reducing symmetry and stabilizing unusual shapes determined, at equilibrium, by the minimal bending-energy configurations. By contrast, when subjected to a hypotonic bath, walls of giant vesicles lose their thermal undulation, accumulate mechanical tension, and, beyond a threshold swelling, exhibit remarkable oscillatory swell-burst cycles, with the latter characterized by damped, periodic oscillations in vesicle size, membrane tension, and phase behavior. This cyclical pattern of the osmotic influx of water, pressure, membrane tension, pore formation, and solute efflux suggests quasi-homeostatic self-regulatory behavior allowing vesicular compartments produced from simple molecular components, namely, water, osmolytes, and lipids, to sense and regulate their microenvironment in a negative feedback loop. PMID:26866787

  7. Adhesion-Induced Phase Behavior of Two-Component Membranes and Vesicles

    PubMed Central

    Rouhiparkouhi, Tahereh; Weikl, Thomas R.; Discher, Dennis E.; Lipowsky, Reinhard

    2013-01-01

    The interplay of adhesion and phase separation is studied theoretically for two-component membranes that can phase separate into two fluid phases such as liquid-ordered and liquid-disordered phases. Many adhesion geometries provide two different environments for these membranes and then partition the membranes into two segments that differ in their composition. Examples are provided by adhering vesicles, by hole- or pore-spanning membranes, and by membranes supported by chemically patterned surfaces. Generalizing a lattice model for binary mixtures to these adhesion geometries, we show that the phase behavior of the adhering membranes depends, apart from composition and temperature, on two additional parameters, the area fraction of one membrane segment and the affinity contrast between the two segments. For the generic case of non-vanishing affinity contrast, the adhering membranes undergo two distinct phase transitions and the phase diagrams in the composition/temperature plane have a generic topology that consists of two two-phase coexistence regions separated by an intermediate one-phase region. As a consequence, phase separation and domain formation is predicted to occur separately in each of the two membrane segments but not in both segments simultaneously. Furthermore, adhesion is also predicted to suppress the phase separation process for certain regions of the phase diagrams. These generic features of the adhesion-induced phase behavior are accessible to experiment. PMID:23340655

  8. The localization of chitin synthase in membranous vesicles (chitosomes) in Neurospora crassa.

    PubMed

    Sietsma, J H; Beth Din, A; Ziv, V; Sjollema, K A; Yarden, O

    1996-07-01

    Polyclonal anti-chitin synthase antibodies raised against the Saccharomyces cerevisiae CHS2 gene product were used to identify and localize chitin synthase in the filamentous ascomycete Neurospora crassa. A single band of approximately 110 kDa was observed in Western blots of total protein extracts of N. crassa, probed with these antibodies. However, several additional bands were labelled when membrane fraction proteins (microsomes) were probed. Histo-immunochemical localization of chitin synthase confirmed that the polypeptide is compartmentalized in membranous vesicles (chitosomes), which are abundant in the vicinity of the hyphal tip. TEM analysis did not reveal chitin synthase in the plasma membrane. However, dense labelling of membrane-associated chitin synthase was observed by light-microscopic analysis of N. crassa protoplasts and at young hyphal tips. PMID:8757723

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

    PubMed

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

    2007-06-01

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

  10. Quantitative and Rapid Estimation of H+ Fluxes in Membrane Vesicles 1

    PubMed Central

    Jennings, Ian R.; Rea, Philip A.; Leigh, Roger A.; Sanders, Dale

    1988-01-01

    Proton transport is often visualized in membrane vesicles by use of fluorescent monoamines which accumulate in acidic intravesicular compartments and undergo concentration-dependent fluorescence quenching. Software for an IBM microcomputer is described which permits logging and editing of changes in fluorescence monitored by a Perkin-Elmer LS-5 luminescence spectrometer. An accurate estimate of the instantaneous rate of fluorescence quenching or recovery is then facilitated by least squares fitting of fluorescence data to a nonlinear function. The software is tested with tonoplast vesicles from Beta vulgaris. Quenching of acridine orange fluorescence by ATP-driven (primary) transport and relaxation of quenching by Na+/H+ antiport can both be fitted with single exponential functions. Initial rates of ATP- and Na+ -dependent fluorescence changes are derived and can be used for Km determinations. The method constitutes a simple and efficient alternative to manual analysis of analog fluorescence traces and results in a reliable quantitative measurement of the relative rate of proton transport in membrane vesicle preparations. PMID:16666064

  11. Glucose transport by brush-border membrane vesicles after proximal resection or ileo-jejunal transposition in the rat.

    PubMed Central

    Menge, H; Murer, H; Robinson, J W

    1978-01-01

    1. The functional properties of the ileal mucosa following jejunal resection or interposition in the jejunum were studied with the help of membrane vesicles. 2. Despite the development of functional and morphological features in the mucosa which are more generally associated with the jejunum than with the ileum, examination of brush-border vesicles derived from ileal and jejunal enterocytes from resected or transposed intestines reveals that the functional characteristics of apical membranes isolated from the different regions of the intestine are maintained after these surgical manoeuvres. Vesicles from control ileum develop an 'overshoot' uptake of glucose that is 3-4 times smaller than that of jejunal vesicles, a difference that is still observed in membranes prepared from mucosa of ileal loops following proximal resection or interposition in the jejunum. Images Fig. 3 PMID:625015

  12. Basolateral chloride current in human airway epithelia.

    PubMed

    Itani, Omar A; Lamb, Fred S; Melvin, James E; Welsh, Michael J

    2007-10-01

    Electrolyte transport by airway epithelia regulates the quantity and composition of liquid covering the airways. Previous data indicate that airway epithelia can absorb NaCl. At the apical membrane, cystic fibrosis transmembrane conductance regulator (CFTR) provides a pathway for Cl(-) absorption. However, the pathways for basolateral Cl(-) exit are not well understood. Earlier studies, predominantly in cell lines, have reported that the basolateral membrane contains a Cl(-) conductance. However, the properties have varied substantially in different epithelia. To better understand the basolateral Cl(-) conductance in airway epithelia, we studied primary cultures of well-differentiated human airway epithelia. The basolateral membrane contained a Cl(-) current that was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The current-voltage relationship was nearly linear, and the halide selectivity was Cl(-) > Br(-) > I(-). Several signaling pathways increased the current, including elevation of cellular levels of cAMP, activation of protein kinase C (PKC), and reduction of pH. In contrast, increasing cell Ca(2+) and inducing cell swelling had no effect. The basolateral Cl(-) current was present in both cystic fibrosis (CF) and non-CF airway epithelia. Likewise, airway epithelia from wild-type mice and mice with disrupted genes for ClC-2 or ClC-3 all showed similar Cl(-) currents. These data suggest that the basolateral membrane of airway epithelia possesses a Cl(-) conductance that is not due to CFTR, ClC-2, or ClC-3. Its regulation by cAMP and PKC signaling pathways suggests that coordinated regulation of Cl(-) conductance in both apical and basolateral membranes may be important in controlling transepithelial Cl(-) movement. PMID:17660331

  13. A Chemical Controller of SNARE-Driven Membrane Fusion That Primes Vesicles for Ca(2+)-Triggered Millisecond Exocytosis.

    PubMed

    Heo, Paul; Yang, Yoosoo; Han, Kyu Young; Kong, Byoungjae; Shin, Jong-Hyeok; Jung, Younghoon; Jeong, Cherlhyun; Shin, Jaeil; Shin, Yeon-Kyun; Ha, Taekjip; Kweon, Dae-Hyuk

    2016-04-01

    Membrane fusion is mediated by the SNARE complex which is formed through a zippering process. Here, we developed a chemical controller for the progress of membrane fusion. A hemifusion state was arrested by a polyphenol myricetin which binds to the SNARE complex. The arrest of membrane fusion was rescued by an enzyme laccase that removes myricetin from the SNARE complex. The rescued hemifusion state was metastable and long-lived with a decay constant of 39 min. This membrane fusion controller was applied to delineate how Ca(2+) stimulates fusion-pore formation in a millisecond time scale. We found, using a single-vesicle fusion assay, that such myricetin-primed vesicles with synaptotagmin 1 respond synchronously to physiological concentrations of Ca(2+). When 10 μM Ca(2+) was added to the hemifused vesicles, the majority of vesicles rapidly advanced to fusion pores with a time constant of 16.2 ms. Thus, the results demonstrate that a minimal exocytotic membrane fusion machinery composed of SNAREs and synaptotagmin 1 is capable of driving membrane fusion in a millisecond time scale when a proper vesicle priming is established. The chemical controller of SNARE-driven membrane fusion should serve as a versatile tool for investigating the differential roles of various synaptic proteins in discrete fusion steps. PMID:26987363

  14. Copper directs ATP7B to the apical domain of hepatic cells via basolateral endosomes.

    PubMed

    Nyasae, Lydia K; Schell, Michael J; Hubbard, Ann L

    2014-12-01

    Physiologic Cu levels regulate the intracellular location of the Cu ATPase ATP7B. Here, we determined the routes of Cu-directed trafficking of endogenous ATP7B in the polarized hepatic cell line WIF-B and in the liver in vivo. Copper (10 µm) caused ATP7B to exit the trans-Golgi network (TGN) in vesicles, which trafficked via large basolateral endosomes to the apical domain within 1 h. Although perturbants of luminal acidification had little effect on the TGN localization of ATP7B in low Cu, they blocked delivery to the apical membrane in elevated Cu. If the vesicular proton-pump inhibitor bafilomycin-A1 (Baf) was present with Cu, ATP7B still exited the TGN, but accumulated in large endosomes located near the coverslip, in the basolateral region. Baf washout restored ATP7B trafficking to the apical domain. If ATP7B was staged apically in high Cu, Baf addition promoted the accumulation of ATP7B in subapical endosomes, indicating a blockade of apical recycling, with concomitant loss of ATP7B at the apical membrane. The retrograde pathway to the TGN, induced by Cu removal, was far less affected by Baf than the anterograde (Cu-stimulated) case. Overall, loss of acidification-impaired Cu-regulated trafficking of ATP7B at two main sites: (i) sorting and exit from large basolateral endosomes and (ii) recycling via endosomes near the apical membrane. PMID:25243755

  15. 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

  16. Bacteriolytic effect of membrane vesicles from Pseudomonas aeruginosa on other bacteria including pathogens: conceptually new antibiotics.

    PubMed Central

    Kadurugamuwa, J L; Beveridge, T J

    1996-01-01

    Pseudomonas aeruginosa releases membrane vesicles (MVs) filled with periplasmic components during normal growth, and the quantity of these vesicles can be increased by brief exposure to gentamicin. Natural and gentamicin-induced membrane vesicles (n-MVs and g-MVs, respectively) are subtly different from one another, but both contain several important virulence factors, including hydrolytic enzyme factors (J. L. Kadurugamuwa and T. J. Beveridge, J. Bacteriol. 177:3998-4008, 1995). Peptidoglycan hydrolases (autolysins) were detected in both MV types, especially a periplasmic 26-kDa autolysin whose expression has been related to growth phase (Z. Li, A. J. Clarke, and T. J. Beveridge, J. Bacteriol. 178:2479-2488, 1996). g-MVs possessed slightly higher autolysin activity and, at the same time, small quantities of gentamicin. Both MV types hydrolyzed isolated gram-positive and gram-negative murein sacculi and were also capable of hydrolyzing several glycyl peptides. Because the MVs were bilayered, they readily fused with the outer membrane of gram-negative bacteria. They also adhered to the cell wall of gram-positive bacteria. g-MVs were more effective in lysing other bacteria because, in addition to the autolysins, they also contained small amounts of gentamicin. The bactericidal activity was 2.5 times the MIC of gentamicin, which demonstrates the synergistic effect of the antibiotic with the autolysins. n-MVs were capable of killing cultures of P. aeruginosa with permeability resistance against gentamicin, indicating that the fusion of n-MV to the outer membrane liberated autolysins into the periplasm, where they degraded the peptidoglycan and lysed the cells. g-MVs had even greater killing power since they liberated both gentamicin and autolysins into these resistant cells. These findings may help develop a conceptually new group of antibiotics designed to be effective against hard-to-kill bacteria. PMID:8631663

  17. 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

  18. Intake of silica nanoparticles by giant lipid vesicles: influence of particle size and thermodynamic membrane state

    PubMed Central

    Strobl, Florian G; Seitz, Florian; Westerhausen, Christoph; Reller, Armin; Torrano, Adriano A; Bräuchle, Christoph

    2014-01-01

    Summary The uptake of nanoparticles into cells often involves their engulfment by the plasma membrane and a fission of the latter. Understanding the physical mechanisms underlying these uptake processes may be achieved by the investigation of simple model systems that can be compared to theoretical models. Here, we present experiments on a massive uptake of silica nanoparticles by giant unilamellar lipid vesicles (GUVs). We find that this uptake process depends on the size of the particles as well as on the thermodynamic state of the lipid membrane. Our findings are discussed in the light of several theoretical models and indicate that these models have to be extended in order to capture the interaction between nanomaterials and biological membranes correctly. PMID:25671142

  19. 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.

  20. 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.

  1. Peptide hormone release monitored from single vesicles in "membrane lawns" of differentiated male pituitary cells: SNAREs and fusion pore widening.

    PubMed

    Stenovec, Matjaž; Gonçalves, Paula P; Zorec, Robert

    2013-03-01

    In this study we used live-cell immunocytochemistry and confocal microscopy to study the release from a single vesicle in a simplified system called membrane lawns. The lawns were prepared by exposing differentiated pituitary prolactin (PRL)-secreting cells to a hypoosmotic shear stress. The density of the immunolabeled ternary soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) complexes that bind complexin was approximately 10 times lower than the PRL-positive, lawn-resident vesicles; this indicates that some but not all vesicles are associated with ternary SNARE complexes. However, lawn-resident PRL vesicles colocalized relatively well with particular SNARE proteins: synaptobrevin 2 (35%), syntaxin 1 (22%), and 25-kDa synaptosome associated protein (6%). To study vesicle discharge, we prepared lawn-resident vesicles, derived from atrial natriuretic peptide tagged with emerald fluorescent protein (ANP.emd)-transfected cells, which label vesicles. These maintained the structural passage to the exterior because approximately 40% of ANP.emd-loaded vesicles were labeled by extracellular PRL antibodies. Cargo release from the lawn-resident vesicles, monitored by the decline in the ANP.emd fluorescence intensity, was similar to that in intact cells. It is likely that SNARE proteins are required for calcium-dependent release from these vesicles. This is because the expression of the dominant-negative SNARE peptide, which interferes with SNARE complex formation, reduced the number of PRL-positive spots per cell (PRL antibodies placed extracellularly) significantly, from 58 ± 9 to 4 ± 2. In dominant-negative SNARE-treated cells, the PRL-positive area was reduced from 0.259 ± 0.013 to 0.123 ± 0.014 μm(2), which is consistent with a hindered vesicle luminal access for extracellular PRL antibodies. These results indicate that vesicle discharge is regulated by SNARE-mediated fusion pore widening. PMID:23372020

  2. 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

  3. 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

  4. Ypq3p-dependent histidine uptake by the vacuolar membrane vesicles of Saccharomyces cerevisiae.

    PubMed

    Manabe, Kunio; Kawano-Kawada, Miyuki; Ikeda, Koichi; Sekito, Takayuki; Kakinuma, Yoshimi

    2016-06-01

    The vacuolar membrane proteins Ypq1p, Ypq2p, and Ypq3p of Saccharomyces cerevisiae are known as the members of the PQ-loop protein family. We found that the ATP-dependent uptake activities of arginine and histidine by the vacuolar membrane vesicles were decreased by ypq2Δ and ypq3Δ mutations, respectively. YPQ1 and AVT1, which are involved in the vacuolar uptake of lysine/arginine and histidine, respectively, were deleted in addition to ypq2Δ and ypq3Δ. The vacuolar membrane vesicles isolated from the resulting quadruple deletion mutant ypq1Δypq2Δypq3Δavt1Δ completely lost the uptake activity of basic amino acids, and that of histidine, but not lysine and arginine, was evidently enhanced by overexpressing YPQ3 in the mutant. These results suggest that Ypq3p is specifically involved in the vacuolar uptake of histidine in S. cerevisiae. The cellular level of Ypq3p-HA(3) was enhanced by depletion of histidine from culture medium, suggesting that it is regulated by the substrate. PMID:26928127

  5. Protein 4.1N is required for translocation of inositol 1,4,5-trisphosphate receptor type 1 to the basolateral membrane domain in polarized Madin-Darby canine kidney cells.

    PubMed

    Zhang, Songbai; Mizutani, Akihiro; Hisatsune, Chihiro; Higo, Takayasu; Bannai, Hiroko; Nakayama, Tomohiro; Hattori, Mitsuharu; Mikoshiba, Katsuhiko

    2003-02-01

    Protein 4.1N was identified as a binding molecule for the C-terminal cytoplasmic tail of inositol 1,4,5-trisphosphate receptor type 1 (IP(3)R1) using a yeast two-hybrid system. 4.1N and IP(3)R1 associate in both subconfluent and confluent Madin-Darby canine kidney (MDCK) cells, a well studied tight polarized epithelial cell line. In subconfluent MDCK cells, 4.1N is distributed in the cytoplasm and the nucleus; IP(3)R1 is localized in the cytoplasm. In confluent MDCK cells, both 4.1N and IP(3)R1 are predominantly translocated to the basolateral membrane domain, whereas 4.1R, the prototypical homologue of 4.1N, is localized at the tight junctions (Mattagajasingh, S. N., Huang, S. C., Hartenstein, J. S., and Benz, E. J., Jr. (2000) J. Biol. Chem. 275, 30573-30585), and other endoplasmic reticulum marker proteins are still present in the cytoplasm. Moreover, the 4.1N-binding region of IP(3)R1 is necessary and sufficient for the localization of IP(3)R1 at the basolateral membrane domain. A fragment of the IP(3)R1-binding region of 4.1N blocks the localization of co-expressed IP(3)R1 at the basolateral membrane domain. These data indicate that 4.1N is required for IP(3)R1 translocation to the basolateral membrane domain in polarized MDCK cells. PMID:12444087

  6. SNARE and regulatory proteins induce local membrane protrusions to prime docked vesicles for fast calcium-triggered fusion

    PubMed Central

    Bharat, Tanmay A M; Malsam, Jörg; Hagen, Wim J H; Scheutzow, Andrea; Söllner, Thomas H; Briggs, John A G

    2014-01-01

    Synaptic vesicles fuse with the plasma membrane in response to Ca2+ influx, thereby releasing neurotransmitters into the synaptic cleft. The protein machinery that mediates this process, consisting of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and regulatory proteins, is well known, but the mechanisms by which these proteins prime synaptic membranes for fusion are debated. In this study, we applied large-scale, automated cryo-electron tomography to image an in vitro system that reconstitutes synaptic fusion. Our findings suggest that upon docking and priming of vesicles for fast Ca2+-triggered fusion, SNARE proteins act in concert with regulatory proteins to induce a local protrusion in the plasma membrane, directed towards the primed vesicle. The SNAREs and regulatory proteins thereby stabilize the membrane in a high-energy state from which the activation energy for fusion is profoundly reduced, allowing synchronous and instantaneous fusion upon release of the complexin clamp. PMID:24493260

  7. 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

  8. Decoupled energy stable schemes for phase-field vesicle membrane model

    NASA Astrophysics Data System (ADS)

    Chen, Rui; Ji, Guanghua; Yang, Xiaofeng; Zhang, Hui

    2015-12-01

    We consider the numerical approximations of the classical phase-field vesicle membrane models proposed a decade ago in Du et al. (2004) [6]. We first reformulate the model derived from an energetic variational formulation into a form which is suitable for numerical approximation, and establish the energy dissipation law. Then, we develop a stabilized, decoupled, time discretization scheme for the coupled nonlinear system. The scheme is unconditionally energy stable and leads to linear and decoupled elliptic equations to be solved at each time step. Stability analysis and ample numerical simulations are presented thereafter.

  9. 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

  10. Transport of leucine, isoleucine and valine by luminal membrane vesicles from rabbit proximal tubule.

    PubMed

    Jørgensen, K E; Kragh-Hansen, U; Sheikh, M I

    1990-03-01

    1. Transport of L- and D-isomers of leucine, isoleucine and valine by luminal membrane vesicles prepared from either the convoluted part (pars convoluta) or the straight part (pars recta) of rabbit proximal tubule was studied by a rapid filtration technique and by a spectrophotometric method using a potential-sensitive carbocyanine dye. 2. Both types of renal membrane vesicle take up the amino acids in a Na(+)-dependent, H(+)-independent and electrogenic manner. The L-isomers are transported with higher affinities than their corresponding D-forms, of which only D-leucine is taken up to a significant extent. 3. Membrane vesicles prepared from pars convoluta take up the L-amino acids by a single and common system. Filtration studies showed that the Km values for L-leucine and L-valine transport are, on average, 0.23 and 0.83 mM, respectively. The values of KA (the concentration of amino acid producing a half-maximal optical response) are comparable to those of Km, namely 0.18 mM for L-leucine and 0.60 mM for L-valine. KA for L-isoleucine transport was found to be 0.19 mM. D-Leucine is taken up by the same system but with a much lower affinity (KA = 7.2 mM). 4. Membrane vesicles prepared from pars recta possess two, and probably common, transport systems for the L-isomers of the amino acids. The average Michaelis-Menten constants were as follows: L-leucine, K1m = 0.17 mM, K2m = 6.5 mM; L-valine, K1m = 0.19 mM, K2m = 11.5 mM. The KA values were: L-leucine, K1A = 0.12 mM, K2A = 7.4 mM; L-valine, K1A = 0.18 mM, K2A = 10.0 mM; L-isoleucine, K1A = 0.17 mM, K2A = 9.0 mM. D-Leucine is taken up by a low-affinity system only (KA = 6.5 mM), which seems to be the same as the low-affinity system transporting the L-forms of the amino acids. PMID:2352186

  11. 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

  12. 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

  13. LRRK2 Affects Vesicle Trafficking, Neurotransmitter Extracellular Level and Membrane Receptor Localization

    PubMed Central

    Spissu, Ylenia; Sanna, Giovanna; Xiong, Yulan; Dawson, Ted M.; Dawson, Valina L.; Galioto, Manuela; Rocchitta, Gaia; Biosa, Alice; Serra, Pier Andrea; Carri, Maria Teresa; Crosio, Claudia; Iaccarino, Ciro

    2013-01-01

    The leucine-rich repeat kinase 2 (LRRK2) gene was found to play a role in the pathogenesis of both familial and sporadic Parkinson’s disease (PD). LRRK2 encodes a large multi-domain protein that is expressed in different tissues. To date, the physiological and pathological functions of LRRK2 are not clearly defined. In this study we have explored the role of LRRK2 in controlling vesicle trafficking in different cellular or animal models and using various readouts. In neuronal cells, the presence of LRRK2G2019S pathological mutant determines increased extracellular dopamine levels either under basal conditions or upon nicotine stimulation. Moreover, mutant LRRK2 affects the levels of dopamine receptor D1 on the membrane surface in neuronal cells or animal models. Ultrastructural analysis of PC12-derived cells expressing mutant LRRK2G2019S shows an altered intracellular vesicle distribution. Taken together, our results point to the key role of LRRK2 to control vesicle trafficking in neuronal cells. PMID:24167564

  14. Interaction of miltefosine with intercellular membranes of stratum corneum and biomimetic lipid vesicles.

    PubMed

    Alonso, Lais; Mendanha, Sebastião Antônio; Marquezin, Cássia Alessandra; Berardi, Marina; Ito, Amando Siuiti; Acuña, A Ulises; Alonso, Antonio

    2012-09-15

    Miltefosine (MT) is an alkylphospholipid approved for breast cancer metastasis and visceral leishmaniasis treatments, although the respective action mechanisms at the molecular level remain poorly understood. In this work, the interaction of miltefosine with the lipid component of stratum corneum (SC), the uppermost skin layer, was studied by electron paramagnetic resonance (EPR) spectroscopy of several fatty acid spin-labels. In addition, the effect of miltefosine on (i) spherical lipid vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and (ii) lipids extracted from SC was also investigated, by EPR and time-resolved polarized fluorescence methods. In SC of neonatal Wistar rats, 4% (w/w) miltefosine give rise to a large increase of the fluidity of the intercellular membranes, in the temperature range from 6 to about 50°C. This effect becomes negligible at temperatures higher that ca. 60°C. In large unilamelar vesicles of DPPC no significant changes could be observed with a miltefosine concentration 25% molar, in close analogy with the behavior of biomimetic vesicles prepared with bovine brain ceramide, behenic acid and cholesterol. In these last samples, a 25 mol% molar concentration of miltefosine produced only a modest decrease in the bilayer fluidity. Although miltefosine is not a feasible skin permeation enhancer due to its toxicity, the information provided in this work could be of utility in the development of a MT topical treatment of cutaneous leishmaniasis. PMID:22692081

  15. Branched phospholipids render lipid vesicles more susceptible to membrane-active peptides.

    PubMed

    Mitchell, Natalie J; Seaton, Pamela; Pokorny, Antje

    2016-05-01

    Iso- and anteiso-branched lipids are abundant in the cytoplasmic membranes of bacteria. Their function is assumed to be similar to that of unsaturated lipids in other organisms - to maintain the membrane in a fluid state. However, the presence of terminally branched membrane lipids is likely to impact other membrane properties as well. For instance, lipid acyl chain structure has been shown to influence the activity of antimicrobial peptides. Moreover, the development of resistance to antimicrobial agents in Staphylococcus aureus is accompanied by a shift in the fatty acid composition toward a higher fraction of anteiso-branched lipids. Little is known about how branched lipids and the location of the branch point affect the activity of membrane-active peptides. We hypothesized that bilayers containing lipids with low phase transition temperatures would tend to exclude peptides and be less susceptible to peptide-induced perturbation than those made from higher temperature melting lipids. To test this hypothesis, we synthesized a series of asymmetric phospholipids that only differ in the type of fatty acid esterified at the sn-2 position of the lipid glycerol backbone. We tested the influence of acyl chain structure on peptide activity by measuring the kinetics of release from dye-encapsulated lipid vesicles made from these synthetic lipids. The results were compared to those obtained using vesicles made from S. aureus and Staphylococcus sciuri membrane lipid extracts. Anteiso-branched phospholipids, which melt at very low temperatures, produced lipid vesicles that were only slightly less susceptible to peptide-induced dye release than those made from the iso-branched isomer. However, liposomes made from bacterial phospholipid extracts were generally much more resistant to peptide-induced perturbation than those made from any of the synthetic lipids. The results suggest that the increase in the fraction of anteiso-branched fatty acids in antibiotic-resistant strains

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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

  1. Proteomic analysis of the mosquito Aedes aegypti midgut brush border membrane vesicles

    PubMed Central

    Popova-Butler, Alexandra; Dean, Donald H.

    2009-01-01

    We analyzed brush border membrane vesicle proteins from isolated midguts of the mosquito Aedes aegypti, by two proteomic methods: two-dimensional gel electrophoresis (isoelectric focusing and SDS-PAGE) and a shotgun two-dimensional liquid chromatographic (LS/LS) approach based on multidimensional protein identification technology (MudPIT). We were interested in the most abundant proteins of the apical brush border midgut membrane. About 400 spots were detected on 2D gels and 39 spots were cored and identified by mass spectrometry. 86 proteins were identified by MudPIT. Three proteins, arginine kinase, putative allergen and actin are shown to be the most predominant proteins in the sample. The total number of 36 proteins detected by both methods represents the most abundant proteins in the BBMV. PMID:19133270

  2. 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

  3. 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.

  4. 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

  5. Decoration of Outer Membrane Vesicles with Multiple Antigens by Using an Autotransporter Approach

    PubMed Central

    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.

    2014-01-01

    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

  6. Characterization of tryptophan transport in human placental brush-border membrane vesicles.

    PubMed Central

    Ganapathy, M E; Leibach, F H; Mahesh, V B; Howard, J C; Devoe, L D; Ganapathy, V

    1986-01-01

    The characteristics of tryptophan uptake in isolated human placental brush-border membrane vesicles were investigated. Tryptophan uptake in these vesicles was predominantly Na+-independent. Uptake of tryptophan as measured with short incubations occurred exclusively by a carrier-mediated process, but significant binding of this amino acid to the membrane vesicles was observed with longer incubations. The carrier-mediated system obeyed Michaelis-Menten kinetics, with an apparent affinity constant of 12.7 +/- 1.0 microM and a maximal velocity of 91 +/- 5 pmol/15 s per mg of protein. The kinetic constants were similar in the presence and absence of a Na+ gradient. Competition experiments showed that tryptophan uptake was effectively inhibited by many neutral amino acids except proline, hydroxyproline and 2-(methylamino)isobutyric acid. The inhibitory amino acids included aromatic amino acids as well as other system-1-specific amino acids (system 1 refers to the classical L system, according to the most recent nomenclature of amino acid transport systems). The transport system showed very low affinity for D-isomers, was not affected by phloretin or glucose but was inhibited by p-azidophenylalanine and N-ethylmaleimide. The uptake rates were only minimally affected by change in pH over the range 4.5-8.0. Tryptophan uptake markedly responded to trans-stimulation, and the amino acids capable of causing trans-stimulation included all amino acids with system-1-specificity. The patterns of inhibition of uptake of tryptophan and leucine by various amino acids were very similar. We conclude that system t, which is specific for aromatic amino acids, is absent from human placenta and that tryptophan transport in this tissue occurs via system 1, which has very broad specificity. PMID:3800932

  7. 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.

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

    PubMed

    Reidl, Joachim

    2016-01-01

    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

  9. Spatial and temporal electroselection patterns in electric field stimulation of polarized luminescence from photosynthetic membrane vesicles

    PubMed Central

    Rosemberg, Yosef; Rozen, Philip; Malkin, Shmuel; Korenstein, Rafi

    1992-01-01

    Electroselection processes of charge recombination are manifested in the study of electric field induced polarized emission from photosynthetic membrane vesicles. The study explores the coupled spatial-temporal characteristics of electric field induced charge recombination by examining the dependence of the integrated polarized emission and the time dependent polarization on electric field strength. The experimental results were fitted to theoretical models by computer simulations employing empirical parameters. Simulation of the dependence of the integrated polarized components of emission on electric field strength, suggests field-dependent increased ratio between radiative and nonradiative rates of charge recombination. The observation that the initial polarization values are independent of electric field strength supports the assumption that electric field induced emission originates from the pole area and then spreads away from it towards the equator. The propagation rate of this electric field induced charge recombination from the pole area towards the equator is reflected by the decay of polarization which increases upon raising the electric field strength. Simulation of the polarization's decay, based on a calculated angle of 26.3 ± 0.4° between the transition moment of emission and the plane of the membrane, establishes coupled temporal spatial patterns of electroselection in intramembrane electron transfer invoked by exposing preilluminated photosynthetic vesicles to a homogeneous electric field. PMID:19431835

  10. 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.

  11. 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

  12. 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

  13. Platelet-mediated modulation of adaptive immunity: unique delivery of CD154 signal by platelet-derived membrane vesicles

    PubMed Central

    Sprague, Daniel L.; Elzey, Bennett D.; Crist, Scott A.; Waldschmidt, Thomas J.; Jensen, Robert J.

    2008-01-01

    Although mounting evidence indicates that platelets participate in the modulation of both innate and adaptive immunity, the mechanisms by which platelets exert these effects have not been clearly defined. The study reported herein uses a previously documented adoptive transfer model to investigate the ability of platelet-derived membrane vesicles to communicate activation signals to the B-cell compartment. The findings demonstrate for the first time that platelet-derived membrane vesicles are sufficient to deliver CD154 to stimulate antigen-specific IgG production and modulate germinal center formation through cooperation with responses elicited by CD4+ T cells. The data are consistent with the hypothesis that platelets modulate inflammation and adaptive immunity at sites distant from the location of activation and that platelet-derived membrane vesicles are sufficient to mediate the effect. PMID:18198347

  14. Active transport of maltose in membrane vesicles obtained from Escherichia coli cells producing tethered maltose-binding protein.

    PubMed Central

    Dean, D A; Fikes, J D; Gehring, K; Bassford, P J; Nikaido, H

    1989-01-01

    Attempts to reconstitute periplasmic binding protein-dependent transport activity in membrane vesicles have often resulted in systems with poor and rather inconsistent activity, possibly because of the need to add a large excess of purified binding protein to the vesicles. We circumvented this difficulty by using a mutant which produces a precursor maltose-binding protein that is translocated across the cytoplasmic membrane but is not cleaved by the signal peptidase (J. D. Fikes and P. J. Bassford, Jr., J. Bacteriol. 169:2352-2359, 1987). The protein remains tethered to the cytoplasmic membrane, presumably through the hydrophobic signal sequence, and we show here that the spheroplasts and membrane vesicles prepared from this mutant catalyze active maltose transport without the addition of purified maltose-binding protein. In vesicles, the transport requires electron donors, such as ascorbate and phenazine methosulfate or D-lactate. However, inhibition by dicyclohexylcarbodiimide and stimulation of transport by the inculsion of ADP or ATP in the intravesicular space suggest that ATP (or compounds derived from it) is involved in the energization of the transport. The transport activity of intact cells can be recovered without much inactivation in the vesicles, and their high activity and ease of preparation will be useful in studies of the mechanism of the binding protein-dependent transport process. Images PMID:2644203

  15. The oxidative activities of membrane vesicles from Bacillus caldolyticus. Energy-dependence of succinate oxidation.

    PubMed

    Dawson, A G; Chappell, J B

    1978-02-15

    1. The properties of membrane vesicles from the extreme thermophile Bacillus caldolyticus were investigated. 2. Vesicles prepared by exposure of spheroplasts to ultrasound contained cytochromes a, b and c, and at 50 degrees C they rapidly oxidized NADH and ascorbate in the presence of tetramethyl-p-phenylenediamine. Succinate and l-malate were oxidized more slowly, and dl-lactate, l-alanine and glycerol 1-phosphate were not oxidized. 3. In the absence of proton-conducting uncouplers the oxidation of NADH was accompanied by a net translocation of H(+) into the vesicles. Hydrolysis of ATP by a dicyclohexylcarbodi-imide-sensitive adenosine triphosphatase was accompanied by a similarly directed net translocation of H(+). 4. Uncouplers (carbonyl cyanide p-trifluoromethoxyphenylhydrazone or valinomycin plus NH(4) (+)) prevented net H(+) translocation but stimulated ATP hydrolysis, NADH oxidation and ascorbate oxidation. The last result suggested an energy-conserving site in the respiratory chain between cytochrome c and oxygen. 5. Under anaerobic conditions the reduction of cytochrome b by ascorbate (with tetramethyl-p-phenylenediamine) was stimulated by ATP hydrolysis, indicating an energy-conserving site between cytochrome b and cytochrome c. However, no reduction of NAD(+) supported by oxidation of succinate, malate or ascorbate occurred, neither did it with these substrates in the presence of ATP under anaerobic conditions, suggesting that there was no energy-conserving site between NADH and cytochrome b. 6. Succinate oxidation, in contrast with that of NADH and ascorbate, was strongly inhibited by uncouplers and stimulated by ATP hydrolysis. These effects were not observed when phenazine methosulphate, which transfers electrons from succinate dehydrogenase directly to oxygen, was present. It was concluded that in these vesicles the oxidation of succinate was energy-dependent and that the reoxidation of reduced succinate dehydrogenase was dependent on the outward

  16. 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

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. Integrated light-scattering spectroscopy, a sensitive probe for peptide-vesicle binding: application to the membrane-bound colicin E1 channel peptide.

    PubMed Central

    Strawbridge, K. B.; Palmer, L. R.; Merrill, A. R.; Hallett, F. R.

    1995-01-01

    Integrated light-scattering (ILS) spectroscopy was used to monitor the binding of the colicin E1 channel peptide to POPC:POPG large unilamellar vesicles (LUV; 60:40, mol:mol) at acidic pH (3.5). Binding conditions were chosen such that nearly all of the channel peptide was bound to the vesicles with little free peptide remaining in solution. The increase in vesicle size upon the insertion of the channel peptide was measured by performing a discrete inversion technique on data obtained from an ILS spectrometer. Vesicle size number distributions were determined for five different systems having peptide/vesicle ratios of approximately 0, 77, 154, 206, and 257. The experiment was repeated four times (twice at two different vesicle concentrations) to determine reproducibility. The relative changes in vesicle radius upon peptide binding to the membrane vesicles was remarkably reproducible even though these changes represented only a few nanometers. A comparison of vesicle size number distributions in the absence of bound peptide was made between ILS and dynamic light scattering (DLS) data and showed similar results. However, DLS was incapable of detecting the small changes due to peptide-induced vesicle swelling. The membrane-bound volume of the colicin E1 channel peptide was approximately 177 +/- 22 nm3. These data indicate that in the absence of a membrane potential (closed channel state) the colicin E1 channel peptide inserts into the membrane resulting in a significant displacement of the lipid bilayer as evidenced from the dose-dependent increase in the vesicle radius. These results indicate that ILS spectroscopy is a sensitive sizing technique that is capable of detecting relatively small changes in membrane vesicles and may have a wide application in the determination of peptide binding to membrane vesicles. Images FIGURE 2 PMID:7711234

  5. 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

  6. Proteomic characterization of the whole secretome of Legionella pneumophila and functional analysis of outer membrane vesicles.

    PubMed

    Galka, Frank; Wai, Sun Nyunt; Kusch, Harald; Engelmann, Susanne; Hecker, Michael; Schmeck, Bernd; Hippenstiel, Stefan; Uhlin, Bernt Eric; Steinert, Michael

    2008-05-01

    Secretion of effector molecules is one of the major mechanisms by which the intracellular human pathogen Legionella pneumophila interacts with host cells during infection. Specific secretion machineries which are responsible for the subfraction of secreted proteins (soluble supernatant proteins [SSPs]) and the production of bacterial outer membrane vesicles (OMVs) both contribute to the protein composition of the extracellular milieu of this lung pathogen. Here we present comprehensive proteome reference maps for both SSPs and OMVs. Protein identification and assignment analyses revealed a total of 181 supernatant proteins, 107 of which were specific to the SSP fraction and 33 of which were specific to OMVs. A functional classification showed that a large proportion of the identified OMV proteins are involved in the pathogenesis of Legionnaires' disease. Zymography and enzyme assays demonstrated that the SSP and OMV fractions possess proteolytic and lipolytic enzyme activities which may contribute to the destruction of the alveolar lining during infection. Furthermore, it was shown that OMVs do not kill host cells but specifically modulate their cytokine response. Binding of immunofluorescently stained OMVs to alveolar epithelial cells, as visualized by confocal laser scanning microscopy, suggested that there is delivery of a large and complex group of proteins and lipids in the infected tissue in association with OMVs. On the basis of these new findings, we discuss the relevance of protein sorting and compartmentalization of virulence factors, as well as environmental aspects of the vesicle-mediated secretion. PMID:18250176

  7. 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.

  8. Presence of a sodium-translocating ATPase in membrane vesicles of the homoacetogenic bacterium Acetobacterium woodii.

    PubMed

    Heise, R; Müller, V; Gottschalk, G

    1992-06-01

    Inverted membrane vesicles of the homoacetogenic bacterium Acetobacterium woodii catalyzed the hydrolysis of ATP with a rate of 100-150 nmol.min-1.mg protein-1. The ATPase was stimulated 1.4-1.6-fold by NaCl and inhibited by N,N'-dicyclohexylcarbodiimide tributyltin or azide. The degree of inhibition caused by F0-directed but not F1-directed inhibitors was affected by the Na+ concentration in the medium. These experiments indicated the presence of a sodium-translocating ATPase. This was verified by transport studies. Upon addition of ATP to inverted vesicles, 22Na+ was actively transported into the intravesicular space up to a 24-fold accumulation. Na+ transport was inhibited by the sodium ionophore N,N,N',N',-tetracyclohexyl-1,2-phenyl-enedioxydiacetamide but stimulated by valinomycin with potassium whereas the protonophore 3,5,-di-tert-butyl-4-hydroxybenzylidenemalonitrile was without effect. N,N'-dicyclohexylcarbodiimide and tributyltin inhibited 22Na+ transport. These experiments are in accordance with a primary electrogenic Na+ transport as catalyzed by a F1F0-ATPase. PMID:1534543

  9. Analysis of bacteria-derived outer membrane vesicles using tunable resistive pulse sensing

    NASA Astrophysics Data System (ADS)

    Bogomolny, Evgeny; Hong, Jiwon; Blenkiron, Cherie; Simonov, Denis; Dauros, Priscila; Swift, Simon; Phillips, Anthony; Willmott, Geoff R.

    2015-03-01

    Accurate characterization of submicron particles within biological fluids presents a major challenge for a wide range of biomedical research. Detection, characterization and classification are difficult due to the presence of particles and debris ranging from single molecules up to particles slightly smaller than cells. Especial interest arises from extracellular vesicles (EVs) which are known to play a pivotal role in cell-signaling in multicellular organisms. Tunable resistive pulse sensing (TRPS) is increasingly proving to be a useful tool for high throughput particle-by-particle analysis of EVs and other submicron particles. This study examines the capability of TRPS for characterization of EVs derived from bacteria, also called outer membrane vesicles (OMVs). Measurement of a size distribution (124 +/- 3 nm modal diameter) and concentration (lower bound 7.4 x 109 mL-1) are demonstrated using OMVs derived from uropathogenic Escherichia coli. Important aspects of measurement are discussed, including sample preparation and size selection. Application of TRPS to study EVs could assist the development of these particles in clinical diagnostics and therapeutics.

  10. 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.

  11. 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. PMID:25165491

  12. 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

  13. Cytotoxic effects of Kingella kingae outer membrane vesicles on human cells

    PubMed Central

    Maldonado, R; Wei, R; Kachlany, SC; Kazi, M; Balashova, NV

    2011-01-01

    Kingella kingae is an emerging pathogen causing osteoarticular infections in pediatric patients. Electron microscopy of K. kingae clinical isolates revealed the heterogeneously-sized membranous structures blebbing from the outer membrane that were classified as outer membrane vesicles (OMVs). OMVs purified from the secreted fraction of a septic arthritis K. kingae isolate were characterized. Among several major proteins, K. kingae OMVs contained virulence factors RtxA toxin and PilC2 pilus adhesin. RtxA was also found secreted as a soluble protein in the extracellular environment indicating that the bacterium may utilize different mechanisms for the toxin delivery. OMVs were shown to be hemolytic and possess some leukotoxic activity while high leukotoxicity was detected in the non-hemolytic OMV-free component of the secreted fraction. OMVs were internalized by human osteoblasts and synovial cells. Upon interaction with OMVs, the cells produced increased levels of human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleuskin 6 (IL-6) suggesting that these cytokines might be involved in the signaling response of infected joint and bone tissues during natural K. kingae infection. This study is the first report of OMV production by K. kingae and demonstrates that OMVs are a complex virulence factor of the organism causing cytolytic and inflammatory effects on host cells. PMID:21443941

  14. Phospholipid-cationic lipid interactions: influences on membrane and vesicle properties.

    PubMed

    Campbell, R B; Balasubramanian, S V; Straubinger, R M

    2001-05-01

    Liposomes composed of synthetic dialkyl cationic lipids and zwitterionic phospholipids such as dioleoylphosphatidylethanolamine have been studied extensively as vehicles for gene delivery, but the broader potentials of these cationic liposomes for drug delivery have not. An understanding of phospholipid-cationic lipid interactions is essential for rational development of this potential. We evaluated the effect of the cationic lipid DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium) on liposome physical properties such as size and membrane domain structure. DSC (differential scanning calorimetry) showed progressive decrease and broadening of the phase transition temperature of dipalmitoylphosphatidylcholine (DPPC) with increasing fraction of DOTAP, in the range of 0.4-20 mol%. Laurdan (6-dodecanolyldimethylamino-naphthalene), a fluorescent probe of membrane domain structure, showed that DOTAP and DPPC remained miscible at all ratios tested. DOTAP reduced the size of spontaneously-forming PC-containing liposomes, regardless of the acyl chain length and degree of saturation. The anionic lipid DOPG (dioleoylphosphatidylglycerol) had similar effects on DPPC membrane fluidity and size. However, DOTAP/DOPC (50/50) vesicles were taken up avidly by OVCAR-3 human ovarian tumor cells, in contrast to DOPG/DOPC (50/50) liposomes. Overall, DOTAP exerts potent effects on bilayer physical properties, and may provide advantages for drug delivery. PMID:11334622

  15. 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.

  16. Perinuclear Localization of Internalized Outer Membrane Vesicles Carrying Active Cytolethal Distending Toxin from Aggregatibacter actinomycetemcomitans

    PubMed Central

    Rompikuntal, Pramod Kumar; Thay, Bernard; Khan, Muhammad Khanzeb; Alanko, Jonna; Penttinen, Anna-Maija; Asikainen, Sirkka; Wai, Sun Nyunt

    2012-01-01

    Aggregatibacter actinomycetemcomitans is implicated in aggressive forms of periodontitis. Similarly to several other Gram-negative species, this organism produces and excretes a cytolethal distending toxin (CDT), a genotoxin associated with cell distention, G2 cell cycle arrest, and/or apoptosis in many mammalian cell types. In this study, we have identified A. actinomycetemcomitans outer membrane vesicles (OMVs) as a vehicle for simultaneous delivery of multiple proteins, including CDT, into human cells. The OMV proteins were internalized in both HeLa cells and human gingival fibroblasts (HGF) via a mechanism of OMV fusion with lipid rafts in the plasma membrane. The active toxin unit, CdtB, was localized inside the nucleus of the intoxicated cells, whereas OmpA and proteins detected using an antibody specific to whole A. actinomycetemcomitans serotype a cells had a perinuclear distribution. In accordance with a tight association of CdtB with OMVs, vesicles isolated from A. actinomycetemcomitans strain D7SS (serotype a), in contrast to OMVs from a D7SS cdtABC mutant, induced a cytolethal distending effect on HeLa and HGF cells, indicating that OMV-associated CDT was biologically active. Association of CDT with OMVs was also observed in A. actinomycetemcomitans isolates belonging to serotypes b and c, indicating that OMV-mediated release of CDT may be conserved in A. actinomycetemcomitans. Although the role of A. actinomycetemcomitans OMVs in periodontal disease has not yet been elucidated, our present data suggest that OMVs could deliver biologically active CDT and additional virulence factors into susceptible cells of the periodontium. PMID:22025516

  17. Characteristics of glutamine transport in dog jejunal brush-border membrane vesicles.

    PubMed

    Bulus, N M; Abumrad, N N; Ghishan, F K

    1989-07-01

    The present study characterizes glutamine transport across brush-border membrane vesicles (BBMV) prepared from dog jejunum. The purity of these vesicles was demonstrated by a 20-fold enrichment of leucine aminopeptidase, a marker for BBM. Glutamine uptake was found to occur into an osmotically active space with no membrane binding and to exhibit temperature and pH dependence (optimal uptake at pH 7-7.5). Glutamine uptake was driven by an inwardly directed Na+ gradient with a distinct overshoot not observed under K+ gradient. Lithium could not substitute for Na+ as a stimulator of glutamine uptake. Na+-dependent glutamine uptake was not inhibited by methylaminoisobutyric acid, a typical substrate for system A, and was found to be electrogenic and saturable with a Km of 0.97 +/- 0.58 mM and a Vmax of 3.93 +/- 0.99 nmol.mg protein-1.10 s-1. A Na+-glutamine coupling ratio of 1:1 could be demonstrated by a plot of Hill transformation. Na+-independent glutamine uptake was found to be electroneutral and saturable with a Km of 3.70 +/- 0.66 mM and a Vmax of 2.70 +/- 1.55 nmol.mg protein-1.10 s-1. Inhibition studies confirmed the presence of a Na+-dependent as well as a Na+-independent carrier for glutamine uptake. We conclude that glutamine uptake across dog BBMV occurs via two transport systems: a Na+-dependent high-affinity system similar to the neutral brush-border system and a Na+-independent lower-affinity system similar to system L. PMID:2750912

  18. 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

  19. 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

  20. Role of tetanus neurotoxin insensitive vesicle-associated membrane protein in membrane domains transport and homeostasis

    PubMed Central

    Molino, Diana; Nola, Sébastien; Lam, Sin Man; Verraes, Agathe; Proux-Gillardeaux, Véronique; Boncompain, Gaëlle; Perez, Franck; Wenk, Markus; Shui, Guanghou; Danglot, Lydia; Galli, Thierry

    2015-01-01

    Biological membranes in eukaryotes contain a large variety of proteins and lipids often distributed in domains in plasma membrane and endomembranes. Molecular mechanisms responsible for the transport and the organization of these membrane domains along the secretory pathway still remain elusive. Here we show that vesicular SNARE TI-VAMP/VAMP7 plays a major role in membrane domains composition and transport. We found that the transport of exogenous and endogenous GPI-anchored proteins was altered in fibroblasts isolated from VAMP7-knockout mice. Furthermore, disassembly and reformation of the Golgi apparatus induced by Brefeldin A treatment and washout were impaired in VAMP7-depleted cells, suggesting that loss of VAMP7 expression alters biochemical properties and dynamics of the Golgi apparatus. In addition, lipid profiles from these knockout cells indicated a defect in glycosphingolipids homeostasis. We conclude that VAMP7 is required for effective transport of GPI–anchored proteins to cell surface and that VAMP7-dependent transport contributes to both sphingolipids and Golgi homeostasis. PMID:26196023

  1. 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

  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. Measuring H(+) Pumping and Membrane Potential Formation in Sealed Membrane Vesicle Systems.

    PubMed

    Wielandt, Alex Green; Palmgren, Michael G; Fuglsang, Anja Thoe; Günther-Pomorski, Thomas; Justesen, Bo Højen

    2016-01-01

    The activity of enzymes involved in active transport of matter across lipid bilayers can conveniently be assayed by measuring their consumption of energy, such as ATP hydrolysis, while it is more challenging to directly measure their transport activities as the transported substrate is not converted into a product and only moves a few nanometers in space. Here, we describe two methods for the measurement of active proton pumping across lipid bilayers and the concomitant formation of a membrane potential, applying the dyes 9-amino-6-chloro-2-methoxyacridine (ACMA) and oxonol VI. The methods are exemplified by assaying transport of the Arabidopsis thaliana plasma membrane H(+)-ATPase (proton pump), which after heterologous expression in Saccharomyces cerevisiae and subsequent purification has been reconstituted in proteoliposomes. PMID:26695032

  4. Electrical properties and fusion dynamics of in vitro membrane vesicles derived from separate parts of the contractile vacuole complex of Paramecium multimicronucleatum.

    PubMed

    Sugino, Kazuyuki; Tominaga, Takashi; Allen, Richard D; Naitoh, Yutaka

    2005-10-01

    The contractile vacuole complex of Paramecium multimicronucleatum transforms into membrane-bound vesicles on excision from the cell. The I-V relationship was linear in a voltage range of -80 to +80 mV in all vesicles, despite being derived from different parts of the contractile vacuole complex. No voltage-gated unit currents were observed in membrane patches from the vesicles. Vesicles derived from the radial arm showed a membrane potential of >10 mV, positive with reference to the cytosol, while those derived from the contractile vacuole showed a residual (<5 mV) membrane potential. The electrogenic V-ATPases in the decorated spongiome are responsible for the positive potential, and Cl- leakage channels are responsible for the residual potential. The specific resistance of the vesicle membrane (approximately 6 kOmega cm2) increased, while the membrane potential shifted in a negative direction when the vesicle rounded. An increase in the membrane tension (to approximately 5 x 10(-3) N m(-1)) is assumed to reduce the Cl- leakage conductance. It is concluded that neither voltage- nor mechano-sensitive ion channels are involved in the control of the fluid segregation and membrane dynamics that govern fluid discharge cycles in the contractile vacuole complex. The membrane vesicles shrank when the external osmolarity was increased, and swelled when the osmolarity was decreased, implying that the contractile vacuole complex membrane is water permeable. The water permeability of the membrane was 4-20 x 10(-7) microm s(-1) Pa(-1). The vesicles containing radial arm membrane swelled after initially shrinking when exposed to higher external osmolarity, implying that the V-ATPases energize osmolyte transport mechanisms that remain functional in the vesicle membrane. The vesicles showed an abrupt (<30 ms), slight, slackening after rounding to the maximum extent. Similar slackening was also observed in the contractile vacuoles in situ before the opening of the contractile

  5. 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+).

  6. 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.

  7. 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

  8. 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

  9. Characterization and vaccine potential of outer membrane vesicles produced by Haemophilus parasuis

    DOE PAGESBeta

    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. Intranasal Immunization with Nontypeable Haemophilus influenzae Outer Membrane Vesicles Induces Cross-Protective Immunity in Mice

    PubMed Central

    Roier, Sandro; Leitner, Deborah R.; Iwashkiw, Jeremy; Schild-Prüfert, Kristina; Feldman, Mario F.; Krohne, Georg; Reidl, Joachim; Schild, Stefan

    2012-01-01

    Abstract Haemophilus influenzae is a Gram-negative human-restricted bacterium that can act as a commensal and a pathogen of the respiratory tract. Especially nontypeable H. influenzae (NTHi) is a major threat to public health and is responsible for several infectious diseases in humans, such as pneumonia, sinusitis, and otitis media. Additionally, NTHi strains are highly associated with exacerbations in patients suffering from chronic obstructive pulmonary disease. Currently, there is no licensed vaccine against NTHi commercially available. Thus, this study investigated the utilization of outer membrane vesicles (OMVs) as a potential vaccine candidate against NTHi infections. We analyzed the immunogenic and protective properties of OMVs derived from various NTHi strains by means of nasopharyngeal immunization and colonization studies with BALB/c mice. The results presented herein demonstrate that an intranasal immunization with NTHi OMVs results in a robust and complex humoral and mucosal immune response. Immunoprecipitation revealed the most important immunogenic proteins, such as the heme utilization protein, protective surface antigen D15, heme binding protein A, and the outer membrane proteins P1, P2, P5 and P6. The induced immune response conferred not only protection against colonization with a homologous NTHi strain, which served as an OMV donor for the immunization mixtures, but also against a heterologous NTHi strain, whose OMVs were not part of the immunization mixtures. These findings indicate that OMVs derived from NTHi strains have a high potential to act as a vaccine against NTHi infections. PMID:22880074

  11. Structural modifications of outer membrane vesicles to refine them as vaccine delivery vehicles

    PubMed Central

    Kim, Sang-Hyun; Kim, Keun-Su; Lee, Sang-Rae; Kim, Ekyune; Kim, Myeong-Su; Lee, Eun-Young; Gho, Yong Song; Kim, Jung-Woo; Bishop, Russell E.; Chang, Kyu-Tae

    2016-01-01

    In an effort to devise a safer and more effective vaccine delivery system, outer membrane vesicles (OMVs) were engineered to have properties of intrinsically low endotoxicity sufficient for the delivery of foreign antigens. Our strategy involved mutational inactivation of the MsbB (LpxM) lipid A acyltransferase to generate OMVs of reduced endotoxicity from Escherichia coli (E. coli) O157:H7. The chromosomal tagging of a foreign FLAG epitope within an OmpA-fused protein was exploited to localize the FLAG epitope in the OMVs produced by the E. coli mutant having the defined msbB and the ompA::FLAG mutations. It was confirmed that the desired fusion protein (OmpA::FLAG) was expressed and destined to the outer membrane (OM) of the E. coli mutant from which the OMVs carrying OmpA::FLAG are released during growth. A luminal localization of the FLAG epitope within the OMVs was inferred from its differential immunoprecipitation and resistance to proteolytic degradation. Thus, by using genetic engineering-based approaches, the native OMVs were modified to have both intrinsically low endotoxicity and a foreign epitope tag to establish a platform technology for development of multifunctional vaccine delivery vehicles. PMID:19695218

  12. Proteomic characterization of the outer membrane vesicle of Pseudomonas putida KT2440.

    PubMed

    Choi, Chi-Won; Park, Edmond Changkyun; Yun, Sung Ho; Lee, Sang-Yeop; Lee, Yeol Gyun; Hong, Yeonhee; Park, Kyeong Ryang; Kim, Sang-Hyun; Kim, Gun-Hwa; Kim, Seung Il

    2014-10-01

    Outer membrane vesicles (OMVs) are produced by various pathogenic Gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. In this study, we isolated OMVs from a representative soil bacterium, Pseudomonas putida KT2440, which has a biodegradative activity toward various aromatic compounds. Proteomic analysis identified the outer membrane proteins (OMPs) OprC, OprD, OprE, OprF, OprH, OprG, and OprW as major components of the OMV of P. putida KT2440. The production of OMVs was dependent on the nutrient availability in the culture media, and the up- or down-regulation of specific OMPs was observed according to the culture conditions. In particular, porins (e.g., benzoate-specific porin, BenF-like porin) and enzymes (e.g., catechol 1,2-dioxygenase, benzoate dioxygenase) for benzoate degradation were uniquely found in OMVs prepared from P. putida KT2440 that were cultured in media containing benzoate as the energy source. OMVs of P. putida KT2440 showed low pathological activity toward cultured cells that originated from human lung cells, which suggests their potential as adjuvants or OMV vaccine carriers. Our results suggest that the protein composition of the OMVs of P. putida KT2440 reflects the characteristics of the total proteome of P. putida KT2440. PMID:25198519

  13. 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.

  14. 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

  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. 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

  17. 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.

  18. 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

  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. 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

  1. Temporin L: antimicrobial, haemolytic and cytotoxic activities, and effects on membrane permeabilization in lipid vesicles.

    PubMed Central

    Rinaldi, Andrea C; Mangoni, Maria Luisa; Rufo, Anna; Luzi, Carla; Barra, Donatella; Zhao, Hongxia; Kinnunen, Paavo K J; Bozzi, Argante; Di Giulio, Antonio; Simmaco, Maurizio

    2002-01-01

    The temporins are a family of small, linear antibiotic peptides with intriguing biological properties. We investigated the antibacterial, haemolytic and cytotoxic activities of temporin L (FVQWFSKFLGRIL-NH2), isolated from the skin of the European red frog Rana temporaria. The peptide displayed the highest activity of temporins studied to date, against both human erythrocytes and bacterial and fungal strains. At variance with other known temporins, which are mainly active against Gram-positive bacteria, temporin L was also active against Gram-negative strains such as Pseudomonas aeruginosa A.T.C.C. 15692 and Escherichia coli D21 at concentrations comparable with those that are microbiocidal to Gram-positive bacteria. In addition, temporin L was cytotoxic to three different human tumour cell lines (Hut-78, K-562 and U-937), causing a necrosis-like cell death, although sensitivity to the peptide varied markedly with the specific cell line tested. A study of the interaction of temporin L with liposomes of different lipid compositions revealed that the peptide causes perturbation of bilayer integrity of both neutral and negatively charged membranes, as revealed by the release of a vesicle-encapsulated fluorescent marker, and that the action of the peptide is modulated to some extent by membrane lipid composition. In particular, the presence of negatively charged lipids in the model bilayer inhibits the lytic power of temporin L. We also show that the release of fluorescent markers caused by temporin L is size-dependent and that the peptide does not have a detergent-like effect on the membrane, suggesting that perturbation of bilayer organization takes place on a local scale, i.e. through the formation of pore-like openings. PMID:12133008

  2. The theory of semipermeable vesicles and membranes: An integral-equation approach. II. Donnan equilibrium

    NASA Astrophysics Data System (ADS)

    Zhou, Yaoqi; Stell, George

    1988-12-01

    Integral equations that yield the charge and density profiles are derived for a Donnan system, in which an ionic solution is separated into two regions by a semipermeable membrane (SPM) or a spherical semipermeable vesicle (SPV). These equations are obtained from the Ornstein-Zernike (OZ) equation. We show how quantitative results can be obtained from either the mean spherical approximation (MSA) closure or the hypernetted-chain (HNC) closure for profiles. Use is made of bulk-correlation input obtained by means of the Debye-Hückel approximation, the MSA approximation, or the HNC approximation. The resulting approximations will be referred as MSA/DH, HNC/DH, MSA/MSA, etc. The system on which we focus contains three charged hard-sphere species: cation, anion, and a large ion (a protein or polymer ion) separated by a plane SPM, through which the large ion cannot pass, and to one side of which all large ions are confined, or a spherical SPV, outside of which the large ions are confined. Analytical expressions for the bulk density ratio between the two sides of a plane membrane as well as the membrane potential in various approximations are obtained. Results obtained from these expresssions are compared with the results obtained by equating electrochemical potentials. A new contact-value theorem is provided for the plane SPM system. Analytical solutions for the charge profile and the potential profile in the MSA/DH approximation are obtained. It turns out that results obtained in the HNC/DH approximation are exactly the same as those obtained by using 1D nonlinear Poisson-Boltzmann equations if the repulsive cores of the macroions are neglected.

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. Further characterization of ATP-initiated calcification by matrix vesicles isolated from rachitic rat cartilage. Membrane perturbation by detergents and deposition of calcium pyrophosphate by rachitic matrix vesicles.

    PubMed

    Hsu, H H; Camacho, N P; Anderson, H C

    1999-01-12

    Although membrane associated enzymes such as ATPase, alkaline phosphatase, and NTP pyrophosphohydrolase in matrix vesicles (MVs) may underlie the mechanisms of ATP-promoted calcification, prior to the current investigation, the role of the MV membrane in calcification had not been addressed. In this study, various perturbations were introduced to the MV membrane in in vitro calcification systems to determine ideal conditions for ATP-initiated calcification by MVs isolated from rachitic rat epiphyseal cartilage. Membrane integrity appears to be required, since the rupture of the vesicular membrane by vigorously mixing with 10% butanol abolished calcification. In contrast, a mild treatment of MVs with low concentrations (e.g., 0.01%, which is much below the critical concentration for micelle formation) of either neutral Triton X-100 or anionic deoxycholate stimulated calcification by >2-fold, without inducing obvious changes in vesicular appearance. Fourier transform infrared spectroscopic studies were done to identify the mineral phase formed in these experiments. For the first time, rachitic MVs were shown to induce the formation of a calcium pyrophosphate dihydrate-like phase after their exposure to calcifying medium with 1 mM ATP. The integration of spectral areas indicated that calcification was enhanced by Triton X-100. The detergent effect was reversible and appeared to be not mediated through activation of ATPase, alkaline phosphatase, or ATP pyrophosphohydrolase. In contrast to neutral Triton X-100 and anionic deoxycholate, cationic cetyltrimethylammonium bromide inhibited both ATPase activity (I50=10 microM) and ATP-initiated calcification. These observations suggest that membrane perturbations can affect calcification and that the presence of NTP-pyrophosphohydrolase in MVs may play a role in the deposition of CaPPi in rachitic cartilage. PMID:9889389

  8. 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

  9. Flocculation of Escherichia coli Cells in Association with Enhanced Production of Outer Membrane Vesicles

    PubMed Central

    Nguyen, Minh Hong; Yajima, Reiki; Taya, Masahito

    2015-01-01

    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

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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

  15. Long-Distance Delivery of Bacterial Virulence Factors by Pseudomonas aeruginosa Outer Membrane Vesicles

    PubMed Central

    Bomberger, Jennifer M.; MacEachran, Daniel P.; Coutermarsh, Bonita A.; Ye, Siying; O'Toole, George A.; Stanton, Bruce A.

    2009-01-01

    Bacteria use a variety of secreted virulence factors to manipulate host cells, thereby causing significant morbidity and mortality. We report a mechanism for the long-distance delivery of multiple bacterial virulence factors, simultaneously and directly into the host cell cytoplasm, thus obviating the need for direct interaction of the pathogen with the host cell to cause cytotoxicity. We show that outer membrane–derived vesicles (OMV) secreted by the opportunistic human pathogen Pseudomonas aeruginosa deliver multiple virulence factors, including β-lactamase, alkaline phosphatase, hemolytic phospholipase C, and Cif, directly into the host cytoplasm via fusion of OMV with lipid rafts in the host plasma membrane. These virulence factors enter the cytoplasm of the host cell via N-WASP–mediated actin trafficking, where they rapidly distribute to specific subcellular locations to affect host cell biology. We propose that secreted virulence factors are not released individually as naked proteins into the surrounding milieu where they may randomly contact the surface of the host cell, but instead bacterial derived OMV deliver multiple virulence factors simultaneously and directly into the host cell cytoplasm in a coordinated manner. PMID:19360133

  16. 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.

  17. 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

  18. Bacterial Outer Membrane Vesicles Mediate Cytosolic Localization of LPS and Caspase-11 Activation.

    PubMed

    Vanaja, Sivapriya Kailasan; Russo, Ashley J; Behl, Bharat; Banerjee, Ishita; Yankova, Maya; Deshmukh, Sachin D; Rathinam, Vijay A K

    2016-05-19

    Sensing of lipopolysaccharide (LPS) in the cytosol triggers caspase-11 activation and is central to host defense against Gram-negative bacterial infections and to the pathogenesis of sepsis. Most Gram-negative bacteria that activate caspase-11, however, are not cytosolic, and the mechanism by which LPS from these bacteria gains access to caspase-11 in the cytosol remains elusive. Here, we identify outer membrane vesicles (OMVs) produced by Gram-negative bacteria as a vehicle that delivers LPS into the cytosol triggering caspase-11-dependent effector responses in vitro and in vivo. OMVs are internalized via endocytosis, and LPS is released into the cytosol from early endosomes. The use of hypovesiculating bacterial mutants, compromised in their ability to generate OMVs, reveals the importance of OMVs in mediating the cytosolic localization of LPS. Collectively, these findings demonstrate a critical role for OMVs in enabling the cytosolic entry of LPS and, consequently, caspase-11 activation during Gram-negative bacterial infections. PMID:27156449

  19. 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

  20. 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

  1. 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

  2. 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

  3. Isolation, Characterization and Biological Properties of Membrane Vesicles Produced by the Swine Pathogen Streptococcus suis

    PubMed Central

    Haas, Bruno; Grenier, Daniel

    2015-01-01

    Streptococcus suis, more particularly serotype 2, is a major swine pathogen and an emerging zoonotic agent worldwide that mainly causes meningitis, septicemia, endocarditis, and pneumonia. Although several potential virulence factors produced by S. suis have been identified in the last decade, the pathogenesis of S. suis infections is still not fully understood. In the present study, we showed that S. suis produces membrane vesicles (MVs) that range in diameter from 13 to 130 nm and that appear to be coated by capsular material. A proteomic analysis of the MVs revealed that they contain 46 proteins, 9 of which are considered as proven or suspected virulence factors. Biological assays confirmed that S. suis MVs possess active subtilisin-like protease (SspA) and DNase (SsnA). S. suis MVs degraded neutrophil extracellular traps, a property that may contribute to the ability of the bacterium to escape the host defense response. MVs also activated the nuclear factor-kappa B (NF-κB) signaling pathway in both monocytes and macrophages, inducing the secretion of pro-inflammatory cytokines, which may in turn contribute to increase the permeability of the blood brain barrier. The present study brought evidence that S. suis MVs may play a role as a virulence factor in the pathogenesis of S. suis infections, and given their composition be an excellent candidate for vaccine development. PMID:26110524

  4. 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

  5. A new Vibrio cholerae sRNA modulates colonization and affects release of outer membrane vesicles

    PubMed Central

    Song, Tianyan; Mika, Franziska; Lindmark, Barbro; Liu, Zhi; Schild, Stefan; Bishop, Anne; Zhu, Jun; Camilli, Andrew; Johansson, Jörgen; Vogel, Jörg; Wai, Sun Nyunt

    2008-01-01

    We discovered a new small non-coding RNA (sRNA) gene, vrrA of Vibrio cholerae O1 strain A1552. A vrrA mutant overproduces OmpA porin, and we demonstrate that the 140 nt VrrA RNA represses ompA translation by base-pairing with the 5′ region of the mRNA. The RNA chaperone Hfq is not stringently required for VrrA action, but expression of the vrrA gene requires the membrane stress sigma factor, σE, suggesting that VrrA acts on ompA in response to periplasmic protein folding stress. We also observed that OmpA levels inversely correlated with the number of outer membrane vesicles (OMVs), and that VrrA increased OMV production comparable to loss of OmpA. VrrA is the first sRNA known to control OMV formation. Moreover, a vrrA mutant showed a fivefold increased ability to colonize the intestines of infant mice as compared with the wild type. There was increased expression of the main colonization factor of V. cholerae, the toxin co-regulated pili, in the vrrA mutant as monitored by immunoblot detection of the TcpA protein. VrrA overproduction caused a distinct reduction in the TcpA protein level. Our findings suggest that VrrA contributes to bacterial fitness in certain stressful environments, and modulates infection of the host intestinal tract. PMID:18681937

  6. Surface display of a borrelial lipoprotein on meningococcal outer membrane vesicles.

    PubMed

    Salverda, Merijn L M; Meinderts, Sanne M; Hamstra, Hendrik-Jan; Wagemakers, Alex; Hovius, Joppe W R; van der Ark, Arno; Stork, Michiel; van der Ley, Peter

    2016-02-17

    Outer Membrane Vesicles (OMVs) are gaining attention as vaccine candidates. The successful expression of heterologous antigens in OMVs, with the OMV functioning both as adjuvant and delivery vehicle, has greatly enhanced their vaccine potential. Since there are indications that surface exposed antigens might induce a superior immune response, targeting of heterologous antigens to the OMV surface is of special interest. Several systems for surface display of heterologous antigens on OMVs have been developed. However, these systems have not been used to display lipidated membrane-associated proteins known as lipoproteins, which are emerging as key targets for protective immunity. We were therefore interested to see whether we could express a foreign lipoprotein on the outer surface of OMVs. When outer surface protein A (OspA), a borrelial surface-exposed lipoprotein, was expressed in meningococci, it was found that although OspA was present in OMVs, it was no longer surface-exposed. Therefore, a set of fusions of OspA to different regions of factor H binding protein (fHbp), a meningococcal surface-exposed lipoprotein, were designed and tested for their surface-exposure. An N-terminal part of fHbp was found to be necessary for the successful surface display of OspA on meningococcal OMVs. When mice were immunized with this set of OMVs, an OspA-specific antibody response was only elicited by OMVs with clearly surface-exposed OspA, strengthening the idea that the exact positioning of an antigen in the OMV affects the immune response. This method for the surface display of heterologous lipoproteins on OMVs is a step forward in the development of OMVs as a vaccine platform. PMID:26801064

  7. A splice-isoform of vesicle-associated membrane protein-1 (VAMP-1) contains a mitochondrial targeting signal.

    PubMed

    Isenmann, S; Khew-Goodall, Y; Gamble, J; Vadas, M; Wattenberg, B W

    1998-07-01

    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

  8. Heterogeneity of glutamatergic and GABAergic release machinery in cerebral cortex: analysis of synaptogyrin, vesicle-associated membrane protein, and syntaxin.

    PubMed

    Bragina, L; Giovedì, S; Barbaresi, P; Benfenati, F; Conti, F

    2010-02-01

    To define whether cortical glutamatergic and GABAergic release machineries can be differentiated on the basis of the nature and amount of proteins they express, we studied the degree of co-localization of synaptogyrin (SGYR) 1 and 3, vesicle-associated membrane protein (VAMP) 1 and 2, syntaxin (STX) 1A and 1B in vesicular glutamate transporter (VGLUT)1-, VGLUT2- and vesicular GABA transporter (VGAT)-positive (+) puncta and synaptic vesicles in the rat cerebral cortex. Co-localization studies showed that SGYR1 and 3 were expressed in about 90% of VGLUT1+, 70% of VGLUT2+ and 80% of VGAT+ puncta; VAMP1 was expressed in approximately 45% of VGLUT1+, 55% of VGLUT2+, and 80% of VGAT+ puncta; VAMP2 in about 95% of VGLUT1+, 75% of VGLUT2+, and 80% of VGAT+ puncta; STX1A in about 65% of VGLUT1+, 30% of VGLUT2+, and 3% of VGAT+ puncta, and STX1B in approximately 45% of VGLUT1+, 35% of VGLUT2+, and 70% of VGAT+ puncta. Immunoisolation studies showed that while STX1A was completely segregated and virtually absent from VGAT synaptic vesicles, STX1B, VAMP1/VAMP2, SGYR1/SGYR3 showed a similar pattern with the highest expression in VGLUT1 immunoisolated vesicles and the lowest in VGAT immunoisolated vesicles. Moreover, we studied the localization of STX1B at the electron microscope and found that a population of axon terminals forming symmetric synapses were STX1B-positive.These results extend our previous observations on the differential expression of presynaptic proteins involved in neurotransmitter release in GABAergic and glutamatergic terminals and indicate that heterogeneity of glutamatergic and GABAergic release machinery can be contributed by both the presence or absence of a given protein in a nerve terminal and the amount of protein expressed by synaptic vesicles. PMID:19909789

  9. 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.

  10. 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

  11. 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

  12. A Burkholderia pseudomallei outer membrane vesicle vaccine provides protection against lethal sepsis.

    PubMed

    Nieves, Wildaliz; Petersen, Hailey; Judy, Barbara M; Blumentritt, Carla A; Russell-Lodrigue, Kasi; Roy, Chad J; Torres, Alfredo G; Morici, Lisa A

    2014-05-01

    The environmental Gram-negative encapsulated bacillus Burkholderia pseudomallei is the causative agent of melioidosis, a disease associated with high morbidity and mortality rates in areas of Southeast Asia and northern Australia in which the disease is endemic. B. pseudomallei is also classified as a tier I select agent due to the high level of lethality of the bacterium and its innate resistance to antibiotics, as well as the lack of an effective vaccine. Gram-negative bacteria, including B. pseudomallei, secrete outer membrane vesicles (OMVs) which are enriched with multiple protein, lipid, and polysaccharide antigens. Previously, we demonstrated that immunization with multivalent B. pseudomallei-derived OMVs protects highly susceptible BALB/c mice against an otherwise lethal aerosol challenge. In this work, we evaluated the protective efficacy of OMV immunization against intraperitoneal challenge with a heterologous strain because systemic infection with phenotypically diverse environmental B. pseudomallei strains poses another hazard and a challenge to vaccine development. We demonstrated that B. pseudomallei OMVs derived from strain 1026b afforded significant protection against septicemic infection with B. pseudomallei strain K96243. OMV immunization induced robust OMV-, lipopolysaccharide-, and capsular polysaccharide-specific serum IgG (IgG1, IgG2a, and IgG3) and IgM antibody responses. OMV-immune serum promoted bacterial killing in vitro, and passive transfer of B. pseudomallei OMV immune sera protected naive mice against a subsequent challenge. These results indicate that OMV immunization provides antibody-mediated protection against acute, rapidly lethal sepsis in mice. B. pseudomallei-derived OMVs may represent an efficacious multivalent vaccine strategy against melioidosis. PMID:24671550

  13. 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

  14. 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

  15. Legionella pneumophila-Derived Outer Membrane Vesicles Promote Bacterial Replication in Macrophages.

    PubMed

    Jung, Anna Lena; Stoiber, Cornelia; Herkt, Christina E; Schulz, Christine; Bertrams, Wilhelm; Schmeck, Bernd

    2016-04-01

    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

  16. 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

  17. Impact of Reducing Complement Inhibitor Binding on the Immunogenicity of Native Neisseria meningitidis Outer Membrane Vesicles

    PubMed Central

    Daniels-Treffandier, Helene; de Nie, Karlijn; Marsay, Leanne; Dold, Christina; Sadarangani, Manish; Reyes-Sandoval, Arturo; Langford, Paul R.; Wyllie, David; Hill, Fergal; Pollard, Andrew J.; Rollier, Christine S.

    2016-01-01

    Neisseria meningitidis recruits host human complement inhibitors to its surface to down-regulate complement activation and enhance survival in blood. We have investigated whether such complement inhibitor binding occurs after vaccination with native outer membrane vesicles (nOMVs), and limits immunogenicity of such vaccines. To this end, nOMVs reactogenic lipopolysaccharide was detoxified by deletion of the lpxl1 gene (nOMVlpxl1). nOMVs unable to bind human complement factor H (hfH) were generated by additional deletions of the genes encoding factor H binding protein (fHbp) and neisserial surface protein A (NspA) (nOMVdis). Antibody responses elicited in mice with nOMVdis were compared to those elicited with nOMVlpxl1 in the presence of hfH. Results demonstrate that the administration of human fH to mice immunized with fHbp containing OMVlpxl1 decreased immunogenicity against fHbp (but not against the OMV as a whole). The majority of the OMV-induced bactericidal immune response (OMVlpxl1 or OMVdis) was versus PorA. Despite a considerable reduction of hfH binding to nOMVdis, and the absence of the vaccine antigen fHbp, immunogenicity in mice was not different from nOMVlpxl1, in the absence or presence of hfH (serum bactericidal titers of 1:64 vs 1:128 after one dose in the nOMVdis and nOMVlpxl1–immunized groups respectively). Therefore, partial inhibition of fH binding did not enhance immunity in this model. PMID:26871712

  18. Impact of Reducing Complement Inhibitor Binding on the Immunogenicity of Native Neisseria meningitidis Outer Membrane Vesicles.

    PubMed

    Daniels-Treffandier, Helene; de Nie, Karlijn; Marsay, Leanne; Dold, Christina; Sadarangani, Manish; Reyes-Sandoval, Arturo; Langford, Paul R; Wyllie, David; Hill, Fergal; Pollard, Andrew J; Rollier, Christine S

    2016-01-01

    Neisseria meningitidis recruits host human complement inhibitors to its surface to down-regulate complement activation and enhance survival in blood. We have investigated whether such complement inhibitor binding occurs after vaccination with native outer membrane vesicles (nOMVs), and limits immunogenicity of such vaccines. To this end, nOMVs reactogenic lipopolysaccharide was detoxified by deletion of the lpxl1 gene (nOMVlpxl1). nOMVs unable to bind human complement factor H (hfH) were generated by additional deletions of the genes encoding factor H binding protein (fHbp) and neisserial surface protein A (NspA) (nOMVdis). Antibody responses elicited in mice with nOMVdis were compared to those elicited with nOMVlpxl1 in the presence of hfH. Results demonstrate that the administration of human fH to mice immunized with fHbp containing OMVlpxl1 decreased immunogenicity against fHbp (but not against the OMV as a whole). The majority of the OMV-induced bactericidal immune response (OMVlpxl1 or OMVdis) was versus PorA. Despite a considerable reduction of hfH binding to nOMVdis, and the absence of the vaccine antigen fHbp, immunogenicity in mice was not different from nOMVlpxl1, in the absence or presence of hfH (serum bactericidal titers of 1:64 vs 1:128 after one dose in the nOMVdis and nOMVlpxl1-immunized groups respectively). Therefore, partial inhibition of fH binding did not enhance immunity in this model. PMID:26871712

  19. Variation among Staphylococcus aureus membrane vesicle proteomes affects cytotoxicity of host cells.

    PubMed

    Jeon, Hyejin; Oh, Man Hwan; Jun, So Hyun; Kim, Seung Il; Choi, Chi Won; Kwon, Hyo Il; Na, Seok Hyeon; Kim, Yoo Jeong; Nicholas, Asiimwe; Selasi, Gati Noble; Lee, Je Chul

    2016-04-01

    Staphylococcus aureus secretes membrane-derived vesicles (MVs), which can deliver virulence factors to host cells and induce cytopathology. However, the cytopathology of host cells induced by MVs derived from different S. aureus strains has not yet been characterized. In the present study, the cytotoxic activity of MVs from different S. aureus isolates on host cells was compared and the proteomes of S. aureus MVs were analyzed. The MVs purified from S. aureus M060 isolated from a patient with staphylococcal scalded skin syndrome showed higher cytotoxic activity toward host cells than that shown by MVs from three other clinical S. aureus isolates. S. aureus M060 MVs induced HEp-2 cell apoptosis in a dose-dependent manner, but the cytotoxic activity of MVs was completely abolished by treatment with proteinase K. In a proteomic analysis, the MVs from three S. aureus isolates not only carry 25 common proteins, but also carry ≥60 strain-specific proteins. All S. aureus MVs contained δ-hemolysin (Hld), γ-hemolysin, leukocidin D, and exfoliative toxin C, but exfoliative toxin A (ETA) was specifically identified in S. aureus M060 MVs. ETA was delivered to HEp-2 cells via S. aureus MVs. Both rETA and rHld induced cytotoxicity in HEp-2 cells. In conclusion, MVs from clinical S. aureus isolates differ with respect to cytotoxic activity in host cells, and these differences may result from differences in the MV proteomes. Further proteogenomic analysis or mutagenesis of specific genes is necessary to identify cytotoxic factors in S. aureus MVs. PMID:26924795

  20. 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

  1. 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.

  2. 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.

  3. 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 +/.

  4. 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

  5. Sec6 is localized to the plasma membrane of mature synaptic terminals and is transported with secretogranin II-containing vesicles.

    PubMed

    Vik-Mo, E O; Oltedal, L; Hoivik, E A; Kleivdal, H; Eidet, J; Davanger, S

    2003-01-01

    The sec6/8 (exocyst) complex is implicated in targeting of vesicles for regulated exocytosis in various cell types and is believed to play a role in synaptogenesis and brain development. We show that the subunits sec6 and sec8 are present at significant levels in neurons of adult rat brain, and that immunoreactivity for the two subunits has a differential subcellular distribution. We show that in developing as well as mature neurons sec6 is concentrated at the inside of the presynaptic plasma membrane, while sec8 immunoreactivity shows a diffuse cytoplasmic distribution. Among established, strongly synaptophysin-positive neuronal boutons, sec6 displays highly differential concentrations, indicating a role for the complex independent of the ongoing synaptic-vesicle release activity. Sec6 is transported along neurites on secretogranin II-positive vesicles, while sec6-negative/secretogranin II-positive vesicles stay in the cell body. In PC12 cells, sec6-positive vesicles accumulate at the plasma membrane at sites of cell-cell contact. Neuronal induction of the PC12 cells with nerve growth factor shows that sec8 is not freely soluble, but may probably interact with cytoskeletal elements. The complex may facilitate the targeting of membrane material to presynaptic sites and may possibly shuttle vesicles from the cytoskeletal transport machinery to presynaptic membrane sites. Thus, we suggest that the exocyst complex serves to modulate exocytotic activity, by targeting membrane material to its presynaptic destination. PMID:12763070

  6. Large effect of membrane tension on the fluid–solid phase transitions of two-component phosphatidylcholine vesicles

    PubMed Central

    Chen, Dong; Santore, Maria M.

    2014-01-01

    Model phospholipid membranes and vesicles have long provided insight into the nature of confined materials and membranes while also providing a platform for drug delivery. The rich thermodynamic behavior and interesting domain shapes in these membranes have previously been mapped in extensive studies that vary temperature and composition; however, the thermodynamic impact of tension on bilayers has been restricted to recent reports of subtly reduced fluid–fluid transition temperatures. In two-component phosphatidylcholine unilamellar vesicles [1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)], we report a dramatic influence of tension on the fluid–solid transition and resulting phases: At fixed composition, systematic variations in tension produce differently shaped solid domains (striped or irregular hexagons), shift fluid–solid transition temperatures, and produce a triple-point–like intersection of coexistence curves at elevated tensions, about 3 mN/m for 30% DOPC/70% DPPC. Tension therefore represents a potential switch of microstructure in responsive engineered materials; it is an important morphology-determining variable in confined systems, and, in biological membranes, it may provide a means to regulate dynamic structure. PMID:24344297

  7. 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.

  8. 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

  9. 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

  10. Growth Conditions and Cell Cycle Phase Modulate Phase Transition Temperatures in RBL-2H3 Derived Plasma Membrane Vesicles

    PubMed Central

    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 12h. 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

  11. Identification and characterization of a basolateral dicarboxylate/cholate antiport system in rat hepatocytes.

    PubMed

    Boelsterli, U A; Zimmerli, B; Meier, P J

    1995-05-01

    The mechanisms and driving forces for the uptake of the unconjugated bile acid cholate were investigated both in cultured rat hepatocytes and in rat liver basolateral (sinusoidal) plasma membrane (BLPM) vesicles. Determination of initial uptake rates of [3H]cholate (0.1 microM) into cultured hepatocytes confirmed that the majority (75%) of the transmembrane transport was mediated by Na(+)-independent mechanisms. This portion of cholate uptake consisted of a pH-sensitive moiety representing nonionic diffusion, which may become quantitatively important at low pH and high cholate concentrations, as well as of a saturable (Michaelis constant 7.4 microM), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive transport moiety, suggesting the involvement of a carrier. This latter transport system was functionally characterized by 1) inhibition of cellular cholate uptake in the absence of extracellular sodium by the dicarboxylic acid alpha-ketoglutarate (alpha-KG; 1 mM) and by the organic anion p-aminohippurate (PAH; 1 mM); 2) stimulation of cellular cholate uptake by alpha-KG (10 microM) or PAH (1 mM) in the presence of an inwardly directed sodium gradient; 3) lack of sensitivity toward lithium in BLPM vesicles; 4) trans-stimulation of vesicular cholate uptake by alpha-KG or PAH, but not by benzoate; and 5) cis-inhibition of alpha-KG/alpha-KG self-exchange by extravesicular cholate (400 microM), PAH (5 mM), probenecid, or DIDS. Collectively, these data indicate the presence of a Na(+)-dicarboxylate cotransport-coupled organic anion exchanger in the hepatocyte basolateral plasma membrane that may be involved in cholate uptake in the liver.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7762664

  12. Membrane distribution of sodium-hydrogen and chloride-bicarbonate exchangers in crypt and villus cell membranes from rabbit ileum.

    PubMed Central

    Knickelbein, R G; Aronson, P S; Dobbins, J W

    1988-01-01

    Present evidence suggests that in the small intestine, villus cells are primarily absorptive and crypt cells are primarily secretory. In order to further confirm that there are differences in transport properties between villus and crypt cells, we have separated villus from crypt cells, using calcium chelations techniques, and determined the distribution of Na:H and Cl:HCO3 exchange activity on brush border membrane and basolateral membrane preparations from these two cell populations. Separation of cells was determined utilizing alkaline phosphatase and maltase activity as a marker of villus cells and thymidine kinase activity as a marker of crypt cells. Utilizing these techniques, we were able to sequentially collect cells along the villus-crypt axis. Na-stimulated glucose and alanine uptake in brush border membrane vesicles diminished from the villus to the crypt region in the sequentially collected cells fractions, further suggesting separation of these cells. Brush border and basolateral membranes were then prepared from cells from the villus and crypt areas, utilizing a continuous sucrose gradient. In the villus cells, Na:H exchange activity was found associated with both the brush border and basolateral membrane, whereas, in crypt cells, Na:H exchange activity was only found on the basolateral membrane. Cl:HCO3 exchange activity was found only on the brush border membrane, in both villus and crypt cells. These studies suggest functional heterogeneity in ion transport between villus and crypt cells. PMID:2848868

  13. 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

  14. 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.

  15. 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

  16. 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

  17. Release of outer membrane vesicles in Pseudomonas putida as a response to stress caused by cationic surfactants.

    PubMed

    Marisa Heredia, Romina; Sabrina Boeris, Paola; Sebastián Liffourrena, Andrés; Fernanda Bergero, María; Alberto López, Gastón; Inés Lucchesi, Gloria

    2016-05-01

    Pseudomonas putida A (ATCC 12633), a degrader of cationic surfactants, releases outer membrane vesicles (OMVs) when grown with tetradecyltrimethylammonium bromide (TTAB) as the sole carbon, nitrogen and energy source. The OMVs exhibit a bilayer structure and were found to be composed of lipopolysaccharides, proteins and phospholipids (PLs) such as cardiolipin, phosphatidylcholine, phosphatidic acid and phosphatidylglycerol (PG). The OMVs showed a marked increase in the PG content, approximately 43 % higher than the amount registered in the parent cells from which the vesicles were derived. After growth of P. putida with TTAB, the amount of lipoprotein covalently cross-linked to the peptidoglycan showed a twofold decrease when compared with values found after growth without the surfactant [16 ± 2 and 28 ± 3 μg (mg cell envelope protein)- 1, respectively]. This decrease in the amount of lipoprotein can be related to areas of loss of contact between the outer membrane and the peptidoglycan and, therefore, to OMV production. In addition, due to its amphiphilic nature, TTAB can contribute to OMV biogenesis, through a physical mechanism, by induction of the curvature of the membrane. Taking into account that OVMs were produced when the cells were grown under external stress, caused by the surfactant, and that TTAB was detected in the vesicles [48 nmol TTAB (nmol PL)- 1], we concluded that this system of TTAB elimination is a mechanism that P. putida A (ATCC 12633) would utilize for alleviating stress caused by cationic surfactants. PMID:26925774

  18. 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...

  19. Cysteine Depletion Causes Oxidative Stress and Triggers Outer Membrane Vesicle Release by Neisseria meningitidis; Implications for Vaccine Development

    PubMed Central

    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

  20. 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.

  1. On-Demand Formation of Supported Lipid Membrane Arrays by Trehalose-Assisted Vesicle Delivery for SPR Imaging.

    PubMed

    Hinman, Samuel S; Ruiz, Charles J; Drakakaki, Georgia; Wilkop, Thomas E; Cheng, Quan

    2015-08-12

    The fabrication of large-scale, solid-supported lipid bilayer (SLB) arrays has traditionally been an arduous and complex task, primarily due to the need to maintain SLBs within an aqueous environment. In this work, we demonstrate the use of trehalose vitrified phospholipid vesicles that facilitate on-demand generation of microarrays, allowing each element a unique composition, for the label-free and high-throughput analysis of biomolecular interactions by SPR imaging (SPRi). Small, unilamellar vesicles (SUVs) are suspended in trehalose, deposited in a spatially defined manner, with the trehalose vitrifying on either hydrophilic or hydrophobic SPR substrates. SLBs are subsequently spontaneously formed on-demand simply by in situ hydration of the array in the SPR instrument flow cell. The resulting SLBs exhibit high lateral mobility, characteristic of fluidic cellular lipid membranes, and preserve the biological function of embedded cell membrane receptors, as indicated by SPR affinity measurements. Independent fluorescence and SPR imaging studies show that the individual SLBs stay localized at the area of deposition, without any encapsulating matrix, confining coral, or boundaries. The introduced methodology allows individually addressable SLB arrays to be analyzed with excellent label-free sensitivity in a real-time, high-throughput manner. Various protein-ganglioside interactions have been selected as a model system to illustrate discrimination of strong and weak binding responses in SPRi sensorgrams. This methodology has been applied toward generating hybrid bilayer membranes on hydrophobic SPR substrates, demonstrating its versatility toward a range of surfaces and membrane geometries. The stability of the fabricated arrays, over medium to long storage periods, was evaluated and found to be good. The highly efficient and easily scalable nature of the method has the potential to be applied to a variety of label-free sensing platforms requiring lipid membranes for

  2. The effect of acclimation temperature on the fusion kinetics of lipid vesicles derived from endoplasmic reticulum membranes of rainbow trout (Oncorhynchus mykiss) liver.

    PubMed

    Miranda, Estuardo J; Hazel, Jeffrey R

    2002-02-01

    Membrane fusion is an obligatory step in many vital cellular processes. The well-established enrichment of bilayer-destabilizing lipids in membranes of poikilotherms subjected to growth at low temperatures leads to the prediction that such membranes will possess a greater propensity to undergo fusion. This hypothesis was explicitly tested in the present study by determining the kinetics of fusion between small unilamellar vesicles (SUVs) prepared from endoplasmic reticulum (ER) membranes of thermally-acclimated (to 5 and 20 degrees C) rainbow trout (Oncorhynchus mykiss) liver and bovine brain phosphatidylserine (BBPS). At temperatures above 10 degrees C, ER vesicles from 5 degrees C-acclimated trout, fused more rapidly and to a greater extent with BBPS vesicles (by average factors of 1.25- and 1.45-fold, respectively) than ER vesicles of 20 degrees C-acclimated trout. At temperatures >35 degrees C, apparent fusion rates declined while the extent of fusion increased in both acclimation groups. Fusion kinetics were found to be well correlated with and limited by the physical properties and phase state of the BBPS vesicles. These results indicate that dynamic attributes of biological membranes, such as the propensity to undergo fusion, are of potential regulatory significance and are partially conserved when growth or environmental temperature changes. PMID:11818217

  3. Interaction of cholesterol with sphingomyelin in bilayer membranes: Evidence that the hydroxy group of sphingomyelin does not modulate the rate of cholesterol exchange between vesicles

    SciTech Connect

    Chucheng Kan; Zhongshi Ruan; Bittman, R. )

    1991-08-06

    Cholesterol undergoes exchange between membranes containing sphingomyelin at a much slower rate than between membranes lacking sphingomyelin. To investigate the role of the hydroxy group at the 3-position of sphingomyelin in the interaction between sphingomyelin and cholesterol, the authors have measured the rates of (4-{sup 14}C)cholesterol exchange between unilamellar vesicles prepared with N-alkyl group or with hydrogen. Vesicles prepared from 3-deoxy- and 3-0-methyl-N-stearoylsphingomyelin had the same rate of ({sup 14}C) cholesterol desorption. The half-times for exchange from vesicles prepared with 3-0-methyl- and 3-deoxy-N-stearoylsphingomyelins and 10 mol% of cholesterol were only slightly faster than that found from vesicles prepared from N-stearoylsphingomyelin and 10 mol% cholesterol. The rate of cholesterol desorption from vesicles could be accelerated by preparing vesicles from bulky 3-0-alkyl analogues of sphingomyelin. These data indicate that insertion of sterically bulky groups at the 3-position of sphingomyelin in place of hydroxy interferes markedly with the molecular packing of cholesterol and sphingomyelin in bilayer membranes; however, the hydroxy group of sphingomyelin is not critical for the strong interaction of cholesterol with sphingomyelin. These results suggest that van der Waals interactions are more important than hydrogen-bonding interactions involving the hydroxy group in contributing to tight lateral packing of cholesterol with sphingomyelin.

  4. 38,000-DALTON MEMBRANE PROTEIN (P38) PRESENT IN SYNAPTIC VESICLES

    EPA Science Inventory

    A protein with an apparent molecular mass of 38,000 daltons designated p38 was found in synaptic vesicles from rat brain. The subcellular distribution of p38 and some of its properties were determined with the aid of polyclonal and monoclonal antibodies. The subcellular distribut...

  5. Phosphorylation of adenosine in renal brush-border membrane vesicles by an exchange reaction catalysed by adenosine kinase.

    PubMed Central

    Sayós, J; Solsona, C; Mallol, J; Lluis, C; Franco, R

    1994-01-01

    Uptake of [3H]adenosine in brush-border membrane (BBM) vesicles from either rat or pig kidney leads to an accumulation of intravesicular [3H]AMP. The lack of significant levels of ATP and the presence of AMP in BBM indicated that a phosphotransfer between [3H]adenosine and AMP occurs. The phosphotransfer activity is inhibited by iodotubercidin, which suggests that it is performed by adenosine kinase acting in an ATP-independent manner. The existence of a similar phosphotransferase activity was demonstrated in membrane-free extracts from pig kidney. From the compounds tested it was shown that a variety of mononucleotides could act as phosphate donors. The results suggest that phosphotransfer reactions may be physiologically relevant in kidney. PMID:8110185

  6. 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

  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. 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

  10. Basolateral K channels in an insect epithelium. Channel density, conductance, and block by barium

    PubMed Central

    Hanrahan, JW; Wills, NK; Phillips, JE; Lewis, SA

    1986-01-01

    K channels in the basolateral membrane of insect hindgut were studied using current fluctuation analysis and microelectrodes. Locust recta were mounted in Ussing-type chambers containing Cl-free saline and cyclic AMP (cAMP). A transepithelial K current was induced by raising serosal [K] under short-circuit conditions. Adding Ba to the mucosal (luminal) side under these conditions had no effect; however, serosal Ba reversibly inhibited the short-circuit current (Isc), increased transepithelial resistance (Rt), and added a Lorentzian component to power density spectra of the Isc. A nonlinear relationship between corner frequency and serosal [Ba] was observed, which suggests that the rate constant for Ba association with basolateral channels increased as [Ba] was elevated. Microelectrode experiments revealed that the basolateral membrane hyperpolarized when Ba was added: this change in membrane potential could explain the nonlinearity of the 2 pi fc vs. [Ba] relationship if external Ba sensed about three-quarters of the basolateral membrane field. Conventional microelectrodes were used to determine the correspondence between transepithelially measured current noise and basolateral membrane conductance fluctuations, and ion-sensitive microelectrodes were used to measure intracellular K activity (acK). From the relationship between the net electrochemical potential for K across the basolateral membrane and the single channel current calculated from noise analysis, we estimate that the conductance of basolateral K channels is approximately 60 pS, and that there are approximately 180 million channels per square centimeter of tissue area. PMID:2420918

  11. 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.

  12. Development of a cost-effective vaccine candidate with outer membrane vesicles of a tolA-disrupted Shigella boydii strain.

    PubMed

    Mitra, Soma; Sinha, Ritam; Mitobe, Jiro; Koley, Hemanta

    2016-04-01

    Our previous studies on outer membrane vesicles based vaccine development against shigellosis, revealed the inability of Shigella to release significant amount of vesicles naturally, during growth. Disruption of tolA, one of the genes of the Tol-Pal system of Gram negative bacterial membrane, has increased the vesicle release rate of a Shigella boydii type 4 strain to approximately 60% higher. We also noticed the vesicles, released from tolA-disrupted strain captured more OmpA protein and lipopolysaccharide, compared to the vesicles released from its wild type prototype. Six to seven weeks old BALB/c mice, immunized with 25μg of three oral doses of the vesicles, released by tolA mutant, conferred 100% protection against lethal homologous challenge through nasal route, compared to only 60% protection after the same dose of wild type immunogen. Mice, immunized with the vesicles from tolA-mutant, manifested significant secretion of mucosal IgG and IgA. A sharp and significant response of pro-inflammatory cytokines (TNF-α, IL-6, IFN-γ) were also observed in the lung lavage of these groups of mice, within 6h post challenge; but at 24h, these inflammatory cytokines showed the sign of subsidence and the system was taken over by the release of anti-inflammatory cytokines (IL-4 and IL-10). Studies with naïve peritoneal macrophages, proved further, the potency of these vesicles to stimulate nitric oxide and TNF-α, IL-12p70, IL-6 and IL-10 productions in-vitro. The ability of these vesicles to trigger polarization of CD4(+) T cells toward Th1 adaptive immune response, had also been observed along with the presence of anti-inflammatory cytokines in the system. Our study demonstrated, the vesicles from tolA-disrupted Shigella were able to suppress Shigella-mediated inflammation in the host and could balance between inflammation and anti-inflammation, promoting better survival and health of the infected mice. Outer membrane vesicles from tolA-mutant, could be a potential

  13. 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

  14. Segregative clustering of Lo and Ld membrane microdomains induced by local pH gradients in GM1-containing giant vesicles: a lipid model for cellular polarization.

    PubMed

    Staneva, Galya; Puff, Nicolas; Seigneuret, Michel; Conjeaud, Hélène; Angelova, Miglena I

    2012-11-27

    Several cell polarization processes are coupled to local pH gradients at the membrane surface. We have investigated the involvement of a lipid-mediated effect in such coupling. The influence of lateral pH gradients along the membrane surface on lipid microdomain dynamics in giant unilamellar vesicles containing phosphatidylcholine, sphingomyelin, cholesterol, and the ganglioside GM1 was studied. Lo/Ld phase separation was generated by photosensitization. A lateral pH gradient was established along the external membrane surface by acid local microinjection. The gradient promotes the segregation of microdomains: Lo domains within an Ld phase move toward the higher pH side, whereas Ld domains within an Lo phase move toward the lower pH side. This results in a polarization of the vesicle membrane into Lo and Ld phases poles in the axis of the proton source. A secondary effect is inward tubulation in the Ld phase. None of these processes occurs without GM1 or with the analog asialo-GM1. These are therefore related to the acidic character of the GM1 headgroup. LAURDAN fluorescence experiments on large unilamellar vesicles indicated that, with GM1, an increase in lipid packing occurs with decreasing pH, attributed to the lowering of repulsion between GM1 molecules. Packing increase is much higher for Ld phase vesicles than for Lo phase vesicles. It is proposed that the driving forces for domain vectorial segregative clustering and vesicle polarization are related to such differences in packing variations with pH decrease between the Lo and Ld phases. Such pH-driven domain clustering might play a role in cellular membrane polarization processes in which local lateral pH gradients are known to be important, such as migrating cells and epithelial cells. PMID:23121205

  15. 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

  16. Salmonella outer membrane vesicles displaying high densities of pneumococcal antigen at the surface offer protection against colonization.

    PubMed

    Kuipers, Kirsten; Daleke-Schermerhorn, Maria H; Jong, Wouter S P; ten Hagen-Jongman, Corinne M; van Opzeeland, Fred; Simonetti, Elles; Luirink, Joen; de Jonge, Marien I

    2015-04-21

    Bacterial outer membrane vesicles (OMVs) are attractive vaccine formulations because they have intrinsic immunostimulatory properties. In principle, heterologous antigens incorporated into OMVs will elicit specific immune responses, especially if presented at the vesicle surface and thus optimally exposed to the immune system. In this study, we explored the feasibility of our recently developed autotransporter Hbp platform, designed to efficiently and simultaneously display multiple antigens at the surface of bacterial OMVs, for vaccine development. Using two Streptococcus pneumoniae proteins as model antigens, we showed that intranasally administered Salmonella OMVs displaying high levels of antigens at the surface induced strong protection in a murine model of pneumococcal colonization, without the need for a mucosal adjuvant. Importantly, reduction in bacterial recovery from the nasal cavity was correlated with local production of antigen-specific IL-17A. Furthermore, the protective efficacy and the production of antigen-specific IL-17A, and local and systemic IgGs, were all improved at increased concentrations of the displayed antigen. This discovery highlights the importance of an adequate antigen expression system for development of recombinant OMV vaccines. In conclusion, our findings demonstrate the suitability of the Hbp platform for development of a new generation of OMV vaccines, and illustrate the potential of using this approach to develop a broadly protective mucosal pneumococcal vaccine. PMID:25776921

  17. 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

  18. Chalcone inhibitors of the NorA efflux pump in Staphylococcus aureus whole cells and enriched everted membrane vesicles.

    PubMed

    Holler, Jes Gitz; Slotved, Hans-Christian; Mølgaard, Per; Olsen, Carl Erik; Christensen, Søren Brøgger

    2012-07-15

    A library of 117 chalcones was screened for efflux pump inhibitory (EPI) activity against NorA mediated ethidium bromide efflux. Five of the chalcones (5-7, 9, and 10) were active and two chalcones (9 and 10) were equipotent to reserpine with IC(50)-values of 9.0 and 7.7 μM, respectively. Twenty chalcones were subsequently proved to be inhibitors of the NorA efflux pump in everted membrane vesicles. Compounds 5, 7, and 9 synergistically increased the effect of ciprofloxacin on Staphylococcus aureus. Our results suggest that chalcones might be developed into drugs for overcoming multidrug resistance based on efflux transporters of microorganisms. PMID:22682300

  19. The deformation of spherical vesicles with permeable, constant-area membranes: application to the red blood cell.

    PubMed Central

    Parker, K H; Winlove, C P

    1999-01-01

    The deformation of an initially spherical vesicle of radius a with a permeable membrane under extensive forces applied at its poles is calculated as a function of the in-plane shear modulus, H, and the out-of-plane bending modulus, B, using an axisymmetric theory that is valid for large deformations. Suitably nondimensionalized, the results depend upon a single nondimensional parameter, C identical with a(2)H/B. For small deformations, the calculated force-polar strain curves are linear and, under these conditions, the slope of the curve determines only C, not the values of H and B separately. Independent determination of H and B from experimental measurements require deformations that are large enough to produce nonlinear behavior. Simple approximations for large and small C are given, which are applied to experimental measurements on red blood cell ghosts that have been made permeable by treatment with saponin. PMID:10585931

  20. Dipalmitoylphosphatidylcholine/linoleic acid mixed unilamellar vesicles as model membranes for studies on novel free-radical scavengers.

    PubMed

    Castelli, F; Trombetta, D; Tomaino, A; Bonina, F; Romeo, G; Uccella, N; Saija, A

    1997-04-01

    Large unilamellar vesicles (LUVs) are generally accepted to be a suitable model for peroxidation studies. In the present report, dipalmitoylphosphatidylcholine (DPPC)/linoleic acid-mixed LUVs were employed as model membranes to verify the inhibitory effect of tocopherol (an efficient representative antioxidant) against 2,2'-azobis(2-amidinopropane)hydrochloride-induced peroxidation (evaluated by monitoring conjugated diene accumulation). In this model, the appropriate experimental conditions (particularly, liposome composition and peroxidation temperature) were selected following characterization of bilayer physical state, and not only by evaluation of peroxidation rate. Thus, the experiments described provide a routine screening procedure that would be appropriate for assessing the activity profile of novel free-radical scavengers. PMID:9253749

  1. Sodium-dependent transport of neutral amino acids by whole cells and membrane vesicles of Streptococcus bovis, a ruminal bacterium.

    PubMed Central

    Russell, J B; Strobel, H J; Driessen, A J; Konings, W N

    1988-01-01

    Streptococcus bovis JB1 cells were able to transport serine, threonine, or alanine, but only when they were incubated in sodium buffers. If glucose-energized cells were washed in potassium phosphate and suspended in potassium phosphate buffer, there was no detectable uptake. Cells deenergized with 2-deoxyglucose and incubated in sodium phosphate buffer were still able to transport serine, and this result indicated that the chemical sodium gradient was capable of driving transport. However, when the deenergized cells were treated with valinomycin and diluted into sodium phosphate to create both an artificial membrane potential and a chemical sodium gradient, rates of serine uptake were fivefold greater than in cells having only a sodium gradient. If deenergized cells were preloaded with sodium (no membrane potential or sodium gradient), there was little serine transport. Nigericin and monensin, ionophores capable of reversing sodium gradients across membranes, strongly inhibited sodium-dependent uptake of the three amino acids. Membrane vesicles loaded with potassium and diluted into either lithium or choline chloride were unable to transport serine, but rapid uptake was evident if sodium chloride was added to the assay mixture. Serine transport had an extremely poor affinity for sodium, and more than 30 mM was needed for half-maximal rates of uptake. Serine transport was inhibited by an excess of threonine, but an excess of alanine had little effect. Results indicated that S. bovis had separate sodium symport systems for serine or threonine and alanine, and either the membrane potential or chemical sodium gradient could drive uptake. PMID:3136141

  2. 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

  3. 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

  4. 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.

  5. 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

  6. 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...

  7. 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

  8. 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

  9. Human CASK/LIN-2 Binds Syndecan-2 and Protein 4.1 and Localizes to the Basolateral Membrane of Epithelial Cells

    PubMed Central

    Cohen, Alexandra R.; Wood, Daniel F.; Marfatia, Shirin M.; Walther, Zenta; Chishti, Athar H.; Anderson, James Melvin

    1998-01-01

    In Caenorhabditis elegans, mutations in the lin-2 gene inactivate the LET-23 receptor tyrosine kinase/Ras/MAP kinase pathway required for vulval cell differentiation. One function of LIN-2 is to localize LET-23 to the basal membrane domain of vulval precursor cells. LIN-2 belongs to the membrane-associated guanylate kinase family of proteins. We have cloned and characterized the human homolog of LIN-2, termed hCASK, and Northern and Western blot analyses reveal that it is ubiquitously expressed. Indirect immunofluorescence localizes CASK to distinct lateral and/or basal plasma membrane domains in different epithelial cell types. We detect in a yeast two-hybrid screen that the PDZ domain of hCASK binds to the heparan sulfate proteoglycan syndecan-2. This interaction is confirmed using in vitro binding assays and immunofluorescent colocalization. Furthermore, we demonstrate that hCASK binds the actin-binding protein 4.1. Syndecans are known to bind extracellular matrix, and to form coreceptor complexes with receptor tyrosine kinases. We speculate that CASK mediates a link between the extracellular matrix and the actin cytoskeleton via its interaction with syndecan and with protein 4.1. Like other membrane-associated guanylate kinases, its multidomain structure enables it to act as a scaffold at the membrane, potentially recruiting multiple proteins and coordinating signal transduction. PMID:9660868

  10. 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

  11. 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.

  12. Membrane retrieval in the guinea-pig neurohypophysis. Isolation and characterization of secretory vesicles and coated microvesicles after radiolabel incorporation in vivo.

    PubMed

    Saermark, T; Jones, P M; Robinson, I C

    1984-03-01

    We have developed small-scale methods for the isolation and biochemical characterization of subcellular fractions from single guinea-pig posterior-pituitary glands. Secretory vesicles and coated microvesicles produced in this way were of similar purity to those isolated from large amounts of tissue by conventional ultracentrifugation. [35S]Cysteine injected into the hypothalamus was found in the soluble contents of secretory vesicles isolated from the neural lobes 24 h later. High-pressure liquid-chromatographic analysis revealed that the radiolabel was incorporated into the expected neurosecretory products (oxytocin, vasopressin and neurophysin) and also into a biosynthetic intermediate in the vasopressin system. The membranes of secretory vesicles were labelled with [3H]choline 24 h after its hypothalamic injection. Little or no [3H]choline could be demonstrated in coated microvesicles at this time, although these structures were labelled 5 days after injection. Stimulating hormone secretion by chronic dehydration produced a significant fall in [3H]choline content of the secretory-vesicle membranes without any transfer of label into coated microvesicles, suggesting that coated microvesicles are not involved in membrane retrieval in the neurohypophysis. PMID:6712633

  13. Membrane retrieval in the guinea-pig neurohypophysis. Isolation and characterization of secretory vesicles and coated microvesicles after radiolabel incorporation in vivo.

    PubMed Central

    Saermark, T; Jones, P M; Robinson, I C

    1984-01-01

    We have developed small-scale methods for the isolation and biochemical characterization of subcellular fractions from single guinea-pig posterior-pituitary glands. Secretory vesicles and coated microvesicles produced in this way were of similar purity to those isolated from large amounts of tissue by conventional ultracentrifugation. [35S]Cysteine injected into the hypothalamus was found in the soluble contents of secretory vesicles isolated from the neural lobes 24 h later. High-pressure liquid-chromatographic analysis revealed that the radiolabel was incorporated into the expected neurosecretory products (oxytocin, vasopressin and neurophysin) and also into a biosynthetic intermediate in the vasopressin system. The membranes of secretory vesicles were labelled with [3H]choline 24 h after its hypothalamic injection. Little or no [3H]choline could be demonstrated in coated microvesicles at this time, although these structures were labelled 5 days after injection. Stimulating hormone secretion by chronic dehydration produced a significant fall in [3H]choline content of the secretory-vesicle membranes without any transfer of label into coated microvesicles, suggesting that coated microvesicles are not involved in membrane retrieval in the neurohypophysis. Images Fig. 1. PMID:6712633

  14. ΔpH-Dependent Amino Acid Transport into Plasma Membrane Vesicles Isolated from Sugar Beet Leaves

    PubMed Central

    Li, Zhen-Chang; Bush, Daniel R.

    1990-01-01

    Amino acid transport into plasma membrane vesicles isolated from mature sugar beet (Beta vulgaris L. cv Great Western) leaves was investigated. The transport of alanine, leucine, glutamine, glutamate, isoleucine, and arginine was driven by a trans-membrane proton concentration difference. ΔpH-Dependent alanine, leucine, glutamine, and glutamate transport exhibited simple Michaelis-Menten kinetics, and double-reciprocal plots of the data were linear with apparent Km values of 272, 346, 258, and 1981 micromolar, respectively. These results are consistent with carrier mediated transport. ΔpH-Dependent isoleucine and arginine transport exhibited biphasic kinetics, suggesting these amino acids may be transported by at least two transport systems. Symport mediated alanine transport was electrogenic as demonstrated by the effect of membrane potential (ΔΨ) on ΔpH-dependent flux. In the absence of significant charge compensation, a low rate of alanine transport was observed. When ΔΨ was held at 0 millivolt with symmetric potassium concentrations and valinomycin, the rate of flux was stimulated fourfold. In the presence of a negative ΔΨ, alanine transport increased sixfold. These results are consistent with an electrogenic transport process which results in a net flux of positive charge into the vesicles. The effect of changing ΔΨ on the kinetics of alanine transport altered Vmax with no apparent change in Km. Amino acid transport was inhibited by the protein modifier diethyl pyrocarbonate, but was insensitive to N-ethylmaleimide, 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid, p-chloromercuribenzenesulfonic acid, phenylglyoxal, and N,N′-dicyclohexylcarbodiimide. Four amino acid symport systems, two neutral, one acidic, and one basic, were resolved based on inter-amino acid competition experiments. One neutral system appears to be active for all neutral amino acids while the second exhibited a low affinity for isoleucine, threonine, valine, and proline

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. Campylobacter jejuni Outer Membrane Vesicles Play an Important Role in Bacterial Interactions with Human Intestinal Epithelial Cells

    PubMed Central

    Elmi, Abdi; Watson, Eleanor; Sandu, Pamela; Gundogdu, Ozan; Mills, Dominic C.; Inglis, Neil F.; Manson, Erin; Imrie, Lisa; Bajaj-Elliott, Mona; Wren, Brendan W.; Smith, David G. E.

    2012-01-01

    Campylobacter jejuni is the most prevalent cause of food-borne gastroenteritis in the developed world; however, the molecular basis of pathogenesis is unclear. Secretion of virulence factors is a key mechanism by which enteric bacterial pathogens interact with host cells to enhance survival and/or damage the host. However, C. jejuni lacks the virulence-associated secretion systems possessed by other enteric pathogens. Many bacterial pathogens utilize outer membrane vesicles (OMVs) for delivery of virulence factors into host cells. In the absence of prototypical virulence-associated secretion systems, OMVs could be an important alternative for the coordinated delivery of C. jejuni proteins into host cells. Proteomic analysis of C. jejuni 11168H OMVs identified 151 proteins, including periplasmic and outer membrane-associated proteins, but also many determinants known to be important in survival and pathogenesis, including the cytolethal distending toxin (CDT). C. jejuni OMVs contained 16 N-linked glycoproteins, indicating a delivery mechanism by which these periplasm-located yet immunogenic glycoproteins can interact with host cells. C. jejuni OMVs possess cytotoxic activity and induce a host immune response from T84 intestinal epithelial cells (IECs), which was not reduced by OMV pretreatment with proteinase K or polymyxin B prior to coincubation with IECs. Pretreatment of IECs with methyl-beta-cyclodextrin partially blocks OMV-induced host immune responses, indicating a role for lipid rafts in host cell plasma membranes during interactions with C. jejuni OMVs. OMVs isolated from a C. jejuni 11168H cdtA mutant induced interleukin-8 (IL-8) to the same extent as did wild-type OMVs, suggesting OMV induction of IL-8 is independent of CDT. PMID:22966047

  1. 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

  2. Adhesion and membrane tension of single vesicles and living cells using a micropipette-based technique.

    PubMed

    Colbert, M-J; Raegen, A N; Fradin, C; Dalnoki-Veress, K

    2009-10-01

    The fundamental study of the adhesion of cells to each other or to a substrate is a key research topic in cellular biophysics because cell adhesion is important to many biological processes. We report on the adhesion of a model cell, a liposome, and a living HeLa cell to a substrate measured with a novel experimental technique. The cells are held at the end of a micropipette mounted on a micromanipulator and brought into contact with a surface. The adhesion energy and membrane tension are measured directly using the deflection of the micropipette when binding or unbinding the cell from the substrate. Since the force applied on the cells is known throughout the experiment, the technique presented enables the measurement of dynamics such as changes in the adhesion, elasticity, and membrane tension with time. PMID:19777278

  3. 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

  4. Synaptic Vesicle Endocytosis

    PubMed Central

    Saheki, Yasunori; De Camilli, Pietro

    2012-01-01

    Neurons can sustain high rates of synaptic transmission without exhausting their supply of synaptic vesicles. This property relies on a highly efficient local endocytic recycling of synaptic vesicle membranes, which can be reused for hundreds, possibly thousands, of exo-endocytic cycles. Morphological, physiological, molecular, and genetic studies over the last four decades have provided insight into the membrane traffic reactions that govern this recycling and its regulation. These studies have shown that synaptic vesicle endocytosis capitalizes on fundamental and general endocytic mechanisms but also involves neuron-specific adaptations of such mechanisms. Thus, investigations of these processes have advanced not only the field of synaptic transmission but also, more generally, the field of endocytosis. This article summarizes current information on synaptic vesicle endocytosis with an emphasis on the underlying molecular mechanisms and with a special focus on clathrin-mediated endocytosis, the predominant pathway of synaptic vesicle protein internalization. PMID:22763746

  5. Munc13-4 Is a Rab11-binding Protein That Regulates Rab11-positive Vesicle Trafficking and Docking at the Plasma Membrane.

    PubMed

    Johnson, Jennifer L; He, Jing; Ramadass, Mahalakshmi; Pestonjamasp, Kersi; Kiosses, William B; Zhang, Jinzhong; Catz, Sergio D

    2016-02-12

    The small GTPase Rab11 and its effectors control trafficking of recycling endosomes, receptor replenishment and the up-regulation of adhesion and adaptor molecules at the plasma membrane. Despite recent advances in the understanding of Rab11-regulated mechanisms, the final steps mediating docking and fusion of Rab11-positive vesicles at the plasma membrane are not fully understood. Munc13-4 is a docking factor proposed to regulate fusion through interactions with SNAREs. In hematopoietic cells, including neutrophils, Munc13-4 regulates exocytosis in a Rab27a-dependent manner, but its possible regulation of other GTPases has not been explored in detail. Here, we show that Munc13-4 binds to Rab11 and regulates the trafficking of Rab11-containing vesicles. Using a novel Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay, we demonstrate that Munc13-4 binds to Rab11a but not to dominant negative Rab11a. Immunoprecipitation analysis confirmed the specificity of the interaction between Munc13-4 and Rab11, and super-resolution microscopy studies support the interaction of endogenous Munc13-4 with Rab11 at the single molecule level in neutrophils. Vesicular dynamic analysis shows the common spatio-temporal distribution of Munc13-4 and Rab11, while expression of a calcium binding-deficient mutant of Munc13-4 significantly affected Rab11 trafficking. Munc13-4-deficient neutrophils showed normal endocytosis, but the trafficking, up-regulation, and retention of Rab11-positive vesicles at the plasma membrane was significantly impaired. This correlated with deficient NADPH oxidase activation at the plasma membrane in response to Rab11 interference. Our data demonstrate that Munc13-4 is a Rab11-binding partner that regulates the final steps of Rab11-positive vesicle docking at the plasma membrane. PMID:26637356

  6. Determination of membrane cholesterol partition coefficient using a lipid vesicle-cyclodextrin binary system: effect of phospholipid acyl chain unsaturation and headgroup composition.

    PubMed Central

    Niu, Shui-Lin; Litman, Burton J

    2002-01-01

    Lateral domain or raft formation in biological membranes is often discussed in terms of cholesterol-lipid interactions. Preferential interactions of cholesterol with lipids, varying in headgroup and acyl chain unsaturation, were studied by measuring the partition coefficient for cholesterol in unilamellar vesicles. A novel vesicle-cyclodextrin system was used, which precludes the possibility of cross-contamination between donor-acceptor vesicles or the need to modify one of the vesicle populations. Variation in phospholipid headgroup resulted in cholesterol partitioning in the order of sphingomyelin (SM) > phosphatidylserine > phosphatidylcholine (PC) > phosphatidylenthanolamine (PE), spanning a range of partition DeltaG of -1181 cal/mol to +683 cal/mol for SM and PE, respectively. Among the acyl chains examined, the order of cholesterol partitioning was 18:0(stearic acid),18:1n-9(oleic acid) PC > di18:1n-9PC > di18:1n-12(petroselenic acid) PC > di18:2n-6(linoleic acid) PC > 16:0(palmitic acid),22:6n-3(DHA) PC > di18:3n-3(alpha-linolenic acid) PC > di22:6n-3PC with a range in partition DeltaG of 913 cal/mol. Our results suggest that the large differences observed in cholesterol-lipid interactions contribute to the forces responsible for lateral domain formation in plasma membranes. These differences may also be responsible for the heterogeneous cholesterol distribution in cellular membranes, where cholesterol is highly enriched in plasma membranes and relatively depleted in intracellular membranes. PMID:12496107

  7. Free-Flow Electrophoresis of Plasma Membrane Vesicles Enriched by Two-Phase Partitioning Enhances the Quality of the Proteome from Arabidopsis Seedlings.

    PubMed

    de Michele, Roberto; McFarlane, Heather E; Parsons, Harriet T; Meents, Miranda J; Lao, Jeemeng; González Fernández-Niño, Susana M; Petzold, Christopher J; Frommer, Wolf B; Samuels, A Lacey; Heazlewood, Joshua L

    2016-03-01

    The plant plasma membrane is the interface between the cell and its environment undertaking a range of important functions related to transport, signaling, cell wall biosynthesis, and secretion. Multiple proteomic studies have attempted to capture the diversity of proteins in the plasma membrane using biochemical fractionation techniques. In this study, two-phase partitioning was combined with free-flow electrophoresis to produce a population of highly purified plasma membrane vesicles that were subsequently characterized by tandem mass spectroscopy. This combined high-quality plasma membrane isolation technique produced a reproducible proteomic library of over 1000 proteins with an extended dynamic range including plasma membrane-associated proteins. The approach enabled the detection of a number of putative plasma membrane proteins not previously identified by other studies, including peripheral membrane proteins. Utilizing multiple data sources, we developed a PM-confidence score to provide a value indicating association to the plasma membrane. This study highlights over 700 proteins that, while seemingly abundant at the plasma membrane, are mostly unstudied. To validate this data set, we selected 14 candidates and transiently localized 13 to the plasma membrane using a fluorescent tag. Given the importance of the plasma membrane, this data set provides a valuable tool to further investigate important proteins. The mass spectrometry data are available via ProteomeXchange, identifier PXD001795. PMID:26781341

  8. Cytotoxic and Inflammatory Responses Induced by Outer Membrane Vesicle-Associated Biologically Active Proteases from Vibrio cholerae.

    PubMed

    Mondal, Ayan; Tapader, Rima; Chatterjee, Nabendu Sekhar; Ghosh, Amit; Sinha, Ritam; Koley, Hemanta; Saha, Dhira Rani; Chakrabarti, Manoj K; Wai, Sun Nyunt; Pal, Amit

    2016-05-01

    Proteases in Vibrio cholerae have been shown to play a role in its pathogenesis. V. cholerae secretes Zn-dependent hemagglutinin protease (HAP) and calcium-dependent trypsin-like serine protease (VesC) by using the type II secretion system (TIISS). Our present studies demonstrated that these proteases are also secreted in association with outer membrane vesicles (OMVs) and transported to human intestinal epithelial cells in an active form. OMV-associated HAP induces dose-dependent apoptosis in Int407 cells and an enterotoxic response in the mouse ileal loop (MIL) assay, whereas OMV-associated VesC showed a hemorrhagic fluid response in the MIL assay, necrosis in Int407 cells, and an increased interleukin-8 (IL-8) response in T84 cells, which were significantly reduced in OMVs from VesC mutant strain. Our results also showed that serine protease VesC plays a role in intestinal colonization of V. cholerae strains in adult mice. In conclusion, our study shows that V. cholerae OMVs secrete biologically active proteases which may play a role in cytotoxic and inflammatory responses. PMID:26930702

  9. Exploring the Midgut Transcriptome and Brush Border Membrane Vesicle Proteome of the Rice Stem Borer, Chilo suppressalis (Walker)

    PubMed Central

    Peng, Chuanhua; Wang, Xiaoping; Li, Fei; Lin, Yongjun

    2012-01-01

    The rice stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), is one of the most detrimental pests affecting rice crops. The use of Bacillus thuringiensis (Bt) toxins has been explored as a means to control this pest, but the potential for C. suppressalis to develop resistance to Bt toxins makes this approach problematic. Few C. suppressalis gene sequences are known, which makes in-depth study of gene function difficult. Herein, we sequenced the midgut transcriptome of the rice stem borer. In total, 37,040 contigs were obtained, with a mean size of 497 bp. As expected, the transcripts of C. suppressalis shared high similarity with arthropod genes. Gene ontology and KEGG analysis were used to classify the gene functions in C. suppressalis. Using the midgut transcriptome data, we conducted a proteome analysis to identify proteins expressed abundantly in the brush border membrane vesicles (BBMV). Of the 100 top abundant proteins that were excised and subjected to mass spectrometry analysis, 74 share high similarity with known proteins. Among these proteins, Western blot analysis showed that Aminopeptidase N and EH domain-containing protein have the binding activities with Bt-toxin Cry1Ac. These data provide invaluable information about the gene sequences of C. suppressalis and the proteins that bind with Cry1Ac. PMID:22666467

  10. HlyF Produced by Extraintestinal Pathogenic Escherichia coli Is a Virulence Factor That Regulates Outer Membrane Vesicle Biogenesis.

    PubMed

    Murase, Kazunori; Martin, Patricia; Porcheron, Gaëlle; Houle, Sébastien; Helloin, Emmanuelle; Pénary, Marie; Nougayrède, Jean-Philippe; Dozois, Charles M; Hayashi, Tetsuya; Oswald, Eric

    2016-03-01

    Escherichia coli can cause extraintestinal infections in humans and animals. The hlyF gene is epidemiologically associated with virulent strains of avian pathogenic E. coli and human neonatal meningitis-associated E. coli. We demonstrated that culture supernatants of E. coli expressing HlyF induced autophagy in eukaryotic cells. This phenotype coincided with an enhanced production of outer membrane vesicles (OMVs) by bacteria expressing HlyF. The HlyF protein displays a predicted catalytic domain of the short-chain dehydrogenase/reductase superfamily. This conserved domain was involved the ability of HlyF to promote the production of OMVs. The increased production of OMVs was associated with the release of toxins. hlyF was shown to be expressed during extraintestinal infection and to play a role in the virulence of extraintestinal pathogenic E. coli in a chicken model of colibacillosis. This is the first evidence that pathogenic bacteria produce a virulence factor directly involved in the production of OMVs. PMID:26494774

  11. The Pseudomonas aeruginosa extracellular secondary metabolite, Paerucumarin, chelates iron and is not localized to extracellular membrane vesicles.

    PubMed

    Qaisar, Uzma; Kruczek, Cassandra J; Azeem, Muhammed; Javaid, Nasir; Colmer-Hamood, Jane A; Hamood, Abdul N

    2016-08-01

    Proteins encoded by the Pseudomonas aeruginosa pvcA-D operon synthesize a novel isonitrile functionalized cumarin termed paerucumarin. The pvcA-D operon enhances the expression of the P. aeruginosa fimbrial chaperone/usher pathway (cup) genes and this effect is mediated through paerucumarin. Whether pvcA-D and/or paerucumarin affect the expression of other P. aeruginosa genes is not known. In this study, we examined the effect of a mutation in pvcA-D operon the global transcriptome of the P. aeruginosa strain PAO1-UW. The mutation reduced the expression of several ironcontrolled genes including pvdS, which is essential for the expression of the pyoverdine genes. Additional transcriptional studies showed that the pvcA-D operon is not regulated by iron. Exogenously added paerucumarin enhanced pyoverdine production and pvdS expression in PAO1-UW. Iron-chelation experiments revealed that purified paerucumarin chelates iron. However, exogenously added paerucumarin significantly reduced the growth of a P. aeruginosa mutant defective in pyoverdine and pyochelin production. In contrast to other secondary metabolite, Pseudomonas quinolone signal (PQS), paerucumarin is not localized to the P. aeruginosa membrane vesicles. These results suggest that paerucumarin enhances the expression of iron-controlled genes by chelating iron within the P. aeruginosa extracellular environment. Although paerucumarin chelates iron, it does not function as a siderophore. Unlike PQS, paerucumarin is not associated with the P. aeruginosa cell envelope. PMID:27480638

  12. Label-free quantitative mass spectrometry for analysis of protein antigens in a meningococcal group B outer membrane vesicle vaccine.

    PubMed

    Dick, Lawrence W; Mehl, John T; Loughney, John W; Mach, Anna; Rustandi, Richard R; Ha, Sha; Zhang, Lan; Przysiecki, Craig T; Dieter, Lance; Hoang, Van M

    2015-01-01

    The development of a multivalent outer membrane vesicle (OMV) vaccine where each strain contributes multiple key protein antigens presents numerous analytical challenges. One major difficulty is the ability to accurately and specifically quantitate each antigen, especially during early development and process optimization when immunoreagents are limited or unavailable. To overcome this problem, quantitative mass spectrometry methods can be used. In place of traditional mass assays such as enzyme-linked immunosorbent assays (ELISAs), quantitative LC-MS/MS using multiple reaction monitoring (MRM) can be used during early-phase process development to measure key protein components in complex vaccines in the absence of specific immunoreagents. Multiplexed, label-free quantitative mass spectrometry methods using protein extraction by either detergent or 2-phase solvent were developed to quantitate levels of several meningococcal serogroup B protein antigens in an OMV vaccine candidate. Precision was demonstrated to be less than 15% RSD for the 2-phase extraction and less than 10% RSD for the detergent extraction method. Accuracy was 70 to 130% for the method using a 2-phase extraction and 90-110% for detergent extraction. The viability of MS-based protein quantification as a vaccine characterization method was demonstrated and advantages over traditional quantitative methods were evaluated. Implementation of these MS-based quantification methods can help to decrease the development time for complex vaccines and can provide orthogonal confirmation of results from existing antigen quantification techniques. PMID:25997113

  13. A Novel Mechanism of Host-Pathogen Interaction through sRNA in Bacterial Outer Membrane Vesicles

    PubMed Central

    Koeppen, Katja; Hampton, Thomas H.; Jarek, Michael; Scharfe, Maren; Gerber, Scott A.; Mielcarz, Daniel W.; Demers, Elora G.; Dolben, Emily L.; Hammond, John H.; Hogan, Deborah A.; Stanton, Bruce A.

    2016-01-01

    Bacterial outer membrane vesicle (OMV)-mediated delivery of proteins to host cells is an important mechanism of host-pathogen communication. Emerging evidence suggests that OMVs contain differentially packaged short RNAs (sRNAs) with the potential to target host mRNA function and/or stability. In this study, we used RNA-Seq to characterize differentially packaged sRNAs in Pseudomonas aeruginosa OMVs, and to show transfer of OMV sRNAs to human airway cells. We selected one sRNA for further study based on its stable secondary structure and predicted mRNA targets. Our candidate sRNA (sRNA52320), a fragment of a P. aeruginosa methionine tRNA, was abundant in OMVs and reduced LPS-induced as well as OMV-induced IL-8 secretion by cultured primary human airway epithelial cells. We also showed that sRNA52320 attenuated OMV-induced KC cytokine secretion and neutrophil infiltration in mouse lung. Collectively, these findings are consistent with the hypothesis that sRNA52320 in OMVs is a novel mechanism of host-pathogen interaction whereby P. aeruginosa reduces the host immune response. PMID:27295279

  14. Label-free quantitative mass spectrometry for analysis of protein antigens in a meningococcal group B outer membrane vesicle vaccine

    PubMed Central

    Dick Jr, Lawrence W; Mehl, John T; Loughney, John W; Mach, Anna; Rustandi, Richard R; Ha, Sha; Zhang, Lan; Przysiecki, Craig T; Dieter, Lance; Hoang, Van M

    2015-01-01

    ABSTRACT The development of a multivalent outer membrane vesicle (OMV) vaccine where each strain contributes multiple key protein antigens presents numerous analytical challenges. One major difficulty is the ability to accurately and specifically quantitate each antigen, especially during early development and process optimization when immunoreagents are limited or unavailable. To overcome this problem, quantitative mass spectrometry methods can be used. In place of traditional mass assays such as enzyme-linked immunosorbent assays (ELISAs), quantitative LC-MS/MS using multiple reaction monitoring (MRM) can be used during early-phase process development to measure key protein components in complex vaccines in the absence of specific immunoreagents. Multiplexed, label-free quantitative mass spectrometry methods using protein extraction by either detergent or 2-phase solvent were developed to quantitate levels of several meningococcal serogroup B protein antigens in an OMV vaccine candidate. Precision was demonstrated to be less than 15% RSD for the 2-phase extraction and less than 10% RSD for the detergent extraction method. Accuracy was 70 to 130% for the method using a 2-phase extraction and 90–110% for detergent extraction. The viability of MS-based protein quantification as a vaccine characterization method was demonstrated and advantages over traditional quantitative methods were evaluated. Implementation of these MS-based quantification methods can help to decrease the development time for complex vaccines and can provide orthogonal confirmation of results from existing antigen quantification techniques. PMID:25997113

  15. A novel mechanism of generating extracellular vesicles during apoptosis via a beads-on-a-string membrane structure

    PubMed Central

    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.

    2015-01-01

    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. PMID:26074490

  16. Lipopolysaccharide Modifications of a Cholera Vaccine Candidate Based on Outer Membrane Vesicles Reduce Endotoxicity and Reveal the Major Protective Antigen

    PubMed Central

    Leitner, Deborah R.; Feichter, Sandra; Schild-Prüfert, Kristina; Rechberger, Gerald N.; Reidl, Joachim

    2013-01-01

    The causative agent of the life-threatening gastrointestinal infectious disease cholera is the Gram-negative, facultative human pathogen Vibrio cholerae. We recently started to investigate the potential of outer membrane vesicles (OMVs) derived from V. cholerae as an alternative approach for a vaccine candidate against cholera and successfully demonstrated the induction of a long-lasting, high-titer, protective immune response upon immunization with OMVs using the mouse model. In this study, we present immunization data using lipopolysaccharide (LPS)-modified OMVs derived from V. cholerae, which allowed us to improve and identify the major protective antigen of the vaccine candidate. Our results indicate that reduction of endotoxicity can be achieved without diminishing the immunogenic potential of the vaccine candidate by genetic modification of lipid A. Although the protective potential of anti-LPS antibodies has been suggested many times, this is the first comprehensive study that uses defined LPS mutants to characterize the LPS-directed immune response of a cholera vaccine candidate in more detail. Our results pinpoint the O antigen to be the essential immunogenic structure and provide a protective mechanism based on inhibition of motility, which prevents a successful colonization. In a detailed analysis using defined antisera, we can demonstrate that only anti-O antigen antibodies, but not antibodies directed against the major flagellar subunit FlaA or the most abundant outer membrane protein, OmpU, are capable of effectively blocking the motility by binding to the sheathed flagellum and provide protection in a passive immunization assay. PMID:23630951

  17. A novel meningococcal outer membrane vesicle vaccine with constitutive expression of FetA: A phase I clinical trial

    PubMed Central

    Marsay, L.; Dold, C.; Green, C.A.; Rollier, C.S.; Norheim, G.; Sadarangani, M.; Shanyinde, M.; Brehony, C.; Thompson, A.J.; Sanders, H.; Chan, H.; Haworth, K.; Derrick, J.P.; Feavers, I.M.; Maiden, M.C.; Pollard, A.J.

    2015-01-01

    Summary Objectives Outer membrane vesicle (OMV) vaccines are used against outbreaks of capsular group B Neisseria meningitidis (MenB) caused by strains expressing particular PorA outer membrane proteins (OMPs). Ferric enterobactin receptor (FetA) is another variable OMP that induces type-specific bactericidal antibodies, and the combination of judiciously chosen PorA and FetA variants in vaccine formulations is a potential approach to broaden protection of such vaccines. Methods The OMV vaccine MenPF-1 was generated by genetically modifying N. meningitidis strain 44/76 to constitutively express FetA. Three doses of 25 μg or 50 μg of MenPF-1 were delivered intra-muscularly to 52 healthy adults. Results MenPF-1 was safe and well tolerated. Immunogenicity was measured by serum bactericidal assay (SBA) against wild-type and isogenic mutant strains. After 3 doses, the proportion of volunteers with SBA titres ≥1:4 (the putative protective titre) was 98% for the wild-type strain, and 77% for the strain 44/76 FetAonPorAoff compared to 51% in the strain 44/76 FetAoffPorAoff, demonstrating that vaccination with MenPF-1 simultaneously induced FetA and PorA bactericidal antibodies. Conclusion This study provides a proof-of-concept for generating bactericidal antibodies against FetA after OMV vaccination in humans. Prevalence-based choice of PorA and FetA types can be used to formulate a vaccine for broad protection against MenB disease. PMID:25982025

  18. A procedure for the rapid isolation from rat liver of plasma membrane vesicles exhibiting Ca2+-transport and Ca2+-ATPase activities.

    PubMed Central

    Epping, R J; Bygrave, F L

    1984-01-01

    A technique is described for the isolation of a plasma membrane-enriched preparation from a rat liver post-mitochondrial fraction by using discontinuous Percoll density-gradient centrifugation. The procedure is simple, of high reproducibility and yield and requires a total isolation time of only 90 min. The preparation consists almost exclusively of membrane vesicles and is enriched approx. 26-fold in plasma membrane-localized enzymes with minor contamination (less than 10%) with membranes derived mainly from the endoplasmic reticulum and Golgi apparatus. Approx. 20% of the fraction comprises tightly-sealed vesicles in the inverted orientation which are capable of accumulating calcium ions and exhibiting vanadate-insensitive Ca2+-ATPase activity. The properties of these activities, including insensitivity to vanadate, oxalate, and to p-chloromercuribenzoate as well as a lack of requirement for added Mg2+, contrast markedly with the reported properties of Ca2+ transport by the endoplasmic reticulum isolated from rat liver. The technique may have wide application in the study of plasma membrane-associated activities in rat liver, particularly in relation to sinusoidal membrane surface-related events. Images Fig. 2. PMID:6239615

  19. Membrane burdens of chlorinated benzenes lower the main phase transition temperature in dipalmitoyl-phosphatidylcholine vesicles: Implications for toxicity by narcotic chemicals

    SciTech Connect

    Wezel, A.P. van; Cornelissen, G.; Miltenburg, J.K. van; Opperhuizen, A.

    1996-02-01

    In the membrane of an organism that dies due to exposure to narcotic chemicals, the main phase transition temperature (T{sub tr}) of the phospholipids is decreased and the fluidity is increased. The decrease in T{sub tr} depends on the molar concentration of narcotics in the membrane (membrane burden) and is irrespective of the physicochemical properties of the chemicals. If membrane-water partition coefficients, exposure concentrations, and the amount of lipid in the system are known, membrane burdens of narcotic chemicals can be calculated and compared to membrane burdens that yield toxicity. The partition coefficients of a series of chlorobenzenes between phospholipid vesicles and water (K{sub mw}) were measured at different temperatures in a new experimental set-up. K{sub mw}`s were higher in the liquid-crystalline phase than in the gel phase. Partitioning into the el phase was entropy driven, partitioning into the liquid-crystalline phase was driven by entropy and enthalpy. The fluidity change in phospholipid vesicles, after accumulation of chlorobenzenes, was measured from the change in T{sub tr}. The membrane burdens of various chlorobenzenes needed for a lowering of T{sub tr} were comparable (e.g., 20--60 mmol/kg for a decrease of 1.0 C). The membrane burden needed in vivo for lethality by narcotic chemicals such as chlorobenzenes was calculated to be 40--160 mmol/kg membrane. By combining the in vivo and in vitro data, it can be concluded that in organisms that die due to exposure to narcotic chemicals, the fluidity of the membrane is increased.

  20. Cholesterol induces surface localization of polyphenols in model membranes thus enhancing vesicle stability against lysozyme, but reduces protection of distant double bonds from reactive-oxygen species.

    PubMed

    de Athayde Moncorvo Collado, Alejandro; Dupuy, Fernando G; Morero, Roberto D; Minahk, Carlos

    2016-07-01

    The main scope of the present study was to analyze the membrane interaction of members of different classes of polyphenols, i.e. resveratrol, naringenin, epigallocatechin gallate and enterodiol, in model systems of different compositions and phase states. In addition, the possible association between membrane affinity and membrane protection against both lipid oxidation and bilayer-disruptive compounds was studied. Gibbs monolayer experiments indicated that even though polyphenols showed poor surface activity, it readily interacted with lipid films. Actually, a preferential interaction with expanded monolayers was observed, while condensed and cholesterol-containing monolayers decreased the affinity of these phenolic compounds. On the other hand, fluorescence anisotropy studies showed that polyphenols were able to modulate membrane order degree, but again this effect was dependent on the cholesterol concentration and membrane phase state. In fact, cholesterol induced a surface rather than deep into the hydrophobic core localization of phenolic compounds in the membranes. In general, the polyphenolic molecules tested had a better antioxidant activity when they were allowed to get inserted into the bilayers, i.e. in cholesterol-free membranes. On the other hand, a membrane-protective effect against bilayer permeabilizing activity of lysozyme, particularly in the presence of cholesterol, could be assessed. It can be hypothesized that phenolic compounds may protect membrane integrity by loosely covering the surface of lipid vesicles, once cholesterol push them off from the membrane hydrophobic core. However, this cholesterol-driven distribution may lead to a reduced antioxidant activity of linoleic acid double bonds. PMID:27063609

  1. Annexin XIIIb: a novel epithelial specific annexin is implicated in vesicular traffic to the apical plasma membrane

    PubMed Central

    1995-01-01

    The sorting of apical and basolateral proteins into vesicular carriers takes place in the trans-Golgi network (TGN) in MDCK cells. We have previously analyzed the protein composition of immunoisolated apical and basolateral transport vesicles and have now identified a component that is highly enriched in apical vesicles. Isolation of the encoding cDNA revealed that this protein, annexin XIIIb, is a new isoform of the epithelial specific annexin XIII sub-family which includes the previously described intestine-specific annexin (annexin XIIIa; Wice, B. M., and J. I. Gordon. 1992. J. Cell Biol. 116:405-422). Annexin XIIIb differs from annexin XIIIa in that it contains a unique insert of 41 amino acids in the NH2 terminus and is exclusively expressed in dog intestine and kidney. Immunofluorescence microscopy demonstrated that annexin XIIIb was localized to the apical plasma membrane and underlying punctate structures. Since annexins have been suggested to play a role in membrane-membrane interactions in exocytosis and endocytosis, we investigated whether annexin XIIIb is involved in delivery to the apical cell surface. To this aim we used permeabilized MDCK cells and a cytosol-dependent in vitro transport assay. Antibodies specific for annexin XIIIb significantly inhibited the transport of influenza virus hemagglutinin from the TGN to the apical plasma membrane while the transport of vesicular stomatitis virus glycoprotein to the basolateral cell surface was unaffected. We propose that annexin XIIIb plays a role in vesicular transport to the apical plasma membrane in MDCK cells. PMID:7896870

  2. A novel approach for purification and selective capture of membrane vesicles of the periodontopathic bacterium, Porphyromonas gingivalis: membrane vesicles bind to magnetic beads coated with epoxy groups in a noncovalent, species-specific manner.

    PubMed

    Nakao, Ryoma; Kikushima, Kenji; Higuchi, Hideo; Obana, Nozomu; Nomura, Nobuhiko; Bai, Dongying; Ohnishi, Makoto; Senpuku, Hidenobu

    2014-01-01

    Membrane vesicles (MVs) of Porphyromonas gingivalis are regarded as an offensive weapon of the bacterium, leading to tissue deterioration in periodontal disease. Therefore, isolation of highly purified MVs is indispensable to better understand the pathophysiological role of MVs in the progression of periodontitis. MVs are generally isolated by a conventional method based on ultracentrifugation of the bacterial culture supernatant. However, the resulting MVs are often contaminated with co-precipitating bacterial appendages sheared from the live bacteria. Here, we report an intriguing property of P. gingivalis MVs--their ability to bind superparamagnetic beads coated with epoxy groups (SB-Epoxy). Analysis of fractions collected during the purification revealed that all MVs of five tested P. gingivalis stains bound to SB-Epoxy. In contrast, free fimbriae in the crude MV preparation did not bind to the SB-Epoxy. The SB-Epoxy-bound MVs were easily dissociated from the SB-Epoxy using a mild denaturation buffer. These results suggest that the surface chemistry conferred by epoxy on the beads is responsible for the binding, which is mediated by noncovalent bonds. Both the structural integrity and purity of the isolated MVs were confirmed by electron microscopy. The isolated MVs also caused cell detachment from culture dishes at a physiologically relevant concentration. Assays of competitive binding between the SB-Epoxy and mixtures of MVs from five bacterial species demonstrated that only P. gingivalis MVs could be selectively eliminated from the mixtures. We suggest that this novel approach enables efficient purification and selective elimination of P. gingivalis MVs. PMID:24830438

  3. Highly purified bile-canalicular vesicles and lateral plasma membranes isolated from rat liver on Nycodenz gradients. Biochemical and immunolocalization studies.

    PubMed Central

    Ali, N; Aligue, R; Evans, W H

    1990-01-01

    1. A liver canalicular plasma-membrane fraction enriched 115-155-fold in five marker enzymes relative to the tissue homogenate was obtained by sonication of liver plasma membranes followed by fractionation in iso-osmotic Nycodenz gradients. 2. Two lateral-plasma membrane fractions were also collected by this procedure; the lighter-density fraction was still associated with canalicular membranes, as assessed by enzymic and polypeptide analysis. 3. The polypeptide composition of the domain-defined plasma-membrane fractions was evaluated. It was demonstrated by immunoblotting that the 41 kDa alpha-subunit of the inhibitory G-protein, associated in high relative amounts with canalicular plasma-membrane fractions, was partially lost in the last stage of purification; however, this subunit was retained by lateral plasma membranes. 4. Antibodies to the proteins of bile-canalicular vesicles were shown to localize to the hepatocyte surface in thin liver sections examined by immunofluorescent and immuno-gold electron microscopy. Two subsets of antigens were identified, one present on both sinusoidal and canalicular plasma-membrane domains and another, by using antisera pre-absorbed with sinusoidal plasma membranes, that was confined to the bile-canalicular domain. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:2222412

  4. Munc18-1 domain-1 controls vesicle docking and secretion by interacting with syntaxin-1 and chaperoning it to the plasma membrane

    PubMed Central

    Han, Gayoung A.; Malintan, Nancy T.; Saw, Ner Mu Nar; Li, Lijun; Han, Liping; Meunier, Frederic A.; Collins, Brett M.; Sugita, Shuzo

    2011-01-01

    Munc18-1 plays pleiotropic roles in neurosecretion by acting as 1) a molecular chaperone of syntaxin-1, 2) a mediator of dense-core vesicle docking, and 3) a priming factor for soluble N-ethylmaleimide–sensitive factor attachment protein receptor–mediated membrane fusion. However, how these functions are executed and whether they are correlated remains unclear. Here we analyzed the role of the domain-1 cleft of Munc18-1 by measuring the abilities of various mutants (D34N, D34N/M38V, K46E, E59K, K46E/E59K, K63E, and E66A) to bind and chaperone syntaxin-1 and to restore the docking and secretion of dense-core vesicles in Munc18-1/-2 double-knockdown cells. We identified striking correlations between the abilities of these mutants to bind and chaperone syntaxin-1 with their ability to restore vesicle docking and secretion. These results suggest that the domain-1 cleft of Munc18-1 is essential for binding to syntaxin-1 and thereby critical for its chaperoning, docking, and secretory functions. Our results demonstrate that the effect of the alleged priming mutants (E59K, D34N/M38V) on exocytosis can largely be explained by their reduced syntaxin-1–chaperoning functions. Finally, our data suggest that the intracellular expression and distribution of syntaxin-1 determines the level of dense-core vesicle docking. PMID:21900502

  5. Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles Are Internalized in Human Host Cells and Trigger NOD1- and NOD2-Dependent NF-κB Activation

    PubMed Central

    Thay, Bernard; Damm, Anna; Kufer, Thomas A.; Wai, Sun Nyunt

    2014-01-01

    Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. We recently demonstrated that outer membrane vesicles (OMVs) disseminated by A. actinomycetemcomitans could deliver multiple proteins, including biologically active cytolethal distending toxin (CDT), into the cytosol of HeLa cells and human gingival fibroblasts (HGF). In the present work, we have used immunoelectron and confocal microscopy analysis and fluorescently labeled vesicles to further investigate mechanisms for A. actinomycetemcomitans OMV-mediated delivery of bacterial antigens to these host cells. Our results supported that OMVs were internalized into the perinuclear region of HeLa cells and HGF. Colocalization analysis revealed that internalized OMVs colocalized with the endoplasmic reticulum and carried antigens, detected using an antibody specific to whole A. actinomycetemcomitans serotype a cells. Consistent with OMV internalization mediating intracellular antigen exposure, the vesicles acted as strong inducers of cytoplasmic peptidoglycan sensor NOD1- and NOD2-dependent NF-κB activation in human embryonic kidney cells. Moreover, NOD1 was the main sensor of OMV-delivered peptidoglycan in myeloid THP1 cells, contributing to the overall inflammatory responses induced by the vesicles. This work reveals a role of A. actinomycetemcomitans OMVs as a trigger of innate immunity via carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs). PMID:25024364

  6. Phase-Field Modeling of Lipid Vesicles With Pores

    NASA Astrophysics Data System (ADS)

    Seifi, Saman; Salac, David

    2013-11-01

    The formation and annihilation of pores in a lipid vesicle membrane is critical to a number of biotechnologies, such as drug delivery. Previous models of vesicle behavior have ignored the influence of topological changes in the vesicle membrane. Here the entire Helfrich model of a vesicle membrane is considered. Topological changes in the vesicle membrane, such as the formation of a pore, are captured through the use of an embedded phase-field model. The numerical method and sample results will be presented.

  7. Acinetobacter baumannii Extracellular OXA-58 Is Primarily and Selectively Released via Outer Membrane Vesicles after Sec-Dependent Periplasmic Translocation

    PubMed Central

    Liao, Yu-Ting; Kuo, Shu-Chen; Chiang, Ming-Hsien; Lee, Yi-Tzu; Sung, Wang-Chou; Chen, You-Hsuan; Fung, Chang-Phone

    2015-01-01

    Carbapenem-resistant Acinetobacter baumannii (CRAb) shelter cohabiting carbapenem-susceptible bacteria from carbapenem killing via extracellular release of carbapenem-hydrolyzing class D β-lactamases, including OXA-58. However, the mechanism of the extracellular release of OXA-58 has not been elucidated. In silico analysis predicted OXA-58 to be translocated to the periplasm via the Sec system. Using cell fractionation and Western blotting, OXA-58 with the signal peptide and C terminus deleted was not detected in the periplasmic and extracellular fractions. Overexpression of enhanced green fluorescent protein fused to the OXA-58 signal peptide led to its periplasmic translocation but not extracellular release, suggesting that OXA-58 is selectively released. The majority of the extracellular OXA-58 was associated with outer membrane vesicles (OMVs). The OMV-associated OXA-58 was detected only in a strain overexpressing OXA-58. The presence of OXA-58 in OMVs was confirmed by a carbapenem inactivation bioassay, proteomic analysis, and transmission electron microscopy. Imipenem treatment increased OMV formation and caused cell lysis, resulting in an increase in the OMV-associated and OMV-independent release of extracellular OXA-58. OMV-independent OXA-58 hydrolyzed nitrocefin more rapidly than OMV-associated OXA-58 but was more susceptible to proteinase K degradation. Rose bengal, an SecA inhibitor, inhibited the periplasmic translocation and OMV-associated release of OXA-58 and abolished the sheltering effect of CRAb. This study demonstrated that the majority of the extracellular OXA-58 is selectively released via OMVs after Sec-dependent periplasmic translocation. Addition of imipenem increased both OMV-associated and OMV-independent OXA-58, which may have different biological roles. SecA inhibitor could abolish the carbapenem-sheltering effect of CRAb. PMID:26369971

  8. A native outer membrane vesicle vaccine confers protection against meningococcal colonization in human CEACAM1 transgenic mice

    PubMed Central

    Johswich, Kay O.; Gray-Owen, Scott D.; Granoff, Dan M.

    2015-01-01

    Background The effect of protein-based meningococcal vaccines on prevention of nasopharyngeal colonization has been difficult to investigate experimentally because a reliable animal colonization model did not exist. Methods Human CEACAM1 transgenic mice, which can be colonized by meningococci, were immunized IP with one of two meningococcal native outer membrane vesicle (NOMV) vaccines prepared from mutants with attenuated endotoxin (lpxL1 knockout) and over-expressed sub-family B Factor H-binding proteins (FHbp). Animals were challenged intranasally two weeks after the third dose with wild-type strain H44/76, or were treated IP with anti-NOMV serum before and during the bacterial challenge. Results The NOMV-1 vaccine, prepared from the serogroup B H44/76 mutant, elicited ~40-fold higher serum bactericidal antibody titers against the wild-type H44/76 challenge strain than the NOMV-2 vaccine prepared from a heterologous serogroup W mutant strain with different PorA and FHbp amino acid sequence variants. Compared to aluminum hydroxide-immunized control mice, the efficacy for prevention of any H44/76 colonization was 93% (95% confidence interval, 52-99, P<0.0001) for the NOMV-1 vaccine, and 19% (−3-36, P=0.23) for NOMV-2. NOMV-2-vaccinated mice had a 5.6-fold decrease in geometric mean CFU of bacteria per animal in tracheal washes compared to control mice (P=0.007). The efficacy of passive administration of serum from NOMV-1-vaccinated mice to immunologically naïve mice against colonization was 44% (17-61; P=0.002). Conclusions Both NOMV vaccines protected against meningococcal colonization but there was greater protection by the NOMV-1 vaccine with antigens matched with the challenge strain. Meningococcal vaccines that target protein antigens have potential to decrease colonization. PMID:25662856

  9. Pseudomonas aeruginosa Outer Membrane Vesicles Triggered by Human Mucosal Fluid and Lysozyme Can Prime Host Tissue Surfaces for Bacterial Adhesion

    PubMed Central

    Metruccio, Matteo M. E.; Evans, David J.; Gabriel, Manal M.; Kadurugamuwa, Jagath L.; Fleiszig, Suzanne M. J.

    2016-01-01

    Pseudomonas aeruginosa is a leading cause of human morbidity and mortality that often targets epithelial surfaces. Host immunocompromise, or the presence of indwelling medical devices, including contact lenses, can predispose to infection. While medical devices are known to accumulate bacterial biofilms, it is not well understood why resistant epithelial surfaces become susceptible to P. aeruginosa. Many bacteria, including P. aeruginosa, release outer membrane vesicles (OMVs) in response to stress that can fuse with host cells to alter their function. Here, we tested the hypothesis that mucosal fluid can trigger OMV release to compromise an epithelial barrier. This was tested using tear fluid and corneal epithelial cells in vitro and in vivo. After 1 h both human tear fluid, and the tear component lysozyme, greatly enhanced OMV release from P. aeruginosa strain PAO1 compared to phosphate buffered saline (PBS) controls (∼100-fold). Transmission electron microscopy (TEM) and SDS-PAGE showed tear fluid and lysozyme-induced OMVs were similar in size and protein composition, but differed from biofilm-harvested OMVs, the latter smaller with fewer proteins. Lysozyme-induced OMVs were cytotoxic to human corneal epithelial cells in vitro and murine corneal epithelium in vivo. OMV exposure in vivo enhanced Ly6G/C expression at the corneal surface, suggesting myeloid cell recruitment, and primed the cornea for bacterial adhesion (∼4-fold, P < 0.01). Sonication disrupted OMVs retained cytotoxic activity, but did not promote adhesion, suggesting the latter required OMV-mediated events beyond cell killing. These data suggest that mucosal fluid induced P. aeruginosa OMVs could contribute to loss of epithelial barrier function during medical device-related infections. PMID:27375592

  10. Cellular Immune Responses in Humans Induced by Two Serogroup B Meningococcal Outer Membrane Vesicle Vaccines Given Separately and in Combination.

    PubMed

    Oftung, Fredrik; Korsvold, Gro Ellen; Aase, Audun; Næss, Lisbeth M

    2016-04-01

    MenBvac and MeNZB are safe and efficacious outer membrane vesicle (OMV) vaccines against serogroup B meningococcal disease. Antibody responses have previously been investigated in a clinical trial with these two OMV vaccines given separately (25 μg/dose) or in combination (12.5 and 12.5 μg/dose) in three doses administered at 6-week intervals. Here, we report the results from analyzing cellular immune responses against MenBvac and MeNZB OMVs in terms of antigen-specific CD4(+)T cell proliferation and secretion of cytokines. The proliferative CD4(+)T cell responses to the combined vaccine were of the same magnitude as the homologous responses observed for each individual vaccine. The results also showed cross-reactivity in the sense that both vaccine groups receiving separate vaccines responded to both homologous and heterologous OMV antigen when assayed for antigen-specific cellular proliferation. In addition, a multiplex bead array assay was used to analyze the presence of Th1 and Th2 cytokines in cell culture supernatants. The results showed that gamma interferon, interleukin-4 (IL-4), and IL-10 responses could be detected as a result of vaccination with both the MenBvac and the MeNZB vaccines given separately, as well as when given in combination. With respect to cross-reactivity, the cytokine results paralleled the observations made for proliferation. In conclusion, the results demonstrate that cross-reactive cellular immune responses involving both Th1 and Th2 cytokines can be induced to the same extent by different tailor-made OMV vaccines given either separately or in combination with half the dose of each vaccine. PMID:26865595

  11. Neisseria meningitidis Native Outer Membrane Vesicles Containing Different Lipopolysaccharide Glycoforms as Adjuvants for Meningococcal and Nonmeningococcal Antigens

    PubMed Central

    Nagaputra, Jerry C.; Sadarangani, Manish; Hoe, J. Claire; Mehta, Ojas Hrakesh; Norheim, Gunnstein; Saleem, Muhammad; Chan, Hannah; Derrick, Jeremy P.; Feavers, Ian; Pollard, Andrew J.; Moxon, E. Richard

    2014-01-01

    We evaluated the adjuvant effect of a modified glycoform of lipopolysaccharide (LPS) (LgtB-LpxL1) compared to that of the nonmodified glycoform Lpxl1 serogroup B meningococcal H44/76 native outer membrane vesicles (nOMVs) on immune responses to vaccination with the recombinant meningococcal protein, rPorA, tetanus toxoid, or meningococcal serogroup C capsular polysaccharide. We used LgtB-LpxL1 LPS because the disruption of the lgtB gene, which results in the exposure of N-acetylglucosamine-galactose-glucose residues in the LPS outer core, has been shown to enhance the activation of human dendritic cells in vitro. The responses were compared to those of a monophosphoryl lipid A (MPL)-based adjuvant and to an aluminum hydroxide suspension. The nOMVs induced blood serum IgG responses against each of the three antigens comparable to those obtained with MPL or aluminum salt. However, nOMVs elicited (i) a lower IgG1/IgG2a ratio against rPorA and (ii) serum bactericidal antibody titers superior to those achieved with aluminum salt, reaching similar titers to those obtained with MPL. Similarly, bactericidal antibody titers induced by immunization with meningococcal serogroup C polysaccharide and nOMVs were similar to those obtained using MPL but were better than those with aluminum salt. Immunization with tetanus toxoid and nOMVs resulted in tetanus toxoid-specific IgG responses similar to those obtained when adjuvanted with aluminum salt. These results highlight the potential utility of meningococcal LpxL1 LPS-containing nOMVs as an adjuvant for recombinant meningococcal protein vaccines and suggest their possible use with a variety of other antigens. PMID:24351756

  12. Heterogeneity of L-alanine transport systems in brush-border membrane vesicles from rat placenta during late gestation.

    PubMed Central

    Alonso-Torre, S R; Serrano, M A; Medina, J M; Alvarado, F

    1992-01-01

    The placental uptake of L-alanine was studied by using purified brush-border membrane vesicles from rat trophoblasts. Saturation curves were carried out at 37 degrees C in buffers containing 100 mM (zero-trans)-NaSCN, -NaCl, -KSCN, -KCl, or -N-methyl-D-glucamine gluconate. The uncorrected uptake results were fitted by non-linear regression analysis to an equation involving one diffusional component either one or two saturable Michaelian transport terms. In the presence of NaCl, two distinct L-alanine transport systems were distinguished, named respectively System 1 (S-1; Vm1 about 760 pmol/s per mg of protein; KT1 = 0.5 mM) and System 2 (S-2; Vm2 about 1700 pmol/s per mg; KT2 = 9 mM). In contrast, in the presence of K+ (KCl = KSCN) or in the absence of any alkali-metal ions (N-methyl-D-glucamine gluconate), only one saturable system was apparent, which we identify as S-2. When Na+ is present, S-1, but not S-2, appears to be rheogenic, since its maximal transport capacity significantly increases in the presence of an inside-negative membrane potential, created either by replacing Cl- with the permeant anion thiocyanate (NaSCN > NaCl) or by applying an appropriate K+ gradient and valinomycin. alpha-(Methylamino)isobutyrate (methyl-AIB) appears to be a substrate of S-1, but not of S-2. For reasons that remain to be explained, however, methyl-AIB inhibits S-2. We conclude that S-1 represents a truly Na(+)-dependent mechanism, where Na+ behaves as an obligatory activator, whereas S-2 cannot discriminate between Na+ and K+, although its activity is higher in the presence of alkali-metal ions than in their absence (Na+ = K+ > N-methyl-D-glucammonium ion). S-2 appears to be fully developed 2 days before birth, whereas S-1 undergoes a capacity-type activation between days 19.5 and 21.5 of gestation, i.e. its apparent Vmax. nearly doubles, whereas its KT remains constant. PMID:1445280

  13. The t-SNAREs syntaxin4 and SNAP23 but not v-SNARE VAMP2 are indispensable to tether GLUT4 vesicles at the plasma membrane in adipocyte

    SciTech Connect

    Kawaguchi, Takayuki; Tamori, Yoshikazu; Kanda, Hajime; Yoshikawa, Mari; Tateya, Sanshiro; Nishino, Naonobu; Kasuga, Masato

    2010-01-15

    SNARE proteins (VAMP2, syntaxin4, and SNAP23) have been thought to play a key role in GLUT4 trafficking by mediating the tethering, docking and subsequent fusion of GLUT4-containing vesicles with the plasma membrane. The precise functions of these proteins have remained elusive, however. We have now shown that depletion of the vesicle SNARE (v-SNARE) VAMP2 by RNA interference in 3T3-L1 adipocytes inhibited the fusion of GLUT4 vesicles with the plasma membrane but did not affect tethering of the vesicles to the membrane. In contrast, depletion of the target SNAREs (t-SNAREs) syntaxin4 or SNAP23 resulted in impairment of GLUT4 vesicle tethering to the plasma membrane. Our results indicate that the t-SNAREs syntaxin4 and SNAP23 are indispensable for the tethering of GLUT4 vesicles to the plasma membrane, whereas the v-SNARE VAMP2 is not required for this step but is essential for the subsequent fusion event.

  14. Membrane Fluidity and Lipid Order in Ternary Giant Unilamellar Vesicles Using a New Bodipy-Cholesterol Derivative

    PubMed Central

    Ariola, Florly S.; Li, Zaiguo; Cornejo, Christine; Bittman, Robert; Heikal, Ahmed A.

    2009-01-01

    Cholesterol-rich, liquid-ordered (Lo) domains are believed to be biologically relevant, and yet detailed knowledge about them, especially in live cells under physiological conditions, is elusive. Although these domains have been observed in model membranes, understanding cholesterol-lipid interactions at the molecular level, under controlled lipid mixing, remains a challenge. Further, although there are a number of fluorescent lipid analogs that partition into liquid-disordered (Ld) domains, the number of such analogs with a high affinity for biologically relevant Lo domains is limited. Here, we use a new Bodipy-labeled cholesterol (Bdp-Chol) derivative to investigate membrane fluidity, lipid order, and partitioning in various lipid phases in giant unilamellar vesicles (GUVs) as a model system. GUVs were prepared from mixtures of various molar fractions of dioleoylphosphatidylcholine, cholesterol, and egg sphingomyelin. The Ld phase domains were also labeled with 1,1′-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI-C12) for comparison. Two-photon fluorescence lifetime and anisotropy imaging of Bdp-Chol are sensitive to lipid phase domains in GUVs. The fluorescence lifetime of Bdp-Chol in liquid-disordered, single-phase GUVs is 5.50 ± 0.08 ns, compared with 4.1 ± 0.4 ns in the presence of DiI-C12. The observed reduction of fluorescence lifetime is attributed to Förster resonance energy transfer between Bdp-Chol (a donor) and DiI-C12 (an acceptor) with an estimated efficiency of 0.25 and donor-acceptor distance of 2.6 ± 0.2 nm. These results also indicate preferential partitioning (Kp = 1.88) of Bdp-Chol into the Lo phase. One-photon, time-resolved fluorescence anisotropy of Bdp-Chol decays as a triexponential in the lipid bilayer with an average rotational diffusion coefficient, lipid order parameter, and membrane fluidity that are sensitive to phase domains. The translational diffusion coefficient of Bdp-Chol, as measured using fluorescence

  15. Streptococcus mutans Extracellular DNA Is Upregulated during Growth in Biofilms, Actively Released via Membrane Vesicles, and Influenced by Components of the Protein Secretion Machinery

    PubMed Central

    Liao, Sumei; Klein, Marlise I.; Heim, Kyle P.; Fan, Yuwei; Bitoun, Jacob P.; Ahn, San-Joon; Burne, Robert A.; Koo, Hyun; Brady, L. Jeannine

    2014-01-01

    Streptococcus mutans, a major etiological agent of human dental caries, lives primarily on the tooth surface in biofilms. Limited information is available concerning the extracellular DNA (eDNA) as a scaffolding matrix in S. mutans biofilms. This study demonstrates that S. mutans produces eDNA by multiple avenues, including lysis-independent membrane vesicles. Unlike eDNAs from cell lysis that were abundant and mainly concentrated around broken cells or cell debris with floating open ends, eDNAs produced via the lysis-independent pathway appeared scattered but in a structured network under scanning electron microscopy. Compared to eDNA production of planktonic cultures, eDNA production in 5- and 24-h biofilms was increased by >3- and >1.6-fold, respectively. The addition of DNase I to growth medium significantly reduced biofilm formation. In an in vitro adherence assay, added chromosomal DNA alone had a limited effect on S. mutans adherence to saliva-coated hydroxylapatite beads, but in conjunction with glucans synthesized using purified glucosyltransferase B, the adherence was significantly enhanced. Deletion of sortase A, the transpeptidase that covalently couples multiple surface-associated proteins to the cell wall peptidoglycan, significantly reduced eDNA in both planktonic and biofilm cultures. Sortase A deficiency did not have a significant effect on membrane vesicle production; however, the protein profile of the mutant membrane vesicles was significantly altered, including reduction of adhesin P1 and glucan-binding proteins B and C. Relative to the wild type, deficiency of protein secretion and membrane protein insertion machinery components, including Ffh, YidC1, and YidC2, also caused significant reductions in eDNA. PMID:24748612

  16. The effect of imipenem and diffusible signaling factors on the secretion of outer membrane vesicles and associated Ax21 proteins in Stenotrophomonas maltophilia

    PubMed Central

    Devos, Simon; Van Oudenhove, Laurence; Stremersch, Stephan; Van Putte, Wouter; De Rycke, Riet; Van Driessche, Gonzalez; Vitse, Jolien; Raemdonck, Koen; Devreese, Bart

    2015-01-01

    Outer membrane vesicles (OMVs) are small nanoscale structures that are secreted by bacteria and that can carry nucleic acids, proteins, and small metabolites. They can mediate intracellular communication and play a role in virulence. In this study, we show that treatment with the β-lactam antibiotic imipenem leads to a dramatic increase in the secretion of outer membrane vesicles in the nosocomial pathogen Stenotrophomonas maltophilia. Proteomic analysis of their protein content demonstrated that the OMVs contain the chromosomal encoded L1 metallo-β-lactamase and L2 serine-β-lactamase. Moreover, the secreted OMVs contain large amounts of two Ax21 homologs, i.e., outer membrane proteins known to be involved in virulence and biofilm formation. We show that OMV secretion and the levels of Ax21 in the OMVs are dependent on the quorum sensing diffusible signal system (DSF). More specific, we demonstrate that the S. maltophilia DSF cis-Δ2-11-methyl-dodecenoic acid and, to a lesser extent, the Burkholderia cenocepacia DSF cis-Δ2-dodecenoic acid, stimulate OMV secretion. By a targeted proteomic analysis, we confirmed that DSF-induced OMVs contain large amounts of the Ax21 homologs, but not the β-lactamases. This work illustrates that both quorum sensing and disturbance of the peptidoglycan biosynthesis provoke the release of OMVs and that OMV content is context dependent. PMID:25926824

  17. The effect of imipenem and diffusible signaling factors on the secretion of outer membrane vesicles and associated Ax21 proteins in Stenotrophomonas maltophilia.

    PubMed

    Devos, Simon; Van Oudenhove, Laurence; Stremersch, Stephan; Van Putte, Wouter; De Rycke, Riet; Van Driessche, Gonzalez; Vitse, Jolien; Raemdonck, Koen; Devreese, Bart

    2015-01-01

    Outer membrane vesicles (OMVs) are small nanoscale structures that are secreted by bacteria and that can carry nucleic acids, proteins, and small metabolites. They can mediate intracellular communication and play a role in virulence. In this study, we show that treatment with the β-lactam antibiotic imipenem leads to a dramatic increase in the secretion of outer membrane vesicles in the nosocomial pathogen Stenotrophomonas maltophilia. Proteomic analysis of their protein content demonstrated that the OMVs contain the chromosomal encoded L1 metallo-β-lactamase and L2 serine-β-lactamase. Moreover, the secreted OMVs contain large amounts of two Ax21 homologs, i.e., outer membrane proteins known to be involved in virulence and biofilm formation. We show that OMV secretion and the levels of Ax21 in the OMVs are dependent on the quorum sensing diffusible signal system (DSF). More specific, we demonstrate that the S. maltophilia DSF cis-Δ2-11-methyl-dodecenoic acid and, to a lesser extent, the Burkholderia cenocepacia DSF cis-Δ2-dodecenoic acid, stimulate OMV secretion. By a targeted proteomic analysis, we confirmed that DSF-induced OMVs contain large amounts of the Ax21 homologs, but not the β-lactamases. This work illustrates that both quorum sensing and disturbance of the peptidoglycan biosynthesis provoke the release of OMVs and that OMV content is context dependent. PMID:25926824

  18. Mutant Native Outer Membrane Vesicles Combined with a Serogroup A Polysaccharide Conjugate Vaccine for Prevention of Meningococcal Epidemics in Africa

    PubMed Central

    Pajon, Rolando; Fergus, Andrew M.; Granoff, Dan M.

    2013-01-01

    Background The meningococcal serogroup A (MenA) polysaccharide conjugate vaccine used in Sub-Saharan Africa does not prevent disease caused by MenW or MenX strains, which also cause epidemics in the region. We investigated the vaccine-potential of native outer membrane vesicles with over-expressed factor H-binding protein (NOMV-fHbp), which targeted antigens in African meningococcal strains, and was combined with a MenA polysaccharide conjugate vaccine. Methodology/Principal Findings The NOMV-fHbp vaccine was prepared from a mutant African MenW strain with PorA P1.5,2, attenuated endotoxin (ΔLpxL1), deleted capsular genes, and over-expressed fHbp in variant group 1. The NOMV-fHbp was adsorbed with Al(OH)3 and used to reconstitute a lyophilized MenA conjugate vaccine, which normally is reconstituted with liquid MenC, Y and W conjugates in a meningococcal quadrivalent conjugate vaccine (MCV4-CRM, Novartis). Mice immunized with the NOMV-fHbp vaccine alone developed serum bactericidal (human complement) activity against 13 of 15 African MenA strains tested; 10 of 10 African MenX strains, 7 of 7 African MenW strains, and 6 of 6 genetically diverse MenB strains with fHbp variant group 1 (including 1 strain from The Gambia). The combination NOMV-fHbp/MenA conjugate vaccine elicited high serum bactericidal titers against the two MenA strains tested that were resistant to bactericidal antibodies elicited by the NOMV-fHbp alone; the combination elicited higher titers against the MenA and MenW strains than those elicited by a control MCV4-CRM vaccine (P<0.05); and high titers against MenX and MenB strains. For most strains, the titers elicited by a control NOMV-fHbp knock out vaccine were <1∶10 except when the strain PorA matched the vaccine (titers >1∶000). Conclusion/Significance The NOMV-fHbp/MenA conjugate vaccine provided similar or higher coverage against MenA and MenW strains than a quadrivalent meningococcal conjugate vaccine, and extended protection against Men

  19. Proline porter II is activated by a hyperosmotic shift in both whole cells and membrane vesicles of Escherichia coli K12.

    PubMed

    Milner, J L; Grothe, S; Wood, J M

    1988-10-15

    Proline porter II is rapidly activated when nongrowing bacteria are subjected to a hyperosmotic shift (Grothe, S., Krogsrud, R. L., McClellan, D. J., Milner, J. L., and Wood, J. M. (1986) J. Bacteriol. 166, 253-259). Proline porter II was active in membrane vesicles prepared from bacteria grown under optimal conditions, nutritional stress, or osmotic stress. That activity was: (i) dependent on the presence of the energy sources phenazine methosulphate plus ascorbate or D-lactate; (ii) observed only when a hyperosmotic shift accompanied the transport measurement; (iii) inhibited by glycine betaine in a manner analogous to that observed in whole cells; and (iv) eliminated by lesions in proP. Membrane vesicles were able to transport serine but not glutamine and serine transport was reduced by the hyperosmotic shift. In whole cells, proline porter II activity was supported by glucose and by D-lactate in a strain defective for proline porters I and III and the F1F0-ATPase. Glucose energized proline uptake was eliminated by carbonyl cyanide m-chlorophenylhydrazone and KCN as was serine uptake. These results suggested that proline porter II was respiration-dependent and probably ion-linked. Activation of proline porter II in whole cells by sucrose or NaCl was sustained over 30 min, whereas activation by glycerol was transient. Proline porter II was activated by NaCl and sucrose with a half-time of approximately 1 min in both whole cells and membrane vesicles. Thus, activation of proline porter II was reversible. It occurred at a rate comparable to that of K+ influx and much more rapid than the genetic regulatory responses that follow a hyperosmotic shift. PMID:3049595

  20. Differential Regulation of the Serotonin Transporter by Vesicle-Associated Membrane Protein 2 in Cells of Neuronal versus Non-Neuronal Origin

    PubMed Central

    Müller, Heidi Kaastrup; Kragballe, Marie; Fjorback, Anja Winther; Wiborg, Ove

    2014-01-01

    The serotonin transporter (SERT) is a key regulator of serotonergic signalling as it mediates the re-uptake of synaptic serotonin into nerve terminals, thereby terminating or modulating its signal. It is well-known that SERT regulation is a dynamic process orchestrated by a wide array of proteins and mechanisms. However, molecular details on possible coordinated regulation of SERT activity and 5-HT release are incomplete. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, interacts with SERT. This was documented in vitro, through GST pull-down assays, by co-immunoprecipitation experiments on heterologous cells and rat hippocampal synaptosomes, and with FRET analysis in live transfected HEK-293 MSR cells. The related isoforms VAMP1 and VAMP3 also physically interact with SERT. However, comparison of the three VAMP isoforms shows that only VAMP2 possesses a functionally distinct role in relation to SERT. VAMP2 influences 5-HT uptake, cell surface expression and the delivery rate of SERT to the plasma membrane differentially in HEK-293 MSR and PC12 cells. Moreover, siRNA-mediated knock-down of endogenous VAMP2 reduces 5-HT uptake in CAD cells stably expressing low levels of heterologous SERT. Deletion and mutant analysis suggest a role for the isoform specific C-terminal domain of VAMP2 in regulating SERT function. Our data identify a novel interaction between SERT and a synaptic vesicle protein and support a link between 5-HT release and re-uptake. PMID:24878716

  1. Differential regulation of the serotonin transporter by vesicle-associated membrane protein 2 in cells of neuronal versus non-neuronal origin.

    PubMed

    Müller, Heidi Kaastrup; Kragballe, Marie; Fjorback, Anja Winther; Wiborg, Ove

    2014-01-01

    The serotonin transporter (SERT) is a key regulator of serotonergic signalling as it mediates the re-uptake of synaptic serotonin into nerve terminals, thereby terminating or modulating its signal. It is well-known that SERT regulation is a dynamic process orchestrated by a wide array of proteins and mechanisms. However, molecular details on possible coordinated regulation of SERT activity and 5-HT release are incomplete. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, interacts with SERT. This was documented in vitro, through GST pull-down assays, by co-immunoprecipitation experiments on heterologous cells and rat hippocampal synaptosomes, and with FRET analysis in live transfected HEK-293 MSR cells. The related isoforms VAMP1 and VAMP3 also physically interact with SERT. However, comparison of the three VAMP isoforms shows that only VAMP2 possesses a functionally distinct role in relation to SERT. VAMP2 influences 5-HT uptake, cell surface expression and the delivery rate of SERT to the plasma membrane differentially in HEK-293 MSR and PC12 cells. Moreover, siRNA-mediated knock-down of endogenous VAMP2 reduces 5-HT uptake in CAD cells stably expressing low levels of heterologous SERT. Deletion and mutant analysis suggest a role for the isoform specific C-terminal domain of VAMP2 in regulating SERT function. Our data identify a novel interaction between SERT and a synaptic vesicle protein and support a link between 5-HT release and re-uptake. PMID:24878716

  2. Alignment of Synaptic Vesicle Macromolecules with the Macromolecules in Active Zone Material that Direct Vesicle Docking

    PubMed Central

    Xu, Jing; Jung, Jae Hoon; Marshall, Robert M.; McMahan, Uel J.

    2013-01-01

    Synaptic vesicles dock at active zones on the presynaptic plasma membrane of a neuron’s axon terminals as a precondition for fusing with the membrane and releasing their neurotransmitter to mediate synaptic impulse transmission. Typically, docked vesicles are next to aggregates of plasma membrane-bound macromolecules called active zone material (AZM). Electron tomography on tissue sections from fixed and stained axon terminals of active and resting frog neuromuscular junctions has led to the conclusion that undocked vesicles are directed to and held at the docking sites by the successive formation of stable connections between vesicle membrane proteins and proteins in different classes of AZM macromolecules. Using the same nanometer scale 3D imaging technology on appropriately stained frog neuromuscular junctions, we found that ∼10% of a vesicle’s luminal volume is occupied by a radial assembly of elongate macromolecules attached by narrow projections, nubs, to the vesicle membrane at ∼25 sites. The assembly’s chiral, bilateral shape is nearly the same vesicle to vesicle, and nubs, at their sites of connection to the vesicle membrane, are linked to macromolecules that span the membrane. For docked vesicles, the orientation of the assembly’s shape relative to the AZM and the presynaptic membrane is the same vesicle to vesicle, whereas for undocked vesicles it is not. The connection sites of most nubs on the membrane of docked vesicles are paired with the connection sites of the different classes of AZM macromolecules that regulate docking, and the membrane spanning macromolecules linked to these nubs are also attached to the AZM macromolecules. We conclude that the luminal assembly of macromolecules anchors in a particular arrangement vesicle membrane macromolecules, which contain the proteins that connect the vesicles to AZM macromolecules during docking. Undocked vesicles must move in a way that aligns this arrangement with the AZM macromolecules for

  3. Electrohydrodynamics Of Multicomponent Vesicles

    NASA Astrophysics Data System (ADS)

    Gera, Prerna; Salac, David

    2015-11-01

    The addition of cholesterol into a lipid membrane induces the formation of distinct domains. These domains try to minimize the overall energy of the system by coalescence and migration. The application of electric fields will induce flow of these membrane domains and influence the rate at which they coarsen. In this work the electrohydrodynamics of multicomponent vesicles is numerically modelled. The method uses a Cahn-Hilliard-Cook model of the lipid domains restricted to a deforming three-dimensional vesicle and will be briefly discussed. Sample results will be presented and compared to experimental observations. This work supported by NSF Grant #1253739.

  4. Size and CT density of iodine-containing ethosomal vesicles obtained by membrane extrusion: potential for use as CT contrast agents.

    PubMed

    Na, Bomin; Choi, Byoung Wook; Kim, Bumsang

    2013-11-01

    Computed tomography (CT) is the primary non-invasive imaging technique used for most patients with suspected liver disease. In order to improve liver-specific imaging properties and prevent toxic effects in patients with compromised renal function, we investigated the encapsulation of iodine within ethosomal vesicles. As a first step in the development of novel contrast agents using ethosomes for CT imaging applications, iodine was entrapped within ethosomes and iodine-containing ethosomes of the desired size were obtained by extrusion using a polycarbonate membrane with a defined pore size. Ethosomes containing iodine showed a relatively high CT density, which decreased when they were extruded, due to the rupture and re-formation of the lipid bilayer of the ethosome. However, when a solution with a high iodine concentration was used as a dispersion media during the extrusion process, the decrease in CT density could be prevented. In addition, ethosomes containing iodine were taken up efficiently by macrophages, which are abundant in the liver, and these ethosomes exhibited no cellular toxicity. These results demonstrate that iodine could be entrapped within ethosomal vesicles, giving the ethosomes a relatively high CT density, and that the extrusion technique used in this study could conveniently and reproducibly produce ethosomal vesicles with a desired size. Therefore, ethosomes containing iodine, as prepared in this study, have potential as contrast agents with applications in CT imaging. PMID:23857905

  5. Basolateral K+ channel involvement in forskolin-activated chloride secretion in human colon.

    PubMed

    McNamara, B; Winter, D C; Cuffe, J E; O'Sullivan, G C; Harvey, B J

    1999-08-15

    1. In this study we investigated the role of basolateral potassium transport in maintaining cAMP-activated chloride secretion in human colonic epithelium. 2. Ion transport was quantified in isolated human colonic epithelium using the short-circuit current technique. Basolateral potassium transport was studied using nystatin permeabilization. Intracellular calcium measurements were obtained from isolated human colonic crypts using fura-2 spectrofluorescence imaging. 3. In intact isolated colonic strips, forskolin and prostaglandin E2 (PGE2) activated an inward transmembrane current (ISC) consistent with anion secretion (for forskolin DeltaISC = 63.8+/-6.2 microA cm(-2), n = 6; for PGE2 DeltaISC = 34.3+/-5.2 microA cm(-2), n = 6). This current was inhibited in chloride-free Krebs solution or by inhibiting basolateral chloride uptake with bumetanide and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid DIDS). 4. The forskolin- and PGE2-induced chloride secretion was inhibited by basolateral exposure to barium (5 mM), tetrapentylammonium (10 microM) and tetraethylammonium (10 mM). 5. The transepithelial current produced under an apical to serosal K+ gradient in nystatin-perforated colon is generated at the basolateral membrane by K+ transport. Forskolin failed to activate this current under conditions of high or low calcium and failed to increase the levels of intracellular calcium in isolated crypts 6. In conclusion, we propose that potassium recycling through basolateral K+ channels is essential for cAMP-activated chloride secretion. PMID:10432355

  6. Two-photon fluorescence microscopy of laurdan generalized polarization domains in model and natural membranes.

    PubMed

    Parasassi, T; Gratton, E; Yu, W M; Wilson, P; Levi, M

    1997-06-01

    Two-photon excitation microscopy shows coexisting regions of different generalized polarization (GP) in phospholipid vesicles, in red blood cells, in a renal tubular cell line, and in purified renal brushborder and basolateral membranes labeled with the fluorescent probe laurdan. The GP function measures the relative water content of the membrane. In the present study we discuss images obtained with polarized laser excitation, which selects different molecular orientations of the lipid bilayer corresponding to different spatial regions. The GP distribution in the gel-phase vesicles is relatively narrow, whereas the GP distribution in the liquid-crystalline phase vesicles (DOPC and DLPC) is broad. Analysis of images obtained with polarized excitation of the liquid-crystalline phase vesicles leads to the conclusion that coexisting regions of different GP must have dimensions smaller than the microscope resolution (approximately 200 nm radially and 600 nm axially). Vesicles of an equimolar mixture of DOPC and DPPC show coexisting rigid and fluid domains (high GP and low GP), but the rigid domains, which are preferentially excited by polarized light, have GP values lower than the pure gel-phase domains. Cholesterol strongly modifies the domain morphology. In the presence of 30 mol% cholesterol, the broad GP distribution of the DOPC/DPPC equimolar sample becomes narrower. The sample is still very heterogeneous, as demonstrated by the separations of GP disjoined regions, which are the result of photoselection of regions of different lipid orientation. In intact red blood cells, microscopic regions of different GP can be resolved, whereas in the renal cells GP domains have dimensions smaller than the microscope resolution. Preparations of renal apical brush border membranes and basolateral membranes show well-resolved GP domains, which may result from a different local orientation, or the domains may reflect a real heterogeneity of these membranes. PMID:9168019

  7. DMSO Enhances TGF-β Activity by Recruiting the Type II TGF-β Receptor From Intracellular Vesicles to the Plasma Membrane.

    PubMed

    Huang, Shuan Shian; Chen, Chun-Lin; Huang, Franklin W; Hou, Wei-Hsien; Huang, Jung San

    2016-07-01

    Dimethyl sulfoxide (DMSO) is used to treat many diseases/symptoms. The molecular basis of the pharmacological actions of DMSO has been unclear. We hypothesized that DMSO exerts some of these actions by enhancing TGF-β activity. Here we show that DMSO enhances TGF-β activity by ∼3-4-fold in Mv1Lu and NMuMG cells expressing Smad-dependent luciferase reporters. In Mv1Lu cells, DMSO enhances TGF-β-stimulated expression of P-Smad2 and PAI-1. It increases cell-surface expression of TGF-β receptors (TβR-I and/or TβR-II) by ∼3-4-fold without altering their cellular levels as determined by (125) I-labeled TGF-β-cross-linking/Western blot analysis, suggesting the presence of large intracellular pools in these cells. Sucrose density gradient ultracentrifugation/Western blot analysis reveals that DMSO induces recruitment of TβR-II (but not TβR-I) from its intracellular pool to plasma-membrane microdomains. It induces more recruitment of TβR-II to non-lipid raft microdomains than to lipid rafts/caveolae. Mv1Lu cells transiently transfected with TβR-II-HA plasmid were treated with DMSO and analyzed by indirect immunofluoresence staining using anti-HA antibody. In these cells, TβR-II-HA is present as a vesicle-like network in the cytoplasm as well as in the plasma membrane. DMSO causes depletion of TβR-II-HA-containing vesicles from the cytoplasm and co-localization of TβR-II-HA and cveolin-1 at the plasma membrane. These results suggest that DMSO, a fusogenic substance, enhances TGF-β activity presumably by inducing fusion of cytoplasmic vesicles (containing TβR-II) and the plasma membrane, resulting in increased localization of TβR-II to non-lipid raft microdomains where canonical signaling occurs. Fusogenic activity of DMSO may play a pivotal role in its pharmacological actions involving membrane proteins with large cytoplasmic pools. J. Cell. Biochem. 117: 1568-1579, 2016. © 2015 Wiley Periodicals, Inc. PMID:26587792

  8. Vesicle-associated membrane protein (VAMP)/synaptobrevin-2 is associated with dense core secretory granules in PC12 neuroendocrine cells.

    PubMed

    Papini, E; Rossetto, O; Cutler, D F

    1995-01-20

    The presence and intracellular distribution of vesicle-associated membrane protein-1 (VAMP-1) and VAMP-2 were investigated in the PC12 neuroendocrine cell line using isotype-specific polyclonal antibodies. VAMP-2 was detected in the total membrane fraction, while VAMP-1 was undetectable. Subcellular fractionation demonstrates that a substantial amount of the VAMP-2 (24-36%) is associated with dense core, catecholamine-containing granules (DCGs). This was confirmed by immunofluorescence microscopy. The L chain of tetanus neurotoxin, known to inhibit granule mediated secretion in permeabilized PC12 cells, as well as botulinum neurotoxins F and G, effectively cleaved DCG-associated VAMP-2. These data demonstrate that VAMP-2 is present on the secretory granules of PC12 cells. PMID:7836399

  9. Myosin IIA participates in docking of Glut4 storage vesicles with the plasma membrane in 3T3-L1 adipocytes

    SciTech Connect

    Chung, Le Thi Kim; Hosaka, Toshio; Harada, Nagakatsu; Jambaldorj, Bayasgalan; Fukunaga, Keiko; Nishiwaki, Yuka; Teshigawara, Kiyoshi; Sakai, Tohru; Nakaya, Yutaka; Funaki, Makoto

    2010-01-01

    In adipocytes and myocytes, insulin stimulation translocates glucose transporter 4 (Glut4) storage vesicles (GSVs) from their intracellular storage sites to the plasma membrane (PM) where they dock with the PM. Then, Glut4 is inserted into the PM and initiates glucose uptake into these cells. Previous studies using chemical inhibitors demonstrated that myosin II participates in fusion of GSVs and the PM and increase in the intrinsic activity of Glut4. In this study, the effect of myosin IIA on GSV trafficking was examined by knocking down myosin IIA expression. Myosin IIA knockdown decreased both glucose uptake and exposures of myc-tagged Glut4 to the cell surface in insulin-stimulated cells, but did not affect insulin signal transduction. Interestingly, myosin IIA knockdown failed to decrease insulin-dependent trafficking of Glut4 to the PM. Moreover, in myosin IIA knockdown cells, insulin-stimulated binding of GSV SNARE protein, vesicle-associated membrane protein 2 (VAMP2) to PM SNARE protein, syntaxin 4 was inhibited. These data suggest that myosin IIA plays a role in insulin-stimulated docking of GSVs to the PM in 3T3-L1 adipocytes through SNARE complex formation.

  10. Cation Activation of the Basolateral Sodium-Potassium Pump in Turtle Colon

    PubMed Central

    Halm, D R; Dawson, D C

    1983-01-01

    The current generated by electrogenic sodium-potassium exchange at the basolateral membrane of the turtle colon can be measured directly in tissues that have been treated with serosal barium (to block the basolateral potassium conductance) and mucosal amphotericin B (to reduce the cation selectivity of the apical membrane). We studied the activation of this pump current by mucosal sodium and serosal potassium, rubidium, cesium, and ammonium. The kinetics of sodium activation were consistent with binding to three independent sites on the cytoplasmic side of the pump. The pump was not activated by cellular lithium ions. The kinetics of serosal cation activation were consistent with binding to two independent sites with the selectivity Rb > K > Cs > NH4. The properties and kinetics of the basolateral Na/K pump in the turtle colon are at least qualitati