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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kelsey, D.R.; Flanagan, T.D.; Young, J.E.

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 effectivenessmore » 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.« less

Fusion of small unilamellar vesicles induced by bovine serum albumin fragments.

PubMed

Garcia, L A; Schenkman, S; Araujo, P S; Chaimovich, H

1983-07-01

The limited pepsin proteolysis products of bovine serum albumin, fragment A (residues 307-586) and fragment B (residues 1-306), induced the fusion of small unilamellar vesicles of egg phosphatidyl choline at concentrations near 5 microM. Fusion was demonstrated and analyzed on the basis of: a) time-dependent changes in absorbance; b) dilution of the fluorescent label 2-(10-(1-pyrene)decanoyl) phosphatidyl choline, incorporated into a small percentage of the vesicles, as measured by the decrease in the excimer to monomer (E/M) ratio; c) increase of the average hydrodynamic radius of the liposomes, estimated by Sepharose 4B filtration, and d) the strict inverse relationship between the size of the liposomes and their E/M ratios. Albumin fragment B, like albumin, induced the formation of large aggregates in which rapid cooperative fusion produced vesicles having a large hydrodynamic radius. Fragment A did not produce large aggregates and the initial fusion products exhibited a hydrodynamic radius. Fragment A did not produce large aggregates and the initial fusion products exhibited a hydrodynamic radius smaller than those obtained with fragment B. Albumin and fragments A and B are fusogenic only at pH below 4.0. These data discussed in terms of a general model for a signal-dependent protein-induced membrane fusion.

Lights on: Dye dequenching reveals polymersome fusion with polymer, lipid and stealth lipid vesicles

DOE PAGES

Henderson, Ian M.; Collins, Aaron M.; Quintana, Hope A.; ...

2015-12-13

In this study, we develop a quantitative dye dequenching technique for the measurement of polymersome fusion, using it to characterize the salt mediated, mechanically-induced fusion of polymersomes with polymer, lipid, and so-called stealth lipid vesicles. While dye dequenching has been used to quantitatively explore liposome fusion in the past, this is the first use of dye dequenching to measure polymersome fusion of which we are aware. In addition to providing quantitative results, dye dequenching is ideal for detecting fusion in instances where DLS results would be ambiguous, such as low yield levels and size ranges outside the capabilities of DLS.more » The dye chosen for this study was a cyanine derivative, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR), which proved to provide excellent data on the extent of polymersome fusion. Using this technique, we have shown the limited fusion capabilities of polymersome/liposome heterofusion, notably DOPC vesicles fusing with polymersomes at half the efficiency of polymersome homofusion and DPPC vesicles showing virtually no fusion. In addition to these key heterofusion experiments, we determined the broad applicability of dye dequenching in measuring kinetic rates of polymersome fusion; and showed that even at elevated temperatures or over multiple weeks' time, no polymersome fusion occurred without agitation. Stealth liposomes formed from DPPC and PEO-functionalized lipid, however, fused with polymersomes and stealth liposomes with relatively high efficiency, lending support to our hypothesis that the response of the PEO corona to salt is a key factor in the fusion process. This last finding suggests that although the conjugation of PEO to lipids increases vesicle biocompatibility and enables their longer circulation times, it also renders the vesicles subject to destabilization under high salt and shear (e.g. in the circulatory system) that may lead to, in this case, fusion.« less

G protein betagamma-subunits activated by serotonin mediate presynaptic inhibition by regulating vesicle fusion properties.

PubMed

Photowala, Huzefa; Blackmer, Trillium; Schwartz, Eric; Hamm, Heidi E; Alford, Simon

2006-03-14

Neurotransmitters are thought to be released as quanta, where synaptic vesicles deliver packets of neurotransmitter to the synaptic cleft by fusion with the plasma membrane. However, synaptic vesicles may undergo incomplete fusion. We provide evidence that G protein-coupled receptors inhibit release by causing such incomplete fusion. 5-hydroxytryptamine (5-HT) receptor signaling potently inhibits excitatory postsynaptic currents (EPSCs) between lamprey reticulospinal axons and their postsynaptic targets by a direct action on the vesicle fusion machinery. We show that 5-HT receptor-mediated presynaptic inhibition, at this synapse, involves a reduction in EPSC quantal size. Quantal size was measured directly by comparing unitary quantal amplitudes of paired EPSCs before and during 5-HT application and indirectly by determining the effect of 5-HT on the relationship between mean-evoked EPSC amplitude and variance. Results from FM dye-labeling experiments indicate that 5-HT prevents full fusion of vesicles. 5-HT reduces FM1-43 staining of vesicles with a similar efficacy to its effect on the EPSC. However, destaining of FM1-43-labeled vesicles is abolished by lower concentrations of 5-HT that leave a substantial EPSC. The use of a water-soluble membrane impermeant quenching agent in the extracellular space reduced FM1-43 fluorescence during stimulation in 5-HT. Thus vesicles contact the extracellular space during inhibition of synaptic transmission by 5-HT. We conclude that 5-HT, via free Gbetagamma, prevents the collapse of synaptic vesicles into the presynaptic membrane.

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

NASA Astrophysics Data System (ADS)

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

2004-07-01

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

The dissimilar effect of diacylglycerols on Ca(2+)-induced phosphatidylserine vesicle fusion.

PubMed Central

Sánchez-Migallón, M P; Aranda, F J; Gómez-Fernández, J C

1995-01-01

We have studied the effect of physiological concentrations of different diacylglycerols on Ca(2+)-induced fusion between phosphatidylserine vesicles. We monitored vesicle fusion as mixing of membrane lipids under conditions where the limiting factor was the aggregation and also in conditions where this aggregation was not the limiting factor. We found that diacylglycerols have a different modulating effect on the Ca(2+)-induced fusion: i) depending on their interfacial conformation, so that 1,2-isomers of diacylglycerols containing unsaturated or short saturated acyl chains stimulated fusion and their 1,3-isomers did not, and ii) depending on their specific type of bilayer interior perturbation, so that diacylglycerols containing unsaturated or short chain saturated acyl chains stimulated fusion but those containing long-chain saturated acyl chains did not. These requirements resembled those required for the diacylglycerol activation of protein kinase C, suggesting that diacylglycerol acts in both the specific activation of this enzyme and the induction of membrane fusion through the same perturbation of lipid structure. We found that polylysine affected the stimulatory role of 1,2-dioleoylglycerol differently, depending on whether aggregation was the limiting factor of fusion. When we studied the effect of very low concentrations of diacylglycerols on the bulk structural properties of phosphatidylserine, we found that they neither significantly perturbed the thermotropic transitions of phosphatidylserine nor affected the interaction of Ca2+ with the phosphate group of phosphatidylserine. The underlying mechanism of fusion between phosphatidylserine vesicles is discussed. PMID:7696508

In vitro fusion of endocytic vesicles is inhibited by cyclin A-cdc2 kinase.

PubMed Central

Woodman, P G; Adamczewski, J P; Hunt, T; Warren, G

1993-01-01

Receptor-mediated endocytosis and recycling are inhibited in mitotic mammalian cells, and previous studies have shown that inhibition of endocytic vesicle fusion in vitro occurs via cyclin B-cdc2 kinase. To test for the ability of cyclin A-cdc2 kinase to inhibit endocytic vesicle fusion, we employed recombinant cyclin A proteins. Addition of cyclin A to interphase extracts activated a histone kinase and markedly reduced the efficiency of endocytic vesicle fusion. By a number of criteria, inhibition of fusion was shown to be due to the action of cyclin A, via the mitosis-specific cdc2 kinase, and not an indirect effect through cyclin B. Two-stage incubations were used to demonstrate that at least one target of cyclin A-cdc2 kinase is a cytosolic component of the fusion apparatus. Reconstitution experiments showed that this component was also modified in mitotic cytosols and was unaffected by N-ethyl maleimide treatment. Images PMID:8334308

In vitro fusion of endocytic vesicles is inhibited by cyclin A-cdc2 kinase.

PubMed

Woodman, P G; Adamczewski, J P; Hunt, T; Warren, G

1993-05-01

Receptor-mediated endocytosis and recycling are inhibited in mitotic mammalian cells, and previous studies have shown that inhibition of endocytic vesicle fusion in vitro occurs via cyclin B-cdc2 kinase. To test for the ability of cyclin A-cdc2 kinase to inhibit endocytic vesicle fusion, we employed recombinant cyclin A proteins. Addition of cyclin A to interphase extracts activated a histone kinase and markedly reduced the efficiency of endocytic vesicle fusion. By a number of criteria, inhibition of fusion was shown to be due to the action of cyclin A, via the mitosis-specific cdc2 kinase, and not an indirect effect through cyclin B. Two-stage incubations were used to demonstrate that at least one target of cyclin A-cdc2 kinase is a cytosolic component of the fusion apparatus. Reconstitution experiments showed that this component was also modified in mitotic cytosols and was unaffected by N-ethyl maleimide treatment.

A Novel Synaptic Vesicle Fusion Path in the Rat Cerebral Cortex: The “Saddle” Point Hypothesis

PubMed Central

Zampighi, Guido A.; Serrano, Raul; Vergara, Julio L.

2014-01-01

We improved freeze-fracture electron microscopy to study synapses in the neuropil of the rat cerebral cortex at ∼2 nm resolution and in three-dimensions. In the pre-synaptic axon, we found that “rods” assembled from short filaments protruding from the vesicle and the plasma membrane connects synaptic vesicles to the membrane of the active zone. We equated these “connector rods” to protein complexes involved in “docking” and “priming” vesicles to the active zone. Depending on their orientation, the “rods” define two synaptic vesicle-fusion paths: When parallel to the plasma membrane, the vesicles hemi-fuse anywhere (“randomly”) in the active zone following the conventional path anticipated by the SNARE hypothesis. When perpendicular to the plasma membrane, the vesicles hemi-fuse at the base of sharp crooks, called “indentations,” that are spaced 75–85 nm center-to-center, arranged in files and contained within gutters. They result from primary and secondary membrane curvatures that intersect at stationary inflection (“saddle”) points. Computer simulations indicate that this novel vesicle-fusion path evokes neurotransmitter concentration domains on the post-synaptic spine that are wider, shallower, and that reach higher average concentrations than the more conventional vesicle fusion path. In the post-synaptic spine, large (∼9× ∼15 nm) rectangular particles at densities of 72±10/ µm2 (170–240/spine) match the envelopes of the homotetrameric GluR2 AMPA-sensitive receptor. While these putative receptors join clusters, called the “post-synaptic domains,” the overwhelming majority of the rectangular particles formed bands in the “non-synaptic” plasma membrane of the spine. In conclusion, in the neuropil of the rat cerebral cortex, curvatures of the plasma membrane define a novel vesicle-fusion path that preconditions specific regions of the active zone for neurotransmitter release. We hypothesize that a change in the

Control of mechanically activated polymersome fusion: Factors affecting fusion

DOE PAGES

Henderson, Ian M.; Paxton, Walter F.

2014-12-15

Previously we have studied the mechanically-activated fusion of extruded (200 nm) polymer vesicles into giant polymersomes using agitation in the presence of salt. In this study we have investigated several factors contributing to this phenomenon, including the effects of (i) polymer vesicle concentration, (ii) agitation speed and duration, and iii) variation of the salt and its concentration. It was found that increasing the concentration of the polymer dramatically increases the production of giant vesicles through the increased collisions of polymersomes. Our investigations also found that increasing the frequency of agitation increased the efficiency of fusion, though ultimately limited the sizemore » of vesicle which could be produced due to the high shear involved. Finally it was determined that salt-mediation of the fusion process was not limited to NaCl, but is instead a general effect facilitated by the presence of solvated ionic compounds, albeit with different salts initiating fusion at different concentration.« less

Vesicle Adhesion and Fusion Studied by Small-Angle X-Ray Scattering.

PubMed

Komorowski, Karlo; Salditt, Annalena; Xu, Yihui; Yavuz, Halenur; Brennich, Martha; Jahn, Reinhard; Salditt, Tim

2018-04-24

We have studied the adhesion state (also denoted by docking state) of lipid vesicles as induced by the divalent ions Ca 2+ or Mg 2+ at well-controlled ion concentration, lipid composition, and charge density. The bilayer structure and the interbilayer distance in the docking state were analyzed by small-angle x-ray scattering. A strong adhesion state was observed for DOPC:DOPS vesicles, indicating like-charge attraction resulting from ion correlations. The observed interbilayer separations of ∼1.6 nm agree quantitatively with the predictions of electrostatics in the strong coupling regime. Although this phenomenon was observed when mixing anionic and zwitterionic (or neutral) lipids, pure anionic membranes (DOPS) with highest charge density σ resulted in a direct phase transition to a multilamellar state, which must be accompanied by rupture and fusion of vesicles. To extend the structural assay toward protein-controlled docking and fusion, we have characterized reconstituted N-ethylmaleimide-sensitive factor attachment protein receptors in controlled proteoliposome suspensions by small-angle x-ray scattering. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Two synaptobrevin molecules are sufficient for vesicle fusion in central nervous system synapses

PubMed Central

Sinha, Raunak; Ahmed, Saheeb; Jahn, Reinhard; Klingauf, Jurgen

2011-01-01

Exocytosis of synaptic vesicles (SVs) during fast synaptic transmission is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly formed by the coil-coiling of three members of this protein family: vesicle SNARE protein, synaptobrevin 2 (syb2), and the presynaptic membrane SNAREs syntaxin-1A and SNAP-25. However, it is controversially debated how many SNARE complexes are minimally needed for SV priming and fusion. To quantify this effective number, we measured the fluorescence responses from single fusing vesicles expressing pHluorin (pHl), a pH-sensitive variant of GFP, fused to the luminal domain of the vesicular SNARE syb2 (spH) in cultured hippocampal neurons lacking endogenous syb2. Fluorescence responses were quantal, with the unitary signals precisely corresponding to single pHluorin molecules. Using this approach we found that two copies of spH per SV fully rescued evoked fusion whereas SVs expressing only one spH were unable to rapidly fuse upon stimulation. Thus, two syb2 molecules and likely two SNARE complexes are necessary and sufficient for SV fusion during fast synaptic transmission. PMID:21844343

Salt-induced aggregation and fusion of dioctadecyldimethylammonium chloride and sodium dihexadecylphosphate vesicles.

PubMed Central

Carmona-Ribeiro, A M; Chaimovich, H

1986-01-01

Small dioctadecyldimethylammonium chloride (DODAC) vesicles prepared by sonication fuse upon addition of NaCl as detected by several methods (electron microscopy, trapped volume determinations, temperature-dependent phase transition curves, and osmometer behavior. In contrast, small sodium dihexadecyl phosphate (DHP) vesicles mainly aggregate upon NaCl addition as shown by electron microscopy and the lack of osmometer behavior. Scatter-derived absorbance changes of small and large DODAC or DHP vesicles as a function of time after salt addition were obtained for a range of NaCl or amphiphile concentration. These changes were interpreted in accordance with a phenomenological model based upon fundamental light-scattering laws and simple geometrical considerations. Short-range hydration repulsion between DODAC (or DHP) vesicles is possibly the main energy barrier for the fusion process. Images FIGURE 2 FIGURE 9 PMID:3779002

Exocytosis from chromaffin cells: hydrostatic pressure slows vesicle fusion

PubMed Central

Stühmer, Walter

2015-01-01

Pressure affects reaction kinetics because chemical transitions involve changes in volume, and therefore pressure is a standard thermodynamic parameter to measure these volume changes. Many organisms live in environments at external pressures other than one atmosphere (0.1 MPa). Marine animals have adapted to live at depths of over 7000 m (at pressures over 70 MPa), and microorganisms living in trenches at over 110 MPa have been retrieved. Here, kinetic changes in secretion from chromaffin cells, measured as capacitance changes using the patch-clamp technique at pressures of up to 20 MPa are presented. It is known that these high pressures drastically slow down physiological functions. High hydrostatic pressure also affects the kinetics of ion channel gating and the amount of current carried by them, and it drastically slows down synaptic transmission. The results presented here indicate a similar change in volume (activation volume) of 390 ± 57 Å3 for large dense-core vesicles undergoing fusion in chromaffin cells and for degranulation of mast cells. It is significantly larger than activation volumes of voltage-gated ion channels in chromaffin cells. This information will be useful in finding possible protein conformational changes during the reactions involved in vesicle fusion and in testing possible molecular dynamic models of secretory processes. PMID:26009771

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

Coupling Between Metabolism and Compartmentalization: Vesicle Growth in the Presence of Dipeptides

NASA Astrophysics Data System (ADS)

Wei, C.; Pohorille, A.

2017-07-01

Extensive molecular dynamics simulations demonstrate low energy pathway for fast fusion of vesicle mediated by membrane-bound hydrophobic dipeptides and facilitated flip-flop transport of fatty acid molecule for transmembrane proton transfer.

An intracellular motif of GLUT4 regulates fusion of GLUT4-containing vesicles.

PubMed

Heyward, Catherine A; Pettitt, Trevor R; Leney, Sophie E; Welsh, Gavin I; Tavaré, Jeremy M; Wakelam, Michael J O

2008-05-20

Insulin stimulates glucose uptake by adipocytes through increasing translocation of the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane. Fusion of GLUT4-containing vesicles at the cell surface is thought to involve phospholipase D activity, generating the signalling lipid phosphatidic acid, although the mechanism of action is not yet clear. Here we report the identification of a putative phosphatidic acid-binding motif in a GLUT4 intracellular loop. Mutation of this motif causes a decrease in the insulin-induced exposure of GLUT4 at the cell surface of 3T3-L1 adipocytes via an effect on vesicle fusion. The potential phosphatidic acid-binding motif identified in this study is unique to GLUT4 among the sugar transporters, therefore this motif may provide a unique mechanism for regulating insulin-induced translocation by phospholipase D signalling.

Otoferlin acts as a Ca2+ sensor for vesicle fusion and vesicle pool replenishment at auditory hair cell ribbon synapses

PubMed Central

Goutman, Juan D; Auclair, Sarah Marie; Boutet de Monvel, Jacques; Tertrais, Margot; Emptoz, Alice; Parrin, Alexandre; Nouaille, Sylvie; Guillon, Marc; Sachse, Martin; Ciric, Danica; Bahloul, Amel; Hardelin, Jean-Pierre; Sutton, Roger Bryan; Avan, Paul; Krishnakumar, Shyam S; Rothman, James E

2017-01-01

Hearing relies on rapid, temporally precise, and sustained neurotransmitter release at the ribbon synapses of sensory cells, the inner hair cells (IHCs). This process requires otoferlin, a six C2-domain, Ca2+-binding transmembrane protein of synaptic vesicles. To decipher the role of otoferlin in the synaptic vesicle cycle, we produced knock-in mice (Otof Ala515,Ala517/Ala515,Ala517) with lower Ca2+-binding affinity of the C2C domain. The IHC ribbon synapse structure, synaptic Ca2+ currents, and otoferlin distribution were unaffected in these mutant mice, but auditory brainstem response wave-I amplitude was reduced. Lower Ca2+ sensitivity and delay of the fast and sustained components of synaptic exocytosis were revealed by membrane capacitance measurement upon modulations of intracellular Ca2+ concentration, by varying Ca2+ influx through voltage-gated Ca2+-channels or Ca2+ uncaging. Otoferlin thus functions as a Ca2+ sensor, setting the rates of primed vesicle fusion with the presynaptic plasma membrane and synaptic vesicle pool replenishment in the IHC active zone. PMID:29111973

Otoferlin acts as a Ca2+ sensor for vesicle fusion and vesicle pool replenishment at auditory hair cell ribbon synapses.

PubMed

Michalski, Nicolas; Goutman, Juan D; Auclair, Sarah Marie; Boutet de Monvel, Jacques; Tertrais, Margot; Emptoz, Alice; Parrin, Alexandre; Nouaille, Sylvie; Guillon, Marc; Sachse, Martin; Ciric, Danica; Bahloul, Amel; Hardelin, Jean-Pierre; Sutton, Roger Bryan; Avan, Paul; Krishnakumar, Shyam S; Rothman, James E; Dulon, Didier; Safieddine, Saaid; Petit, Christine

2017-11-07

Hearing relies on rapid, temporally precise, and sustained neurotransmitter release at the ribbon synapses of sensory cells, the inner hair cells (IHCs). This process requires otoferlin, a six C 2 -domain, Ca 2+ -binding transmembrane protein of synaptic vesicles. To decipher the role of otoferlin in the synaptic vesicle cycle, we produced knock-in mice ( Otof Ala515,Ala517/Ala515,Ala517 ) with lower Ca 2+ -binding affinity of the C 2 C domain. The IHC ribbon synapse structure, synaptic Ca 2+ currents, and otoferlin distribution were unaffected in these mutant mice, but auditory brainstem response wave-I amplitude was reduced. Lower Ca 2+ sensitivity and delay of the fast and sustained components of synaptic exocytosis were revealed by membrane capacitance measurement upon modulations of intracellular Ca 2+ concentration, by varying Ca 2+ influx through voltage-gated Ca 2+ -channels or Ca 2+ uncaging. Otoferlin thus functions as a Ca 2+ sensor, setting the rates of primed vesicle fusion with the presynaptic plasma membrane and synaptic vesicle pool replenishment in the IHC active zone.

Munc13 controls the location and efficiency of dense-core vesicle release in neurons.

PubMed

van de Bospoort, Rhea; Farina, Margherita; Schmitz, Sabine K; de Jong, Arthur; de Wit, Heidi; Verhage, Matthijs; Toonen, Ruud F

2012-12-10

Neuronal dense-core vesicles (DCVs) contain diverse cargo crucial for brain development and function, but the mechanisms that control their release are largely unknown. We quantified activity-dependent DCV release in hippocampal neurons at single vesicle resolution. DCVs fused preferentially at synaptic terminals. DCVs also fused at extrasynaptic sites but only after prolonged stimulation. In munc13-1/2-null mutant neurons, synaptic DCV release was reduced but not abolished, and synaptic preference was lost. The remaining fusion required prolonged stimulation, similar to extrasynaptic fusion in wild-type neurons. Conversely, Munc13-1 overexpression (M13OE) promoted extrasynaptic DCV release, also without prolonged stimulation. Thus, Munc13-1/2 facilitate DCV fusion but, unlike for synaptic vesicles, are not essential for DCV release, and M13OE is sufficient to produce efficient DCV release extrasynaptically.

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.

Presynaptic pH and vesicle fusion in Drosophila larvae neurones.

PubMed

Caldwell, Lesley; Harries, Peter; Sydlik, Sebastian; Schwiening, Christof J

2013-11-01

Both intracellular pH (pHi) and synaptic cleft pH change during neuronal activity yet little is known about how these pH shifts might affect synaptic transmission by influencing vesicle fusion. To address this we imaged pH- and Ca(2+) -sensitive fluorescent indicators (HPTS, Oregon green) in boutons at neuromuscular junctions. Electrical stimulation of motor nerves evoked presynaptic Ca(2+) i rises and pHi falls (∼0.1 pH units) followed by recovery of both Ca(2+) i and pHi. The plasma-membrane calcium ATPase (PMCA) inhibitor, 5(6)-carboxyeosin diacetate, slowed both the calcium recovery and the acidification. To investigate a possible calcium-independent role for the pHi shifts in modulating vesicle fusion we recorded post-synaptic miniature end-plate potential (mEPP) and current (mEPC) frequency in Ca(2+) -free solution. Acidification by propionate superfusion, NH(4)(+) withdrawal, or the inhibition of acid extrusion on the Na(+)/H(+) exchanger (NHE) induced a rise in miniature frequency. Furthermore, the inhibition of acid extrusion enhanced the rise induced by propionate addition and NH(4)(+) removal. In the presence of NH(4)(+), 10 out of 23 cells showed, after a delay, one or more rises in miniature frequency. These findings suggest that Ca(2+) -dependent pHi shifts, caused by the PMCA and regulated by NHE, may stimulate vesicle release. Furthermore, in the presence of membrane permeant buffers, exocytosed acid or its equivalents may enhance release through positive feedback. This hitherto neglected pH signalling, and the potential feedback role of vesicular acid, could explain some important neuronal excitability changes associated with altered pH and its buffering. Copyright © 2013 Wiley Periodicals, Inc.

Fusion of small unilamellar vesicles induced by a serum albumin fragment of molecular weight 9000.

PubMed

Garcia, L A; Araújo, P S; Chaimovich, H

1984-05-16

A peptide (P-9) comprising amino acids 307 to 385 of bovine serum albumin induced the fusion of small unilamellar vesicles of phosphatidylcholine at low pH. Upon acidification P-9 exhibited a ultraviolet differential spectrum characteristic of hydrophilic exposure of chromophores. This conformational change, and the structure of P-9 composed of three amphiphilic helixes , suggested a general working hypothesis for the description of protein-induced membrane fusion.

Full-length trimeric influenza virus hemagglutinin II membrane fusion protein and shorter constructs lacking the fusion peptide or transmembrane domain: Hyperthermostability of the full-length protein and the soluble ectodomain and fusion peptide make significant contributions to fusion of membrane vesicles.

PubMed

Ratnayake, Punsisi U; Prabodha Ekanayaka, E A; Komanduru, Sweta S; Weliky, David P

2016-01-01

Influenza virus is a class I enveloped virus which is initially endocytosed into a host respiratory epithelial cell. Subsequent reduction of the pH to the 5-6 range triggers a structural change of the viral hemagglutinin II (HA2) protein, fusion of the viral and endosomal membranes, and release of the viral nucleocapsid into the cytoplasm. HA2 contains fusion peptide (FP), soluble ectodomain (SE), transmembrane (TM), and intraviral domains with respective lengths of ∼ 25, ∼ 160, ∼ 25, and ∼ 10 residues. The present work provides a straightforward protocol for producing and purifying mg quantities of full-length HA2 from expression in bacteria. Biophysical and structural comparisons are made between full-length HA2 and shorter constructs including SHA2 ≡ SE, FHA2 ≡ FP+SE, and SHA2-TM ≡ SE+TM constructs. The constructs are helical in detergent at pH 7.4 and the dominant trimer species. The proteins are highly thermostable in decylmaltoside detergent with Tm>90 °C for HA2 with stabilization provided by the SE, FP, and TM domains. The proteins are likely in a trimer-of-hairpins structure, the final protein state during fusion. All constructs induce fusion of negatively-charged vesicles at pH 5.0 with much less fusion at pH 7.4. Attractive protein/vesicle electrostatics play a role in fusion, as the proteins are positively-charged at pH 5.0 and negatively-charged at pH 7.4 and the pH-dependence of fusion is reversed for positively-charged vesicles. Comparison of fusion between constructs supports significant contributions to fusion from the SE and the FP with little effect from the TM. Copyright © 2015 Elsevier Inc. All rights reserved.

Structure-activity relationships in the fusion of small unilamellar phosphatidylcholine vesicles induced by a model peptide.

PubMed

da Costa, M H; Chaimovich, H

1997-09-01

Limited proteolysis of fatty acid-free bovine serum albumin by pepsin yields several well characterized peptides, one of which (P9, M(r) 9,000), induces fusion of small unilamellar vesicles (SUV) of phosphatidylcholine at pH 3.6. Circular dichroism (CD) of P9 solutions confirmed that the peptide undergoes a reversible transition between pH 7 and pH 3.6. The spectral changes observed with CD suggest that in the low pH conformation there is a decrease in the alpha-helical contents and an exposure of hydrophobic residues. CD and differential ultraviolet spectroscopy demonstrated that P9 binds to micelles of hexadecylphosphorylcholine and the binding produces changes in the tertiary structure of the peptide. Reduction and carboxymethylation of the two disulfide bridges of P9 produced loss of the ability to induce fusion of SUV, although the reduced peptide binds to vesicles, induces loss of entrapped marker and produces vesicle disruption. In the active form P9 exposes hydrophobic groups, one amphiphilic alpha-helix and requires the integrity of the disulfide bridge-stabilized tertiary structure.

Myosin light chain kinase facilitates endocytosis of synaptic vesicles at hippocampal boutons.

PubMed

Li, Lin; Wu, Xiaomei; Yue, Hai-Yuan; Zhu, Yong-Chuan; Xu, Jianhua

2016-07-01

At nerve terminals, endocytosis efficiently recycles vesicle membrane to maintain synaptic transmission under different levels of neuronal activity. Ca(2+) and its downstream signal pathways are critical for the activity-dependent regulation of endocytosis. An activity- and Ca(2+) -dependent kinase, myosin light chain kinase (MLCK) has been reported to regulate vesicle mobilization, vesicle cycling, and motility in different synapses, but whether it has a general contribution to regulation of endocytosis at nerve terminals remains unknown. We investigated this issue at rat hippocampal boutons by imaging vesicle endocytosis as the real-time retrieval of vesicular synaptophysin tagged with a pH-sensitive green fluorescence protein. We found that endocytosis induced by 200 action potentials (5-40 Hz) was slowed by acute inhibition of MLCK and down-regulation of MLCK with RNA interference, while the total amount of vesicle exocytosis and somatic Ca(2+) channel current did not change with MLCK down-regulation. Acute inhibition of myosin II similarly impaired endocytosis. Furthermore, down-regulation of MLCK prevented depolarization-induced phosphorylation of myosin light chain, an effect shared by blockers of Ca(2+) channels and calmodulin. These results suggest that MLCK facilitates vesicle endocytosis through activity-dependent phosphorylation of myosin downstream of Ca(2+) /calmodulin, probably as a widely existing mechanism among synapses. Our study suggests that MLCK is an important activity-dependent regulator of vesicle recycling in hippocampal neurons, which are critical for learning and memory. The kinetics of vesicle membrane endocytosis at nerve terminals has long been known to depend on activity and Ca(2+) . This study provides evidence suggesting that myosin light chain kinase increases endocytosis efficiency at hippocampal neurons by mediating Ca(2+) /calmodulin-dependent phosphorylation of myosin. The authors propose that this signal cascade may serve as

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

PubMed Central

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

2016-01-01

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

Early steps of supported bilayer formation probed by single vesicle fluorescence assays.

PubMed Central

Johnson, Joseph M; Ha, Taekjip; Chu, Steve; Boxer, Steven G

2002-01-01

We have developed a single vesicle assay to study the mechanisms of supported bilayer formation. Fluorescently labeled, unilamellar vesicles (30-100 nm diameter) were first adsorbed to a quartz surface at low enough surface concentrations to visualize single vesicles. Fusion and rupture events during the bilayer formation, induced by the subsequent addition of unlabeled vesicles, were detected by measuring two-color fluorescence signals simultaneously. Lipid-conjugated dyes monitored the membrane fusion while encapsulated dyes reported on the vesicle rupture. Four dominant pathways were observed, each exhibiting characteristic two-color fluorescence signatures: 1) primary fusion, in which an unlabeled vesicle fuses with a labeled vesicle on the surface, is signified by the dequenching of the lipid-conjugated dyes followed by rupture and final merging into the bilayer; 2) simultaneous fusion and rupture, in which a labeled vesicle on the surface ruptures simultaneously upon fusion with an unlabeled vesicle; 3) no dequenching, in which loss of fluorescence signal from both dyes occur simultaneously with the final merger into the bilayer; and 4) isolated rupture (pre-ruptured vesicles), in which a labeled vesicle on the surface spontaneously undergoes content loss, a process that occurs with high efficiency in the presence of a high concentration of Texas Red-labeled lipids. Vesicles that have undergone content loss appear to be more fusogenic than intact vesicles. PMID:12496104

Ca2+ Dependence of Synaptic Vesicle Endocytosis.

PubMed

Leitz, Jeremy; Kavalali, Ege T

2016-10-01

Ca(2+)-dependent synaptic vesicle recycling is essential for structural homeostasis of synapses and maintenance of neurotransmission. Although, the executive role of intrasynaptic Ca(2+) transients in synaptic vesicle exocytosis is well established, identifying the exact role of Ca(2+) in endocytosis has been difficult. In some studies, Ca(2+) has been suggested as an essential trigger required to initiate synaptic vesicle retrieval, whereas others manipulating synaptic Ca(2+) concentrations reported a modulatory role for Ca(2+) leading to inhibition or acceleration of endocytosis. Molecular studies of synaptic vesicle endocytosis, on the other hand, have consistently focused on the roles of Ca(2+)-calmodulin dependent phosphatase calcineurin and synaptic vesicle protein synaptotagmin as potential Ca(2+) sensors for endocytosis. Most studies probing the role of Ca(2+) in endocytosis have relied on measurements of synaptic vesicle retrieval after strong stimulation. Strong stimulation paradigms elicit fusion and retrieval of multiple synaptic vesicles and therefore can be affected by several factors besides the kinetics and duration of Ca(2+) signals that include the number of exocytosed vesicles and accumulation of released neurotransmitters thus altering fusion and retrieval processes indirectly via retrograde signaling. Studies monitoring single synaptic vesicle endocytosis may help resolve this conundrum as in these settings the impact of Ca(2+) on synaptic fusion probability can be uncoupled from its putative role on synaptic vesicle retrieval. Future experiments using these single vesicle approaches will help dissect the specific role(s) of Ca(2+) and its sensors in synaptic vesicle endocytosis. © The Author(s) 2015.

The Fusion of Membranes and Vesicles: Pathway and Energy Barriers from Dissipative Particle Dynamics

PubMed Central

Grafmüller, Andrea; Shillcock, Julian; Lipowsky, Reinhard

2009-01-01

The fusion of lipid bilayers is studied with dissipative particle dynamics simulations. First, to achieve control over membrane properties, the effects of individual simulation parameters are studied and optimized. Then, a large number of fusion events for a vesicle and a planar bilayer are simulated using the optimized parameter set. In the observed fusion pathway, configurations of individual lipids play an important role. Fusion starts with individual lipids assuming a splayed tail configuration with one tail inserted in each membrane. To determine the corresponding energy barrier, we measure the average work for interbilayer flips of a lipid tail, i.e., the average work to displace one lipid tail from one bilayer to the other. This energy barrier is found to depend strongly on a certain dissipative particle dynamics parameter, and, thus, can be adjusted in the simulations. Overall, three subprocesses have been identified in the fusion pathway. Their energy barriers are estimated to lie in the range 8–15 kBT. The fusion probability is found to possess a maximum at intermediate tension values. As one decreases the tension, the fusion probability seems to vanish before the tensionless membrane state is attained. This would imply that the tension has to exceed a certain threshold value to induce fusion. PMID:19348749

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pszon-Bartosz, Kamila; Hansen, Jesper S.; Technical University of Denmark, Department of Micro- and Nanotechnology, DK-2800 Kongens Lyngby

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 proteinmore » 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.« less

Fusion competent synaptic vesicles persist upon active zone disruption and loss of vesicle docking

PubMed Central

Wang, Shan Shan H.; Held, Richard G.; Wong, Man Yan; Liu, Changliang; Karakhanyan, Aziz; Kaeser, Pascal S.

2016-01-01

In a nerve terminal, synaptic vesicle docking and release are restricted to an active zone. The active zone is a protein scaffold that is attached to the presynaptic plasma membrane and opposed to postsynaptic receptors. Here, we generated conditional knockout mice removing the active zone proteins RIM and ELKS, which additionally led to loss of Munc13, Bassoon, Piccolo, and RIM-BP, indicating disassembly of the active zone. We observed a near complete lack of synaptic vesicle docking and a strong reduction in vesicular release probability and the speed of exocytosis, but total vesicle numbers, SNARE protein levels, and postsynaptic densities remained unaffected. Despite loss of the priming proteins Munc13 and RIM and of docked vesicles, a pool of releasable vesicles remained. Thus, the active zone is necessary for synaptic vesicle docking and to enhance release probability, but releasable vesicles can be localized distant from the presynaptic plasma membrane. PMID:27537483

Phosphatidylinositol 4,5-bisphosphate regulates SNARE-dependent membrane fusion.

PubMed

James, Declan J; Khodthong, Chuenchanok; Kowalchyk, Judith A; Martin, Thomas F J

2008-07-28

Phosphatidylinositol 4,5-bisphosphate (PI 4,5-P(2)) on the plasma membrane is essential for vesicle exocytosis but its role in membrane fusion has not been determined. Here, we quantify the concentration of PI 4,5-P(2) as approximately 6 mol% in the cytoplasmic leaflet of plasma membrane microdomains at sites of docked vesicles. At this concentration of PI 4,5-P(2) soluble NSF attachment protein receptor (SNARE)-dependent liposome fusion is inhibited. Inhibition by PI 4,5-P(2) likely results from its intrinsic positive curvature-promoting properties that inhibit formation of high negative curvature membrane fusion intermediates. Mutation of juxtamembrane basic residues in the plasma membrane SNARE syntaxin-1 increase inhibition by PI 4,5-P(2), suggesting that syntaxin sequesters PI 4,5-P(2) to alleviate inhibition. To define an essential rather than inhibitory role for PI 4,5-P(2), we test a PI 4,5-P(2)-binding priming factor required for vesicle exocytosis. Ca(2+)-dependent activator protein for secretion promotes increased rates of SNARE-dependent fusion that are PI 4,5-P(2) dependent. These results indicate that PI 4,5-P(2) regulates fusion both as a fusion restraint that syntaxin-1 alleviates and as an essential cofactor that recruits protein priming factors to facilitate SNARE-dependent fusion.

Novel interactions of CAPS (Ca2+-dependent activator protein for secretion) with the three neuronal SNARE proteins required for vesicle fusion.

PubMed

Daily, Neil J; Boswell, Kristin L; James, Declan J; Martin, Thomas F J

2010-11-12

CAPS (aka CADPS) is required for optimal vesicle exocytosis in neurons and endocrine cells where it functions to prime the exocytic machinery for Ca(2+)-triggered fusion. Fusion is mediated by trans complexes of the SNARE proteins VAMP-2, syntaxin-1, and SNAP-25 that bridge vesicle and plasma membrane. CAPS promotes SNARE complex formation on liposomes, but the SNARE binding properties of CAPS are unknown. The current work revealed that CAPS exhibits high affinity binding to syntaxin-1 and SNAP-25 and moderate affinity binding to VAMP-2. CAPS binding is specific for a subset of exocytic SNARE protein isoforms and requires membrane integration of the SNARE proteins. SNARE protein binding by CAPS is novel and mediated by interactions with the SNARE motifs in the three proteins. The C-terminal site for CAPS binding on syntaxin-1 does not overlap the Munc18-1 binding site and both proteins can co-reside on membrane-integrated syntaxin-1. As expected for a C-terminal binding site on syntaxin-1, CAPS stimulates SNARE-dependent liposome fusion with N-terminal truncated syntaxin-1 but exhibits impaired activity with C-terminal syntaxin-1 mutants. Overall the results suggest that SNARE complex formation promoted by CAPS may be mediated by direct interactions of CAPS with each of the three SNARE proteins required for vesicle exocytosis.

Novel Interactions of CAPS (Ca2+-dependent Activator Protein for Secretion) with the Three Neuronal SNARE Proteins Required for Vesicle Fusion*

PubMed Central

Daily, Neil J.; Boswell, Kristin L.; James, Declan J.; Martin, Thomas F. J.

2010-01-01

CAPS (aka CADPS) is required for optimal vesicle exocytosis in neurons and endocrine cells where it functions to prime the exocytic machinery for Ca2+-triggered fusion. Fusion is mediated by trans complexes of the SNARE proteins VAMP-2, syntaxin-1, and SNAP-25 that bridge vesicle and plasma membrane. CAPS promotes SNARE complex formation on liposomes, but the SNARE binding properties of CAPS are unknown. The current work revealed that CAPS exhibits high affinity binding to syntaxin-1 and SNAP-25 and moderate affinity binding to VAMP-2. CAPS binding is specific for a subset of exocytic SNARE protein isoforms and requires membrane integration of the SNARE proteins. SNARE protein binding by CAPS is novel and mediated by interactions with the SNARE motifs in the three proteins. The C-terminal site for CAPS binding on syntaxin-1 does not overlap the Munc18-1 binding site and both proteins can co-reside on membrane-integrated syntaxin-1. As expected for a C-terminal binding site on syntaxin-1, CAPS stimulates SNARE-dependent liposome fusion with N-terminal truncated syntaxin-1 but exhibits impaired activity with C-terminal syntaxin-1 mutants. Overall the results suggest that SNARE complex formation promoted by CAPS may be mediated by direct interactions of CAPS with each of the three SNARE proteins required for vesicle exocytosis. PMID:20826818

High Cholesterol Obviates a Prolonged Hemifusion Intermediate in Fast SNARE-Mediated Membrane Fusion

PubMed Central

Kreutzberger, Alex J.B.; Kiessling, Volker; Tamm, Lukas K.

2015-01-01

Cholesterol is essential for exocytosis in secretory cells, but the exact molecular mechanism by which it facilitates exocytosis is largely unknown. Distinguishing contributions from the lateral organization and dynamics of membrane proteins to vesicle docking and fusion and the promotion of fusion pores by negative intrinsic spontaneous curvature and other mechanical effects of cholesterol have been elusive. To shed more light on this process, we examined the effect of cholesterol on SNARE-mediated membrane fusion in a single-vesicle assay that is capable of resolving docking and elementary steps of fusion with millisecond time resolution. The effect of cholesterol on fusion pore formation between synaptobrevin-2 (VAMP-2)-containing proteoliposomes and acceptor t-SNARE complex-containing planar supported bilayers was examined using both membrane and content fluorescent markers. This approach revealed that increasing cholesterol in either the t-SNARE or the v-SNARE membrane favors a mechanism of direct fusion pore opening, whereas low cholesterol favors a mechanism leading to a long-lived (>5 s) hemifusion state. The amount of cholesterol in the target membrane had no significant effect on docking of synaptobrevin vesicles. Comparative studies with α-tocopherol (vitamin E) show that the negative intrinsic spontaneous curvature of cholesterol and its presumed promotion of a very short-lived (<50 ms) lipid stalk intermediate is the main factor that favors rapid fusion pore opening at high cholesterol. This study also shows that this single-vesicle fusion assay can distinguish between hemifusion and full fusion with only a single lipid dye, thereby freeing up a fluorescence channel for the simultaneous measurement of another parameter in fast time-resolved fusion assays. PMID:26200867

An extended model of vesicle fusion at the plasma membrane to estimate protein lateral diffusion from TIRF microscopy images.

PubMed

Basset, Antoine; Bouthemy, Patrick; Boulanger, Jérôme; Waharte, François; Salamero, Jean; Kervrann, Charles

2017-07-24

Characterizing membrane dynamics is a key issue to understand cell exchanges with the extra-cellular medium. Total internal reflection fluorescence microscopy (TIRFM) is well suited to focus on the late steps of exocytosis at the plasma membrane. However, it is still a challenging task to quantify (lateral) diffusion and estimate local dynamics of proteins. A new model was introduced to represent the behavior of cargo transmembrane proteins during the vesicle fusion to the plasma membrane at the end of the exocytosis process. Two biophysical parameters, the diffusion coefficient and the release rate parameter, are automatically estimated from TIRFM image sequences, to account for both the lateral diffusion of molecules at the membrane and the continuous release of the proteins from the vesicle to the plasma membrane. Quantitative evaluation on 300 realistic computer-generated image sequences demonstrated the efficiency and accuracy of the method. The application of our method on 16 real TIRFM image sequences additionally revealed differences in the dynamic behavior of Transferrin Receptor (TfR) and Langerin proteins. An automated method has been designed to simultaneously estimate the diffusion coefficient and the release rate for each individual vesicle fusion event at the plasma membrane in TIRFM image sequences. It can be exploited for further deciphering cell membrane dynamics.

Fast retrieval and autonomous regulation of single spontaneously recycling synaptic vesicles

PubMed Central

Leitz, Jeremy; Kavalali, Ege T

2014-01-01

Presynaptic terminals release neurotransmitters spontaneously in a manner that can be regulated by Ca2+. However, the mechanisms underlying this regulation are poorly understood because the inherent stochasticity and low probability of spontaneous fusion events has curtailed their visualization at individual release sites. Here, using pH-sensitive optical probes targeted to synaptic vesicles, we visualized single spontaneous fusion events and found that they are retrieved extremely rapidly with faster re-acidification kinetics than their action potential-evoked counterparts. These fusion events were coupled to postsynaptic NMDA receptor-driven Ca2+ signals, and at elevated Ca2+ concentrations there was an increase in the number of vesicles that would undergo fusion. Furthermore, spontaneous vesicle fusion propensity in a synapse was Ca2+-dependent but regulated autonomously: independent of evoked fusion probability at the same synapse. Taken together, these results expand classical quantal analysis to incorporate endocytic and exocytic phases of single fusion events and uncover autonomous regulation of spontaneous fusion. DOI: http://dx.doi.org/10.7554/eLife.03658.001 PMID:25415052

Trafficking of astrocytic vesicles in hippocampal slices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Potokar, Maja; Kreft, Marko; Celica Biomedical Center, Technology Park 24, 1000 Ljubljana

2009-12-25

The increasingly appreciated role of astrocytes in neurophysiology dictates a thorough understanding of the mechanisms underlying the communication between astrocytes and neurons. In particular, the uptake and release of signaling substances into/from astrocytes is considered as crucial. The release of different gliotransmitters involves regulated exocytosis, consisting of the fusion between the vesicle and the plasma membranes. After fusion with the plasma membrane vesicles may be retrieved into the cytoplasm and may continue to recycle. To study the mobility implicated in the retrieval of secretory vesicles, these structures have been previously efficiently and specifically labeled in cultured astrocytes, by exposing livemore » cells to primary and secondary antibodies. Since the vesicle labeling and the vesicle mobility properties may be an artifact of cell culture conditions, we here asked whether the retrieving exocytotic vesicles can be labeled in brain tissue slices and whether their mobility differs to that observed in cell cultures. We labeled astrocytic vesicles and recorded their mobility with two-photon microscopy in hippocampal slices from transgenic mice with fluorescently tagged astrocytes (GFP mice) and in wild-type mice with astrocytes labeled by Fluo4 fluorescence indicator. Glutamatergic vesicles and peptidergic granules were labeled by the anti-vesicular glutamate transporter 1 (vGlut1) and anti-atrial natriuretic peptide (ANP) antibodies, respectively. We report that the vesicle mobility parameters (velocity, maximal displacement and track length) recorded in astrocytes from tissue slices are similar to those reported previously in cultured astrocytes.« less

Imaging synaptic vesicle recycling by staining and destaining vesicles with FM dyes.

PubMed

Hoopmann, Peer; Rizzoli, Silvio O; Betz, William J

2012-01-01

The synaptic vesicle is the essential organelle of the synapse. Many approaches for studying synaptic vesicle recycling have been devised, one of which, the styryl (FM) dye, is well suited for this purpose. FM dyes reversibly stain, but do not permeate, membranes; hence they can specifically label membrane-bound organelles. Their quantum yield is drastically higher when bound to membranes than when in aqueous solution. This protocol describes the imaging of synaptic vesicle recycling by staining and destaining vesicles with FM dyes. Nerve terminals are stimulated (electrically or by depolarization with high K(+)) in the presence of dye, their vesicles are then allowed to recycle, and finally dye is washed from the chamber. In neuromuscular junction (NMJ) preparations, movements of the muscle must be inhibited if imaging during stimulation is desired (e.g., by application of curare, a potent acetylcholine receptor inhibitor). The main characteristics of FM dyes are also reviewed here, as are recent FM dye monitoring techniques that have been used to investigate the kinetics of synaptic vesicle fusion.

Presynaptic PICK1 facilitates trafficking of AMPA-receptors between active zone and synaptic vesicle pool.

PubMed

Haglerød, C; Hussain, S; Nakamura, Y; Xia, J; Haug, F-M S; Ottersen, O P; Henley, J M; Davanger, S

2017-03-06

Previous studies have indicated that presynaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs) contribute to the regulation of neurotransmitter release. In hippocampal synapses, the presynaptic surface expression of several AMPAR subunits, including GluA2, is regulated in a ligand-dependent manner. However, the molecular mechanisms underlying the presynaptic trafficking of AMPARs are still unknown. Here, using bright-field immunocytochemistry, western blots, and quantitative immunogold electron microscopy of the hippocampal CA1 area from intact adult rat brain, we demonstrate the association of AMPA receptors with the presynaptic active zone and with small presynaptic vesicles, in Schaffer collateral synapses in CA1 of the hippocampus. Furthermore, we show that GluA2 and protein interacting with C kinase 1 (PICK1) are colocalized at presynaptic vesicles. Similar to postsynaptic mechanisms, overexpression of either PICK1 or pep2m, which inhibit the N-ethylmaleimide sensitive fusion protein (NSF)-GluA2 interaction, decreases the concentration of GluA2 in the presynaptic active zone membrane. These data suggest that the interacting proteins PICK1 and NSF act as regulators of presynaptic GluA2-containing AMPAR trafficking between the active zone and a vesicle pool that may provide the basis of presynaptic components of synaptic plasticity. Copyright © 2017 IBRO. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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

2016-09-01

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

Loss-of-function of the ciliopathy protein Cc2d2a disorganizes the vesicle fusion machinery at the periciliary membrane and indirectly affects Rab8-trafficking in zebrafish photoreceptors

PubMed Central

Gesemann, Matthias; Mateos, José M.; Barmettler, Gery; Forbes, Austin; Ziegler, Urs

2017-01-01

Ciliopathies are human disorders caused by dysfunction of primary cilia, ubiquitous organelles involved in transduction of environmental signals such as light sensation in photoreceptors. Concentration of signal detection proteins such as opsins in the ciliary membrane is achieved by RabGTPase-regulated polarized vesicle trafficking and by a selective barrier at the ciliary base, the transition zone (TZ). Dysfunction of the TZ protein CC2D2A causes Joubert/Meckel syndromes in humans and loss of ciliary protein localization in animal models, including opsins in retinal photoreceptors. The link between the TZ and upstream vesicle trafficking has been little explored to date. Moreover, the role of the small GTPase Rab8 in opsin-carrier vesicle (OCV) trafficking has been recently questioned in a mouse model. Using correlative light and electron microscopy and live imaging in zebrafish photoreceptors, we provide the first live characterization of Rab8-mediated trafficking in photoreceptors in vivo. Our results support a possibly redundant role for both Rab8a/b paralogs in OCV trafficking, based on co-localization of Rab8 and opsins in vesicular structures, and joint movement of Rab8-tagged particles with opsin. We further investigate the role of the TZ protein Cc2d2a in Rab8-mediated trafficking using cc2d2a zebrafish mutants and identify a requirement for Cc2d2a in the latest step of OCV trafficking, namely vesicle fusion. Progressive accumulation of opsin-containing vesicles in the apical portion of photoreceptors lacking Cc2d2a is caused by disorganization of the vesicle fusion machinery at the periciliary membrane with mislocalization and loss of the t-SNAREs SNAP25 and Syntaxin3 and of the exocyst component Exoc4. We further observe secondary defects on upstream Rab8-trafficking with cytoplasmic accumulation of Rab8. Taken together, our results support participation of Rab8 in OCV trafficking and identify a novel role for the TZ protein Cc2d2a in fusion of incoming

Loss-of-function of the ciliopathy protein Cc2d2a disorganizes the vesicle fusion machinery at the periciliary membrane and indirectly affects Rab8-trafficking in zebrafish photoreceptors.

PubMed

Ojeda Naharros, Irene; Gesemann, Matthias; Mateos, José M; Barmettler, Gery; Forbes, Austin; Ziegler, Urs; Neuhauss, Stephan C F; Bachmann-Gagescu, Ruxandra

2017-12-01

Ciliopathies are human disorders caused by dysfunction of primary cilia, ubiquitous organelles involved in transduction of environmental signals such as light sensation in photoreceptors. Concentration of signal detection proteins such as opsins in the ciliary membrane is achieved by RabGTPase-regulated polarized vesicle trafficking and by a selective barrier at the ciliary base, the transition zone (TZ). Dysfunction of the TZ protein CC2D2A causes Joubert/Meckel syndromes in humans and loss of ciliary protein localization in animal models, including opsins in retinal photoreceptors. The link between the TZ and upstream vesicle trafficking has been little explored to date. Moreover, the role of the small GTPase Rab8 in opsin-carrier vesicle (OCV) trafficking has been recently questioned in a mouse model. Using correlative light and electron microscopy and live imaging in zebrafish photoreceptors, we provide the first live characterization of Rab8-mediated trafficking in photoreceptors in vivo. Our results support a possibly redundant role for both Rab8a/b paralogs in OCV trafficking, based on co-localization of Rab8 and opsins in vesicular structures, and joint movement of Rab8-tagged particles with opsin. We further investigate the role of the TZ protein Cc2d2a in Rab8-mediated trafficking using cc2d2a zebrafish mutants and identify a requirement for Cc2d2a in the latest step of OCV trafficking, namely vesicle fusion. Progressive accumulation of opsin-containing vesicles in the apical portion of photoreceptors lacking Cc2d2a is caused by disorganization of the vesicle fusion machinery at the periciliary membrane with mislocalization and loss of the t-SNAREs SNAP25 and Syntaxin3 and of the exocyst component Exoc4. We further observe secondary defects on upstream Rab8-trafficking with cytoplasmic accumulation of Rab8. Taken together, our results support participation of Rab8 in OCV trafficking and identify a novel role for the TZ protein Cc2d2a in fusion of incoming

Endocytic vesicle rupture is a conserved mechanism of cellular invasion by amyloid proteins.

PubMed

Flavin, William P; Bousset, Luc; Green, Zachary C; Chu, Yaping; Skarpathiotis, Stratos; Chaney, Michael J; Kordower, Jeffrey H; Melki, Ronald; Campbell, Edward M

2017-10-01

Numerous pathological amyloid proteins spread from cell to cell during neurodegenerative disease, facilitating the propagation of cellular pathology and disease progression. Understanding the mechanism by which disease-associated amyloid protein assemblies enter target cells and induce cellular dysfunction is, therefore, key to understanding the progressive nature of such neurodegenerative diseases. In this study, we utilized an imaging-based assay to monitor the ability of disease-associated amyloid assemblies to rupture intracellular vesicles following endocytosis. We observe that the ability to induce vesicle rupture is a common feature of α-synuclein (α-syn) assemblies, as assemblies derived from WT or familial disease-associated mutant α-syn all exhibited the ability to induce vesicle rupture. Similarly, different conformational strains of WT α-syn assemblies, but not monomeric or oligomeric forms, efficiently induced vesicle rupture following endocytosis. The ability to induce vesicle rupture was not specific to α-syn, as amyloid assemblies of tau and huntingtin Exon1 with pathologic polyglutamine repeats also exhibited the ability to induce vesicle rupture. We also observe that vesicles ruptured by α-syn are positive for the autophagic marker LC3 and can accumulate and fuse into large, intracellular structures resembling Lewy bodies in vitro. Finally, we show that the same markers of vesicle rupture surround Lewy bodies in brain sections from PD patients. These data underscore the importance of this conserved endocytic vesicle rupture event as a damaging mechanism of cellular invasion by amyloid assemblies of multiple neurodegenerative disease-associated proteins, and suggest that proteinaceous inclusions such as Lewy bodies form as a consequence of continued fusion of autophagic vesicles in cells unable to degrade ruptured vesicles and their amyloid contents.

Planar Supported Membranes with Mobile SNARE Proteins and Quantitative Fluorescence Microscopy Assays to Study Synaptic Vesicle Fusion

PubMed Central

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

2017-01-01

Synaptic vesicle membrane fusion, the process by which neurotransmitter gets released at the presynaptic membrane is mediated by a complex interplay between proteins and lipids. The realization that the lipid bilayer is not just a passive environment where other molecular players like SNARE proteins act, but is itself actively involved in the process, makes the development of biochemical and biophysical assays particularly challenging. We summarize in vitro assays that use planar supported membranes and fluorescence microscopy to address some of the open questions regarding the molecular mechanisms of SNARE-mediated membrane fusion. Most of the assays discussed in this mini-review were developed in our lab over the last 15 years. We emphasize the sample requirements that we found are important for the successful application of these methods. PMID:28360838

Membrane fusion-competent virus-like proteoliposomes and proteinaceous supported bilayers made directly from cell plasma membranes.

PubMed

Costello, Deirdre A; Hsia, Chih-Yun; Millet, Jean K; Porri, Teresa; Daniel, Susan

2013-05-28

Virus-like particles are useful materials for studying virus-host interactions in a safe manner. However, the standard production of pseudovirus based on the vesicular stomatitis virus (VSV) backbone is an intricate procedure that requires trained laboratory personnel. In this work, a new strategy for creating virus-like proteoliposomes (VLPLs) and virus-like supported bilayers (VLSBs) is presented. This strategy uses a cell blebbing technique to induce the formation of nanoscale vesicles from the plasma membrane of BHK cells expressing the hemagglutinin (HA) fusion protein of influenza X-31. These vesicles and supported bilayers contain HA and are used to carry out single particle membrane fusion events, monitored using total internal reflection fluorescence microscopy. The results of these studies show that the VLPLs and VLSBs contain HA proteins that are fully competent to carry out membrane fusion, including the formation of a fusion pore and the release of fluorophores loaded into vesicles. This new strategy for creating spherical and planar geometry virus-like membranes has many potential applications. VLPLs could be used to study fusion proteins of virulent viruses in a safe manner, or they could be used as therapeutic delivery particles to transport beneficial proteins coexpressed in the cells to a target cell. VLSBs could facilitate high throughput screening of antiviral drugs or pathogen-host cell interactions.

Reduced synaptic vesicle protein degradation at lysosomes curbs TBC1D24/sky-induced neurodegeneration.

PubMed

Fernandes, Ana Clara; Uytterhoeven, Valerie; Kuenen, Sabine; Wang, Yu-Chun; Slabbaert, Jan R; Swerts, Jef; Kasprowicz, Jaroslaw; Aerts, Stein; Verstreken, Patrik

2014-11-24

Synaptic demise and accumulation of dysfunctional proteins are thought of as common features in neurodegeneration. However, the mechanisms by which synaptic proteins turn over remain elusive. In this paper, we study Drosophila melanogaster lacking active TBC1D24/Skywalker (Sky), a protein that in humans causes severe neurodegeneration, epilepsy, and DOOR (deafness, onychdystrophy, osteodystrophy, and mental retardation) syndrome, and identify endosome-to-lysosome trafficking as a mechanism for degradation of synaptic vesicle-associated proteins. In fly sky mutants, synaptic vesicles traveled excessively to endosomes. Using chimeric fluorescent timers, we show that synaptic vesicle-associated proteins were younger on average, suggesting that older proteins are more efficiently degraded. Using a genetic screen, we find that reducing endosomal-to-lysosomal trafficking, controlled by the homotypic fusion and vacuole protein sorting (HOPS) complex, rescued the neurotransmission and neurodegeneration defects in sky mutants. Consistently, synaptic vesicle proteins were older in HOPS complex mutants, and these mutants also showed reduced neurotransmission. Our findings define a mechanism in which synaptic transmission is facilitated by efficient protein turnover at lysosomes and identify a potential strategy to suppress defects arising from TBC1D24 mutations in humans. © 2014 Fernandes et al.

Involvement of vesicle coat material in casein secretion and surface regeneration

PubMed Central

1976-01-01

The ultrastructure of the apical zone of lactating rat mammary epithelial cells was studied with emphasis on vesicle coat structures. Typical 40-60 nm ID "coated vesicles" were abundant, frequently associated with the internal filamentous plasma membrane coat or in direct continuity with secretory vesicles (SV) or plasma membrane proper. Bristle coats partially or totally covered membranes of secretory vesicles identified by their casein micelle content. This coat survived SV isolation. Exocytotic fusion of SV membranes and release of the casein micelles was observed. Frequently, regularly arranged bristle coat structures were identified in those regions of the plasma membrane that were involved in exocytotic processes. Both coated and uncoated surfaces of the casein-containing vesicles, as well as typical "coated vesicles", were frequently associated with microtubules and/or microfilaments. We suggest that coat materials of vesicles are related or identical to components of the internal coat of the surface membrane and that new plasma membrane and associated internal coat is produced concomitantly by fusion and integration of bristle coat moieties. Postexocytotic association of secreted casein micelles with the cell surface, mediated by finely filamentous extensions, provided a marker for the integrated vesicle membrane. An arrangement of SV with the inner surface of the plasma membrane is described which is characterized by regularly spaced, heabily stained membrane to membrane cross-bridges (pre-exocytotic attachment plaques). Such membrane-interconnecting elements may represent a form of coat structure important to recognition and interaction of membrane surfaces. PMID:1254641

On the Computing Potential of Intracellular Vesicles

PubMed Central

Mayne, Richard; Adamatzky, Andrew

2015-01-01

Collision-based computing (CBC) is a form of unconventional computing in which travelling localisations represent data and conditional routing of signals determines the output state; collisions between localisations represent logical operations. We investigated patterns of Ca2+-containing vesicle distribution within a live organism, slime mould Physarum polycephalum, with confocal microscopy and observed them colliding regularly. Vesicles travel down cytoskeletal ‘circuitry’ and their collisions may result in reflection, fusion or annihilation. We demonstrate through experimental observations that naturally-occurring vesicle dynamics may be characterised as a computationally-universal set of Boolean logical operations and present a ‘vesicle modification’ of the archetypal CBC ‘billiard ball model’ of computation. We proceed to discuss the viability of intracellular vesicles as an unconventional computing substrate in which we delineate practical considerations for reliable vesicle ‘programming’ in both in vivo and in vitro vesicle computing architectures and present optimised designs for both single logical gates and combinatorial logic circuits based on cytoskeletal network conformations. The results presented here demonstrate the first characterisation of intracelluar phenomena as collision-based computing and hence the viability of biological substrates for computing. PMID:26431435

PI(4,5)P2-binding effector proteins for vesicle exocytosis

PubMed Central

Martin, Thomas F. J.

2014-01-01

PI(4,5)P2 participates directly in priming and possibly fusion steps of Ca2+-triggered vesicle exocytosis. High concentration nanodomains of PI(4,5)P2 reside on the plasma membrane of neuroendocrine cells. A subset of vesicles that co-localize with PI(4,5)P2 domains appear to undergo preferential exocytosis in stimulated cells. PI(4,5)P2 directly regulates vesicle exocytosis by recruiting and activating PI(4,5)P2-binding proteins that regulate SNARE protein function including CAPS, Munc13-1/2, synaptotagmin-1, and other C2 domain-containing proteins. These PI(4,5)P2 effector proteins are coincidence detectors that engage in multiple interactions at vesicle exocytic sites. The SNARE protein syntaxin-1 also binds to PI(4,5)P2, which promotes clustering, but an activating role for PI(4,5)P2 in syntaxin-1 function remains to be fully characterized. Similar principles underlie polarized constitutive vesicle fusion mediated in part by the PI(4,5)P2-binding subunits of the exocyst complex (Sec3, Exo70). Overall, focal vesicle exocytosis occurs at sites landmarked by PI(4,5)P2, which serves to recruit and/or activate multifunctional PI(4,5)P2-binding proteins. PMID:25280637

How neurosecretory vesicles release their cargo.

PubMed

Scalettar, Bethe A

2006-04-01

Neurons and related cell types often contain two major classes of neurosecretory vesicles, synaptic vesicles (SVs) and dense-core granules (DCGs), which store and release distinct cargo. SVs store and release classic neurotransmitters, which facilitate propagation of action potentials across the synaptic cleft, whereas DCGs transport, store, and release hormones, proteins, and neuropeptides, which facilitate neuronal survival, synaptic transmission, and learning. Over the past few years, there has been a major surge in our understanding of many of the key molecular mechanisms underlying cargo release from SVs and DCGs. This surge has been driven largely by the use of fluorescence microscopy (especially total internal reflection fluorescence microscopy) to visualize SVs or DCGs in living cells. This review highlights some of the recent insights into cargo release from neurosecretory vesicles provided by fluorescence microscopy, with emphasis on DCGs.

Vesicles Are Persistent Features of Different Plastids.

PubMed

Lindquist, Emelie; Solymosi, Katalin; Aronsson, Henrik

2016-10-01

Peripheral vesicles in plastids have been observed repeatedly, primarily in proplastids and developing chloroplasts, in which they are suggested to function in thylakoid biogenesis. Previous observations of vesicles in mature chloroplasts have mainly concerned low temperature pretreated plants occasionally treated with inhibitors blocking vesicle fusion. Here, we show that such vesicle-like structures occur not only in chloroplasts and proplastids, but also in etioplasts, etio-chloroplasts, leucoplasts, chromoplasts and even transforming desiccoplasts without any specific pretreatment. Observations are made both in C3 and C4 species, in different cell types (meristematic, epidermis, mesophyll, bundle sheath and secretory cells) and different organs (roots, stems, leaves, floral parts and fruits). Until recently not much focus has been given to the idea that vesicle transport in chloroplasts could be mediated by proteins, but recent data suggest that the vesicle system of chloroplasts has similarities with the cytosolic coat protein complex II system. All current data taken together support the idea of an ongoing, active and protein-mediated vesicle transport not only in chloroplasts but also in other plastids, obviously occurring regardless of chemical modifications, temperature and plastid developmental stage. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Ca-Mediated Electroformation of Cell-Sized Lipid Vesicles

PubMed Central

Tao, Fei; Yang, Peng

2015-01-01

Cell-sized lipid giant unilamellar vesicles (GUVs) are formed when lipid molecules self-assemble to construct a single bilayer compartment with similar morphology to living cells. The physics of self-assembly process is only generally understood and the size distribution of GUVs tends to be very polydisperse. Herein we report a strategy for the production of controlled size distributions of GUVs by a novel mechanism dissecting the mediation ability of calcium (Ca) on the conventional electroformation of GUVs. We finely construct both of the calcium ion (Ca2+) and calcium carbonate (CaCO3) mineral adsorption layers on a lipid film surface respectively during the electroformation of GUVs. It is found that Ca2+ Slip plane polarized by alternating electric field could induce a pattern of electroosmotic flow across the surface, and thus confine the fusion and growth of GUVs to facilitate the formation of uniform GUVs. The model is further improved by directly using CaCO3 that is in situ formed on a lipid film surface, providing a GUV population with narrow polydispersity. The two models deciphers the new biological function of calcium on the birth of cell-like lipid vesicles, and thus might be potentially relevant to the construction of new model to elucidate the cellular development process. PMID:25950604

Ca-mediated electroformation of cell-sized lipid vesicles.

PubMed

Tao, Fei; Yang, Peng

2015-05-07

Cell-sized lipid giant unilamellar vesicles (GUVs) are formed when lipid molecules self-assemble to construct a single bilayer compartment with similar morphology to living cells. The physics of self-assembly process is only generally understood and the size distribution of GUVs tends to be very polydisperse. Herein we report a strategy for the production of controlled size distributions of GUVs by a novel mechanism dissecting the mediation ability of calcium (Ca) on the conventional electroformation of GUVs. We finely construct both of the calcium ion (Ca(2+)) and calcium carbonate (CaCO3) mineral adsorption layers on a lipid film surface respectively during the electroformation of GUVs. It is found that Ca(2+) Slip plane polarized by alternating electric field could induce a pattern of electroosmotic flow across the surface, and thus confine the fusion and growth of GUVs to facilitate the formation of uniform GUVs. The model is further improved by directly using CaCO3 that is in situ formed on a lipid film surface, providing a GUV population with narrow polydispersity. The two models deciphers the new biological function of calcium on the birth of cell-like lipid vesicles, and thus might be potentially relevant to the construction of new model to elucidate the cellular development process.

Proteins on exocytic vesicles mediate calcium-triggered fusion.

PubMed Central

Vogel, S S; Zimmerberg, J

1992-01-01

In many exocytic systems, micromolar concentrations of intracellular Ca2+ trigger fusion. We find that aggregates of secretory granules isolated from sea urchin eggs fuse together when perfused with greater than or equal to 10 microM free Ca2+. Mixing of membrane components was demonstrated by transfer of fluorescent lipophilic dye, and melding of granule contents was seen with differential interference microscopy. A technique based upon light scattering was developed to conveniently detect fusion. Two protein modifiers, trypsin and N-ethylmaleimide, inhibit granule-granule fusion at concentrations similar to those that inhibit granule-plasma membrane fusion. We suggest that molecular machinery sufficient for Ca(2+)-triggered fusion resides on secretory granules as purified and that at least some of these essential components are proteinaceous. Images PMID:1584814

Calcium-dependent regulation of SNARE-mediated membrane fusion by calmodulin.

PubMed

Di Giovanni, Jerome; Iborra, Cécile; Maulet, Yves; Lévêque, Christian; El Far, Oussama; Seagar, Michael

2010-07-30

Neuroexocytosis requires SNARE proteins, which assemble into trans complexes at the synaptic vesicle/plasma membrane interface and mediate bilayer fusion. Ca(2+) sensitivity is thought to be conferred by synaptotagmin, although the ubiquitous Ca(2+)-effector calmodulin has also been implicated in SNARE-dependent membrane fusion. To examine the molecular mechanisms involved, we examined the direct action of calmodulin and synaptotagmin in vitro, using fluorescence resonance energy transfer to assay lipid mixing between target- and vesicle-SNARE liposomes. Ca(2+)/calmodulin inhibited SNARE assembly and membrane fusion by binding to two distinct motifs located in the membrane-proximal regions of VAMP2 (K(D) = 500 nm) and syntaxin 1 (K(D) = 2 microm). In contrast, fusion was increased by full-length synaptotagmin 1 anchored in vesicle-SNARE liposomes. When synaptotagmin and calmodulin were combined, synaptotagmin overcame the inhibitory effects of calmodulin. Furthermore, synaptotagmin displaced calmodulin binding to target-SNAREs. These findings suggest that two distinct Ca(2+) sensors act antagonistically in SNARE-mediated fusion.

Phospholipase A2 activity-dependent and -independent fusogenic activity of Naja nigricollis CMS-9 on zwitterionic and anionic phospholipid vesicles.

PubMed

Chiou, Yi-Ling; Chen, Ying-Jung; Lin, Shinne-Ren; Chang, Long-Sen

2011-11-01

CMS-9, a phospholipase A(2) (PLA(2)) from Naja nigricollis venom, induced the death of human breast cancer MCF-7 cells accompanied with the formation of cell clumps without clear boundaries between cells. Annexin V-FITC staining indicated that abundant phosphatidylserine appeared on the outer membrane of MCF-7 cell clumps, implying the possibility that CMS-9 may promote membrane fusion via anionic phospholipids. To validate this proposition, fusogenic activity of CMS-9 on vesicles composed of zwitterionic phospholipid alone or a combination of zwitterionic and anionic phospholipids was examined. Although CMS-9-induced fusion of zwitterionic phospholipid vesicles depended on PLA(2) activity, CMS-9-induced fusion of vesicles containing anionic phospholipids could occur without the involvement of PLA(2) activity. Membrane-damaging activity of CMS-9 was associated with its fusogenicity. Moreover, CMS-9 induced differently membrane leakage and membrane fusion of vesicles with different compositions. Membrane fluidity and binding capability with phospholipid vesicles were not related to the fusogenicity of CMS-9. However, membrane-bound conformation and mode of CMS-9 depended on phospholipid compositions. Collectively, our data suggest that PLA(2) activity-dependent and -independent fusogenicity of CMS-9 are closely related to its membrane-bound modes and targeted membrane compositions. Copyright © 2011 Elsevier Ltd. All rights reserved.

The Multifaceted Role of SNARE Proteins in Membrane Fusion

PubMed Central

Han, Jing; Pluhackova, Kristyna; Böckmann, Rainer A.

2017-01-01

Membrane fusion is a key process in all living organisms that contributes to a variety of biological processes including viral infection, cell fertilization, as well as intracellular transport, and neurotransmitter release. In particular, the various membrane-enclosed compartments in eukaryotic cells need to exchange their contents and communicate across membranes. Efficient and controllable fusion of biological membranes is known to be driven by cooperative action of SNARE proteins, which constitute the central components of the eukaryotic fusion machinery responsible for fusion of synaptic vesicles with the plasma membrane. During exocytosis, vesicle-associated v-SNARE (synaptobrevin) and target cell-associated t-SNAREs (syntaxin and SNAP-25) assemble into a core trans-SNARE complex. This complex plays a versatile role at various stages of exocytosis ranging from the priming to fusion pore formation and expansion, finally resulting in the release or exchange of the vesicle content. This review summarizes current knowledge on the intricate molecular mechanisms underlying exocytosis triggered and catalyzed by SNARE proteins. Particular attention is given to the function of the peptidic SNARE membrane anchors and the role of SNARE-lipid interactions in fusion. Moreover, the regulatory mechanisms by synaptic auxiliary proteins in SNARE-driven membrane fusion are briefly outlined. PMID:28163686

The Multifaceted Role of SNARE Proteins in Membrane Fusion.

PubMed

Han, Jing; Pluhackova, Kristyna; Böckmann, Rainer A

2017-01-01

Membrane fusion is a key process in all living organisms that contributes to a variety of biological processes including viral infection, cell fertilization, as well as intracellular transport, and neurotransmitter release. In particular, the various membrane-enclosed compartments in eukaryotic cells need to exchange their contents and communicate across membranes. Efficient and controllable fusion of biological membranes is known to be driven by cooperative action of SNARE proteins, which constitute the central components of the eukaryotic fusion machinery responsible for fusion of synaptic vesicles with the plasma membrane. During exocytosis, vesicle-associated v-SNARE (synaptobrevin) and target cell-associated t-SNAREs (syntaxin and SNAP-25) assemble into a core trans-SNARE complex. This complex plays a versatile role at various stages of exocytosis ranging from the priming to fusion pore formation and expansion, finally resulting in the release or exchange of the vesicle content. This review summarizes current knowledge on the intricate molecular mechanisms underlying exocytosis triggered and catalyzed by SNARE proteins. Particular attention is given to the function of the peptidic SNARE membrane anchors and the role of SNARE-lipid interactions in fusion. Moreover, the regulatory mechanisms by synaptic auxiliary proteins in SNARE-driven membrane fusion are briefly outlined.

Structures and mechanisms of vesicle coat components and multisubunit tethering complexes

PubMed Central

Jackson, Lauren P; Kümmel, Daniel; Reinisch, Karin M; Owen, David J

2012-01-01

Eukaryotic cells face a logistical challenge in ensuring prompt and precise delivery of vesicular cargo to specific organelles within the cell. Coat protein complexes select cargo and initiate vesicle formation, while multisubunit tethering complexes participate in the delivery of vesicles to target membranes. Understanding these macromolecular assemblies has greatly benefited from their structural characterization. Recent structural data highlight principles in coat recruitment and uncoating in both the endocytic and retrograde pathways, and studies on the architecture of tethering complexes provide a framework for how they might link vesicles to the respective acceptor compartments and the fusion machinery. PMID:22728063

A Model for Membrane Fusion

NASA Astrophysics Data System (ADS)

Ngatchou, Annita

2010-01-01

Pheochromocytoma is a tumor of the adrenal gland which originates from chromaffin cells and is characterized by the secretion of excessive amounts of neurotransmitter which lead to high blood pressure and palpitations. Pheochromocytoma contain membrane bound granules that store neurotransmitter. The release of these stored molecules into the extracellular space occurs by fusion of the granule membrane with the cell plasma membrane, a process called exocytosis. The molecular mechanism of this membrane fusion is not well understood. It is proposed that the so called SNARE proteins [1] are the pillar of vesicle fusion as their cleavage by clostridial toxin notably, Botulinum neurotoxin and Tetanus toxin abrogate the secretion of neurotransmitter [2]. Here, I describe how physical principles are applied to a biological cell to explore the role of the vesicle SNARE protein synaptobrevin-2 in easing granule fusion. The data presented here suggest a paradigm according to which the movement of the C-terminal of synaptobrevin-2 disrupts the lipid bilayer to form a fusion pore through which molecules can exit.

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons.

PubMed

Villarreal, Seth; Lee, Sung Hoon; Wu, Ling-Gang

2017-09-04

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

CAPS drives trans-SNARE complex formation and membrane fusion through syntaxin interactions.

PubMed

James, Declan J; Kowalchyk, Judith; Daily, Neil; Petrie, Matt; Martin, Thomas F J

2009-10-13

Ca(2+)-dependent activator protein for secretion (CAPS) is an essential factor for regulated vesicle exocytosis that functions in priming reactions before Ca(2+)-triggered fusion of vesicles with the plasma membrane. However, the precise events that CAPS regulates to promote vesicle fusion are unclear. In the current work, we reconstituted CAPS function in a SNARE-dependent liposome fusion assay using VAMP2-containing donor and syntaxin-1/SNAP-25-containing acceptor liposomes. The CAPS stimulation of fusion required PI(4,5)P(2) in acceptor liposomes and was independent of Ca(2+), but Ca(2+) dependence was restored by inclusion of synaptotagmin. CAPS stimulated trans-SNARE complex formation concomitant with the stimulation of full membrane fusion at physiological SNARE densities. CAPS bound syntaxin-1, and CAPS truncations that competitively inhibited syntaxin-1 binding also inhibited CAPS-dependent fusion. The results revealed an unexpected activity of a priming protein to accelerate fusion by efficiently promoting trans-SNARE complex formation. CAPS may function in priming by organizing SNARE complexes on the plasma membrane.

The MARVEL domain protein, Singles Bar, is required for progression past the pre-fusion complex stage of myoblast fusion.

PubMed

Estrada, Beatriz; Maeland, Anne D; Gisselbrecht, Stephen S; Bloor, James W; Brown, Nicholas H; Michelson, Alan M

2007-07-15

Multinucleated myotubes develop by the sequential fusion of individual myoblasts. Using a convergence of genomic and classical genetic approaches, we have discovered a novel gene, singles bar (sing), that is essential for myoblast fusion. sing encodes a small multipass transmembrane protein containing a MARVEL domain, which is found in vertebrate proteins involved in processes such as tight junction formation and vesicle trafficking where--as in myoblast fusion--membrane apposition occurs. sing is expressed in both founder cells and fusion competent myoblasts preceding and during myoblast fusion. Examination of embryos injected with double-stranded sing RNA or embryos homozygous for ethane methyl sulfonate-induced sing alleles revealed an identical phenotype: replacement of multinucleated myofibers by groups of single, myosin-expressing myoblasts at a stage when formation of the mature muscle pattern is complete in wild-type embryos. Unfused sing mutant myoblasts form clusters, suggesting that early recognition and adhesion of these cells are unimpaired. To further investigate this phenotype, we undertook electron microscopic ultrastructural studies of fusing myoblasts in both sing and wild-type embryos. These experiments revealed that more sing mutant myoblasts than wild-type contain pre-fusion complexes, which are characterized by electron-dense vesicles paired on either side of the fusing plasma membranes. In contrast, embryos mutant for another muscle fusion gene, blown fuse (blow), have a normal number of such complexes. Together, these results lead to the hypothesis that sing acts at a step distinct from that of blow, and that sing is required on both founder cell and fusion-competent myoblast membranes to allow progression past the pre-fusion complex stage of myoblast fusion, possibly by mediating fusion of the electron-dense vesicles to the plasma membrane.

Full-Length Trimeric Influenza Virus Hemagglutinin II Membrane Fusion Protein and Shorter Constructs Lacking the Fusion Peptide or Transmembrane Domain: Hyperthermostability of the Full-Length Protein and the Soluble Ectodomain and Fusion Peptide Make Significant Contributions to Fusion of Membrane Vesicles†

PubMed Central

Ratnayake, Punsisi U.; Ekanayaka, E. A. Prabodha; Komanduru, Sweta S.; Weliky, David P.

2015-01-01

Influenza virus is a Class I enveloped virus which is initially endocytosed into a host respiratory epithelial cell. Subsequent reduction of the pH to the 5–6 range triggers a structural change of the viral hemagglutinin II (HA2) protein, fusion of the viral and endosomal membranes, and release of the viral nucleocapsid into the cytoplasm. HA2 contains fusion peptide (FP), soluble ectodomain (SE), transmembrane (TM), and intraviral domains with respective lengths of ~25, ~160, ~25, and ~10 residues. The present work provides a straightforward protocol for producing and purifying mg quantities of full-length HA2 from expression in bacteria. Biophysical and structural comparisons are made between full-length HA2 and shorter constructs including SHA2 ≡ SE, FHA2 ≡ FP + SE, and SHA2-TM ≡ SE + TM constructs. The constructs are helical in detergent at pH 7.4 and the dominant trimer species. The proteins are highly thermostable in decylmaltoside detergent with Tm > 90 °C for HA2 with stabilization provided by the SE, FP, and TM domains. The proteins are likely in a trimer-of-hairpins structure, the final protein state during fusion. All constructs induce fusion of negatively-charged vesicles at pH 5.0 with much less fusion at pH 7.4. Attractive protein/vesicle electrostatics play a role in fusion, as the proteins are positively-charged at pH 5.0 and negatively-charged at pH 7.4 and the pH-dependence of fusion is reversed for positively-charged vesicles. Comparison of fusion between constructs supports significant contributions to fusion from the SE and the FP with little effect from the TM. PMID:26297995

Storage vesicles in neurons are related to Golgi complex alterations in mucopolysaccharidosis IIIB.

PubMed

Vitry, Sandrine; Bruyère, Julie; Hocquemiller, Michaël; Bigou, Stéphanie; Ausseil, Jérôme; Colle, Marie-Anne; Prévost, Marie-Christine; Heard, Jean Michel

2010-12-01

The accumulation of intracellular storage vesicles is a hallmark of lysosomal storage diseases. Neither the identity nor origin of these implicated storage vesicles have yet been established. The vesicles are often considered as lysosomes, endosomes, and/or autophagosomes that are engorged with undigested materials. Our studies in the mouse model of mucopolysaccharidosis type IIIB, a lysosomal storage disease that induces neurodegeneration, showed that large storage vesicles in cortical neurons did not receive material from either the endocytic or autophagy pathway, which functioned normally. Storage vesicles expressed GM130, a Golgi matrix protein, which mediates vesicle tethering in both pre- and cis-Golgi compartments. However, other components of the tethering/fusion complex were not associated with GM130 on storage vesicles, likely accounting for both the resistance of the vesicles to brefeldin A and the alteration of Golgi ribbon architecture, which comprised distended cisterna connected to LAMP1-positive storage vesicles. We propose that alteration in the GM130-mediated control of vesicle trafficking in pre-Golgi and Golgi compartments affects Golgi biogenesis and gives rise to a dead-end storage compartment. Vesicle accumulation, Golgi disorganization, and alterations of other GM130 functions may account for neuron dysfunction and death.

Isolation and characterization of urinary extracellular vesicles: implications for biomarker discovery.

PubMed

Merchant, Michael L; Rood, Ilse M; Deegens, Jeroen K J; Klein, Jon B

2017-12-01

Urine is a valuable diagnostic medium and, with the discovery of urinary extracellular vesicles, is viewed as a dynamic bioactive fluid. Extracellular vesicles are lipid-enclosed structures that can be classified into three categories: exosomes, microvesicles (or ectosomes) and apoptotic bodies. This classification is based on the mechanisms by which membrane vesicles are formed: fusion of multivesicular bodies with the plasma membranes (exosomes), budding of vesicles directly from the plasma membrane (microvesicles) or those shed from dying cells (apoptotic bodies). During their formation, urinary extracellular vesicles incorporate various cell-specific components (proteins, lipids and nucleic acids) that can be transferred to target cells. The rigour needed for comparative studies has fueled the search for optimal approaches for their isolation, purification, and characterization. RNA, the newest extracellular vesicle component to be discovered, has received substantial attention as an extracellular vesicle therapeutic, and compelling evidence suggests that ex vivo manipulation of microRNA composition may have uses in the treatment of kidney disorders. The results of these studies are building the case that urinary extracellular vesicles act as mediators of renal pathophysiology. As the field of extracellular vesicle studies is burgeoning, this Review focuses on primary data obtained from studies of human urine rather than on data from studies of laboratory animals or cultured immortalized cells.

"An estimate of the probability of vesicle exocytosis in a Monte Carlo model of buffered diffusion of calcium channel currents"

NASA Astrophysics Data System (ADS)

Dimcovic, Z. M.; Eagan, T. P.; Kidane, T. K.; Brown, R. W.; Petschek, R. G.; McEnery, M. W.

2001-10-01

The opening of voltage-dependent calcium channels results in an influx of calcium ions promoting the fusion of synaptic vesicles. The fusion leads to release of neurotransmitters, which in turn allow the propagation of nerve impulses. A Monte Carlo model of the diffusion of calcium following its surge into the cell is used to estimate the probability for exocytosis. Besides the calcium absorption by fixed and mobile buffers, key ingredients are the physical size and position of the tethered vesicle and a sensing model for the interaction of the vesicle and calcium. The release probability is compared to previously published studies where the finite vesicle size was not considered. (Supported by NIH MH55747, AHA 96001250, NSF0086643, and a CWRU Presidential Research Initiative grant.)

Freeze-thaw cycles induce content exchange between cell-sized lipid vesicles

NASA Astrophysics Data System (ADS)

Litschel, Thomas; Ganzinger, Kristina A.; Movinkel, Torgeir; Heymann, Michael; Robinson, Tom; Mutschler, Hannes; Schwille, Petra

2018-05-01

Early protocells are commonly assumed to consist of an amphiphilic membrane enclosing an RNA-based self-replicating genetic system and a primitive metabolism without protein enzymes. Thus, protocell evolution must have relied on simple physicochemical self-organization processes within and across such vesicular structures. We investigate freeze-thaw (FT) cycling as a potential environmental driver for the necessary content exchange between vesicles. To this end, we developed a conceptually simple yet statistically powerful high-throughput procedure based on nucleic acid-containing giant unilamellar vesicles (GUVs) as model protocells. GUVs are formed by emulsion transfer in glass bottom microtiter plates and hence can be manipulated and monitored by fluorescence microscopy without additional pipetting and sample handling steps. This new protocol greatly minimizes artefacts, such as unintended GUV rupture or fusion by shear forces. Using DNA-encapsulating phospholipid GUVs fabricated by this method, we quantified the extent of content mixing between GUVs under different FT conditions. We found evidence of nucleic acid exchange in all detected vesicles if fast freezing of GUVs at ‑80 °C is followed by slow thawing at room temperature. In contrast, slow freezing and fast thawing both adversely affected content mixing. Surprisingly, and in contrast to previous reports for FT-induced content mixing, we found that the content is not exchanged through vesicle fusion and fission, but that vesicles largely maintain their membrane identity and even large molecules are exchanged via diffusion across the membranes. Our approach supports efficient screening of prebiotically plausible molecules and environmental conditions, to yield universal mechanistic insights into how cellular life may have emerged.

CAPS and Munc13: CATCHRs that SNARE Vesicles.

PubMed

James, Declan J; Martin, Thomas F J

2013-12-04

CAPS (Calcium-dependent Activator Protein for Secretion, aka CADPS) and Munc13 (Mammalian Unc-13) proteins function to prime vesicles for Ca(2+)-triggered exocytosis in neurons and neuroendocrine cells. CAPS and Munc13 proteins contain conserved C-terminal domains that promote the assembly of SNARE complexes for vesicle priming. Similarities of the C-terminal domains of CAPS/Munc13 proteins with Complex Associated with Tethering Containing Helical Rods domains in multi-subunit tethering complexes (MTCs) have been reported. MTCs coordinate multiple interactions for SNARE complex assembly at constitutive membrane fusion steps. We review aspects of these diverse tethering and priming factors to identify common operating principles.

A STRIPAK complex mediates axonal transport of autophagosomes and dense core vesicles through PP2A regulation

PubMed Central

Neufeld, Thomas P.

2017-01-01

Autophagy plays an essential role in the cellular homeostasis of neurons, facilitating the clearance of cellular debris. This clearance process is orchestrated through the assembly, transport, and fusion of autophagosomes with lysosomes for degradation. The motor protein dynein drives autophagosome motility from distal sites of assembly to sites of lysosomal fusion. In this study, we identify the scaffold protein CKA (connector of kinase to AP-1) as essential for autophagosome transport in neurons. Together with other core components of the striatin-interacting phosphatase and kinase (STRIPAK) complex, we show that CKA associates with dynein and directly binds Atg8a, an autophagosomal protein. CKA is a regulatory subunit of PP2A, a component of the STRIPAK complex. We propose that the STRIPAK complex modulates dynein activity. Consistent with this hypothesis, we provide evidence that CKA facilitates axonal transport of dense core vesicles and autophagosomes in a PP2A-dependent fashion. In addition, CKA-deficient flies exhibit PP2A-dependent motor coordination defects. CKA function within the STRIPAK complex is crucial to prevent transport defects that may contribute to neurodegeneration. PMID:28100687

Self-assembled polyhydroxy fatty acids vesicles: a mechanism for plant cutin synthesis.

PubMed

Heredia-Guerrero, José A; Benítez, José J; Heredia, Antonio

2008-03-01

Despite its biological importance, the mechanism of formation of cutin, the polymeric matrix of plant cuticles, has not yet been fully clarified. Here, for the first time, we show the participation in the process of lipid vesicles formed by the self-assembly of endogenous polyhydroxy fatty acids. The accumulation and fusion of these vesicles (cutinsomes) at the outer part of epidermal cell wall is proposed as the mechanism for early cuticle formation.

Mesenchymal stem cell-derived extracellular vesicles attenuate kidney inflammation.

PubMed

Eirin, Alfonso; Zhu, Xiang-Yang; Puranik, Amrutesh S; Tang, Hui; McGurren, Kelly A; van Wijnen, Andre J; Lerman, Amir; Lerman, Lilach O

2017-07-01

Mesenchymal stem/stromal cells (MSCs) have distinct capability for renal repair, but may have safety concerns. MSC-derived extracellular vesicles emerged as a novel noncellular alternative. Using a porcine model of metabolic syndrome and renal artery stenosis we tested whether extracellular vesicles attenuate renal inflammation, and if this capacity is mediated by their cargo of the anti-inflammatory cytokine interleukin (IL) 10. Pigs with metabolic syndrome were studied after 16 weeks of renal artery stenosis untreated or treated four weeks earlier with a single intrarenal delivery of extracellular vesicles harvested from adipose tissue-derived autologous MSCs. Lean and sham metabolic syndrome animals served as controls (seven each). Five additional pigs with metabolic syndrome and renal artery stenosis received extracellular vesicles with pre-silenced IL10 (IL10 knock-down). Single-kidney renal blood flow, glomerular filtration rate, and oxygenation were studied in vivo and renal injury pathways ex vivo. Retention of extracellular vesicles in the stenotic kidney peaked two days after delivery and decreased thereafter. Four weeks after injection, extracellular vesicle fragments colocalized with stenotic-kidney tubular cells and macrophages, indicating internalization or fusion. Extracellular vesicle delivery attenuated renal inflammation, and improved medullary oxygenation and fibrosis. Renal blood flow and glomerular filtration rate fell in metabolic syndrome and renal artery stenosis compared to metabolic syndrome, but was restored in pigs treated with extracellular vesicles. These renoprotective effects were blunted in pigs treated with IL10-depleted extracellular vesicles. Thus, extracellular vesicle-based regenerative strategies might be useful for patients with metabolic syndrome and renal artery stenosis. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

The effect of spontaneous curvature on a two-phase vesicle

PubMed Central

Cox, Geoffrey; Lowengrub, John

2015-01-01

Vesicles are membrane-bound structures commonly known for their roles in cellular transport and the shape of a vesicle is determined by its surrounding membrane (lipid bilayer). When the membrane is composed of different lipids, it is natural for the lipids of similar molecular structure to migrate towards one another (via spinodal decomposition), creating a multi-phase vesicle. In this article, we consider a two-phase vesicle model which is driven by nature’s propensity to maintain a minimal state of elastic energy. The model assumes a continuum limit, thereby treating the membrane as a closed three-dimensional surface. The main purpose of this study is to reveal the complexity of the Helfrich two-phase vesicle model with non-zero spontaneous curvature and provide further evidence to support the relevance of spontaneous curvature as a modelling parameter. In this paper, we illustrate the complexity of the Helfrich two-phase model by providing multiple examples of undocumented solutions and energy hysteresis. We also investigate the influence of spontaneous curvature on morphological effects and membrane phenomena such as budding and fusion. PMID:26097287

Vesicle Motion during Sustained Exocytosis in Chromaffin Cells: Numerical Model Based on Amperometric Measurements.

PubMed

Jarukanont, Daungruthai; Bonifas Arredondo, Imelda; Femat, Ricardo; Garcia, Martin E

2015-01-01

Chromaffin cells release catecholamines by exocytosis, a process that includes vesicle docking, priming and fusion. Although all these steps have been intensively studied, some aspects of their mechanisms, particularly those regarding vesicle transport to the active sites situated at the membrane, are still unclear. In this work, we show that it is possible to extract information on vesicle motion in Chromaffin cells from the combination of Langevin simulations and amperometric measurements. We developed a numerical model based on Langevin simulations of vesicle motion towards the cell membrane and on the statistical analysis of vesicle arrival times. We also performed amperometric experiments in bovine-adrenal Chromaffin cells under Ba2+ stimulation to capture neurotransmitter releases during sustained exocytosis. In the sustained phase, each amperometric peak can be related to a single release from a new vesicle arriving at the active site. The amperometric signal can then be mapped into a spike-series of release events. We normalized the spike-series resulting from the current peaks using a time-rescaling transformation, thus making signals coming from different cells comparable. We discuss why the obtained spike-series may contain information about the motion of all vesicles leading to release of catecholamines. We show that the release statistics in our experiments considerably deviate from Poisson processes. Moreover, the interspike-time probability is reasonably well described by two-parameter gamma distributions. In order to interpret this result we computed the vesicles' arrival statistics from our Langevin simulations. As expected, assuming purely diffusive vesicle motion we obtain Poisson statistics. However, if we assume that all vesicles are guided toward the membrane by an attractive harmonic potential, simulations also lead to gamma distributions of the interspike-time probability, in remarkably good agreement with experiment. We also show that

Multiple vesicle recycling pathways in central synapses and their impact on neurotransmission

PubMed Central

Kavalali, Ege T

2007-01-01

Short-term synaptic depression during repetitive activity is a common property of most synapses. Multiple mechanisms contribute to this rapid depression in neurotransmission including a decrease in vesicle fusion probability, inactivation of voltage-gated Ca2+ channels or use-dependent inhibition of release machinery by presynaptic receptors. In addition, synaptic depression can arise from a rapid reduction in the number of vesicles available for release. This reduction can be countered by two sources. One source is replenishment from a set of reserve vesicles. The second source is the reuse of vesicles that have undergone exocytosis and endocytosis. If the synaptic vesicle reuse is fast enough then it can replenish vesicles during a brief burst of action potentials and play a substantial role in regulating the rate of synaptic depression. In the last 5 years, we have examined the impact of synaptic vesicle reuse on neurotransmission using fluorescence imaging of synaptic vesicle trafficking in combination with electrophysiological detection of short-term synaptic plasticity. These studies have revealed that synaptic vesicle reuse shapes the kinetics of short-term synaptic depression in a frequency-dependent manner. In addition, synaptic vesicle recycling helps maintain the level of neurotransmission at steady state. Moreover, our studies showed that synaptic vesicle reuse is a highly plastic process as it varies widely among synapses and can adapt to changes in chronic activity levels. PMID:17690145

Ca{sup 2+}-dependent mobility of vesicles capturing anti-VGLUT1 antibodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stenovec, Matjaz; Laboratory of Neuroendocrinology - Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloska 4, 1000 Ljubljana; Kreft, Marko

2007-11-01

Several aspects of secretory vesicle cycle have been studied in the past, but vesicle trafficking in relation to the fusion site is less well understood. In particular, the mobility of recaptured vesicles that traffic back toward the central cytoplasm is still poorly defined. We exposed astrocytes to antibodies against the vesicular glutamate transporter 1 (VGLUT1), a marker of glutamatergic vesicles, to fluorescently label vesicles undergoing Ca{sup 2+}-dependent exocytosis and examined their number, fluorescence intensity, and mobility by confocal microscopy. In nonstimulated cells, immunolabeling revealed discrete fluorescent puncta, indicating that VGLUT1 vesicles, which are approximately 50 nm in diameter, cycle slowlymore » between the plasma membrane and the cytoplasm. When the cytosolic Ca{sup 2+} level was raised with ionomycin, the number and fluorescence intensity of the puncta increased, likely because the VGLUT1 epitopes were more accessible to the extracellularly applied antibodies following Ca{sup 2+}-triggered exocytosis. In nonstimulated cells, the mobility of labeled vesicles was limited. In stimulated cells, many vesicles exhibited directional mobility that was abolished by cytoskeleton-disrupting agents, indicating dependence on intact cytoskeleton. Our findings show that postfusion vesicle mobility is regulated and may likely play a role in synaptic vesicle cycle, and also more generally in the genesis and removal of endocytic vesicles.« less

Diffusional spread and confinement of newly exocytosed synaptic vesicle proteins

PubMed Central

Gimber, Niclas; Tadeus, Georgi; Maritzen, Tanja; Schmoranzer, Jan; Haucke, Volker

2015-01-01

Neurotransmission relies on the calcium-triggered exocytic fusion of non-peptide neurotransmitter-containing small synaptic vesicles (SVs) with the presynaptic membrane at active zones (AZs) followed by compensatory endocytic retrieval of SV membranes. Here, we study the diffusional fate of newly exocytosed SV proteins in hippocampal neurons by high-resolution time-lapse imaging. Newly exocytosed SV proteins rapidly disperse within the first seconds post fusion until confined within the presynaptic bouton. Rapid diffusional spread and confinement is followed by slow reclustering of SV proteins at the periactive endocytic zone. Confinement within the presynaptic bouton is mediated in part by SV protein association with the clathrin-based endocytic machinery to limit diffusional spread of newly exocytosed SV proteins. These data suggest that diffusion, and axonal escape of newly exocytosed vesicle proteins, are counteracted by the clathrin-based endocytic machinery together with a presynaptic diffusion barrier. PMID:26399746

Diffusional spread and confinement of newly exocytosed synaptic vesicle proteins

NASA Astrophysics Data System (ADS)

Gimber, Niclas; Tadeus, Georgi; Maritzen, Tanja; Schmoranzer, Jan; Haucke, Volker

2015-09-01

Neurotransmission relies on the calcium-triggered exocytic fusion of non-peptide neurotransmitter-containing small synaptic vesicles (SVs) with the presynaptic membrane at active zones (AZs) followed by compensatory endocytic retrieval of SV membranes. Here, we study the diffusional fate of newly exocytosed SV proteins in hippocampal neurons by high-resolution time-lapse imaging. Newly exocytosed SV proteins rapidly disperse within the first seconds post fusion until confined within the presynaptic bouton. Rapid diffusional spread and confinement is followed by slow reclustering of SV proteins at the periactive endocytic zone. Confinement within the presynaptic bouton is mediated in part by SV protein association with the clathrin-based endocytic machinery to limit diffusional spread of newly exocytosed SV proteins. These data suggest that diffusion, and axonal escape of newly exocytosed vesicle proteins, are counteracted by the clathrin-based endocytic machinery together with a presynaptic diffusion barrier.

Integration of Synaptic Vesicle Cargo Retrieval with Endocytosis at Central Nerve Terminals

PubMed Central

Cousin, Michael A.

2017-01-01

Central nerve terminals contain a limited number of synaptic vesicles (SVs) which mediate the essential process of neurotransmitter release during their activity-dependent fusion. The rapid and accurate formation of new SVs with the appropriate cargo is essential to maintain neurotransmission in mammalian brain. Generating SVs containing the correct SV cargo with the appropriate stoichiometry is a significant challenge, especially when multiple modes of endocytosis exist in central nerve terminals, which occur at different locations within the nerve terminals. These endocytosis modes include ultrafast endocytosis, clathrin-mediated endocytosis (CME) and activity-dependent bulk endocytosis (ADBE) which are triggered by specific patterns of neuronal activity. This review article will assess the evidence for the role of classical adaptor protein complexes in SV retrieval, discuss the role of monomeric adaptors and how interactions between specific SV cargoes can facilitate retrieval. In addition it will consider the evidence for preassembled plasma membrane cargo complexes and their role in facilitating these endocytosis modes. Finally it will present a unifying model for cargo retrieval at the presynapse, which integrates endocytosis modes in time and space. PMID:28824381

MAL facilitates the incorporation of exocytic uroplakin-delivering vesicles into the apical membrane of urothelial umbrella cells

PubMed Central

Zhou, Ge; Liang, Feng-Xia; Romih, Rok; Wang, Zefang; Liao, Yi; Ghiso, Jorge; Luque-Garcia, Jose L.; Neubert, Thomas A.; Kreibich, Gert; Alonso, Miguel A.; Schaeren-Wiemers, Nicole; Sun, Tung-Tien

2012-01-01

The apical surface of mammalian bladder urothelium is covered by large (500–1000 nm) two-dimensional (2D) crystals of hexagonally packed 16-nm uroplakin particles (urothelial plaques), which play a role in permeability barrier function and uropathogenic bacterial binding. How the uroplakin proteins are delivered to the luminal surface is unknown. We show here that myelin-and-lymphocyte protein (MAL), a 17-kDa tetraspan protein suggested to be important for the apical sorting of membrane proteins, is coexpressed with uroplakins in differentiated urothelial cell layers. MAL depletion in Madin–Darby canine kidney cells did not affect, however, the apical sorting of uroplakins, but it decreased the rate by which uroplakins were inserted into the apical surface. Moreover, MAL knockout in vivo led to the accumulation of fusiform vesicles in mouse urothelial superficial umbrella cells, whereas MAL transgenic overexpression in vivo led to enhanced exocytosis and compensatory endocytosis, resulting in the accumulation of the uroplakin-degrading multivesicular bodies. Finally, although MAL and uroplakins cofloat in detergent-resistant raft fractions, they are associated with distinct plaque and hinge membrane subdomains, respectively. These data suggest a model in which 1) MAL does not play a role in the apical sorting of uroplakins; 2) the propensity of uroplakins to polymerize forming 16-nm particles and later large 2D crystals that behave as detergent-resistant (giant) rafts may drive their apical targeting; 3) the exclusion of MAL from the expanding 2D crystals of uroplakins explains the selective association of MAL with the hinge areas in the uroplakin-delivering fusiform vesicles, as well as at the apical surface; and 4) the hinge-associated MAL may play a role in facilitating the incorporation of the exocytic uroplakin vesicles into the corresponding hinge areas of the urothelial apical surface. PMID:22323295

Complexin and Ca2+ stimulate SNARE-mediated membrane fusion

PubMed Central

Yoon, Tae-Young; Lu, Xiaobind; Diao, Jiajie; Lee, Soo-Min; Ha, Taekjip; Shin, Yeon-Kyun

2008-01-01

Ca2+-triggered, synchronized synaptic vesicle fusion underlies interneuronal communication. Complexin is a major binding partner of the SNARE complex, the core fusion machinery at the presynapse. The physiological data on complexin, however, have been at odds with each other, making delineation of its molecular function difficult. Here we report direct observation of two-faceted functions of complexin using the single-vesicle fluorescence fusion assay and EPR. We show that complexin I has two opposing effects on trans-SNARE assembly: inhibition of SNARE complex formation and stabilization of assembled SNARE complexes. Of note, SNARE-mediated fusion is markedly stimulated by complexin, and it is further accelerated by two orders of magnitude in response to an externally applied Ca2+ wave. We suggest that SNARE complexes, complexins and phospholipids collectively form a complex substrate for Ca2+ and Ca2+-sensing fusion effectors in neurotransmitter release. PMID:18552825

Green fluorescence protein-based content-mixing assay of SNARE-driven membrane fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heo, Paul; Kong, Byoungjae; Jung, Young-Hun

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion by forming a ternary SNARE complex. A minimalist approach utilizing proteoliposomes with reconstituted SNARE proteins yielded a wealth of information pinpointing the molecular mechanism of SNARE-mediated fusion and its regulation by accessory proteins. Two important attributes of a membrane fusion are lipid-mixing and the formation of an aqueous passage between apposing membranes. These two attributes are typically observed by using various fluorescent dyes. Currently available in vitro assay systems for observing fusion pore opening have several weaknesses such as cargo-bleeding, incomplete removal of unencapsulated dyes, and inadequate information regardingmore » the size of the fusion pore, limiting measurements of the final stage of membrane fusion. In the present study, we used a biotinylated green fluorescence protein and streptavidin conjugated with Dylight 594 (DyStrp) as a Föster resonance energy transfer (FRET) donor and acceptor, respectively. This FRET pair encapsulated in each v-vesicle containing synaptobrevin and t-vesicle containing a binary acceptor complex of syntaxin 1a and synaptosomal-associated protein 25 revealed the opening of a large fusion pore of more than 5 nm, without the unwanted signals from unencapsulated dyes or leakage. This system enabled determination of the stoichiometry of the merging vesicles because the FRET efficiency of the FRET pair depended on the molar ratio between dyes. Here, we report a robust and informative assay for SNARE-mediated fusion pore opening. - Highlights: • SNARE proteins drive membrane fusion and open a pore for cargo release. • Biotinylated GFP and DyStrp was used as the reporter pair of fusion pore opening. • Procedure for efficient SNARE reconstitution and reporter encapsulation was established. • The FRET pair reported opening of a large fusion pore bigger than 5�

Nuclear inner membrane fusion facilitated by yeast Jem1p is required for spindle pole body fusion but not for the first mitotic nuclear division during yeast mating.

PubMed

Nishikawa, Shuh-ichi; Hirata, Aiko; Endo, Toshiya

2008-11-01

During mating of budding yeast, Saccharomyces cerevisiae, two haploid nuclei fuse to produce a diploid nucleus. The process of nuclear fusion requires two J proteins, Jem1p in the endoplasmic reticulum (ER) lumen and Sec63p, which forms a complex with Sec71p and Sec72p, in the ER membrane. Zygotes of mutants defective in the functions of Jem1p or Sec63p contain two haploid nuclei that were closely apposed but failed to fuse. Here we analyzed the ultrastructure of nuclei in jem1 Delta and sec71 Delta mutant zygotes using electron microscope with the freeze-substituted fixation method. Three-dimensional reconstitution of nuclear structures from electron microscope serial sections revealed that Jem1p facilitates nuclear inner-membrane fusion and spindle pole body (SPB) fusion while Sec71p facilitates nuclear outer-membrane fusion. Two haploid SPBs that failed to fuse could duplicate, and mitotic nuclear division of the unfused haploid nuclei started in jem1 Delta and sec71 Delta mutant zygotes. This observation suggests that nuclear inner-membrane fusion is required for SPB fusion, but not for SPB duplication in the first mitotic cell division.

Sculpting and fusing biomimetic vesicle networks using optical tweezers.

PubMed

Bolognesi, Guido; Friddin, Mark S; Salehi-Reyhani, Ali; Barlow, Nathan E; Brooks, Nicholas J; Ces, Oscar; Elani, Yuval

2018-05-14

Constructing higher-order vesicle assemblies has discipline-spanning potential from responsive soft-matter materials to artificial cell networks in synthetic biology. This potential is ultimately derived from the ability to compartmentalise and order chemical species in space. To unlock such applications, spatial organisation of vesicles in relation to one another must be controlled, and techniques to deliver cargo to compartments developed. Herein, we use optical tweezers to assemble, reconfigure and dismantle networks of cell-sized vesicles that, in different experimental scenarios, we engineer to exhibit several interesting properties. Vesicles are connected through double-bilayer junctions formed via electrostatically controlled adhesion. Chemically distinct vesicles are linked across length scales, from several nanometres to hundreds of micrometres, by axon-like tethers. In the former regime, patterning membranes with proteins and nanoparticles facilitates material exchange between compartments and enables laser-triggered vesicle merging. This allows us to mix and dilute content, and to initiate protein expression by delivering biomolecular reaction components.

Selective Metal-Ion-Mediated Vesicle Adhesion Based on Dynamic Self-Organization of a Pyrene-Appended Glutamic Acid.

PubMed

Xing, Pengyao; Wang, Yajie; Yang, Minmin; Zhang, Yimeng; Wang, Bo; Hao, Aiyou

2016-07-13

Vesicles with dynamic membranes provide an ideal model system for investigating biological membrane activities, whereby vesicle aggregation behaviors including adhesion, fusion, fission, and membrane contraction/extension have attracted much attention. In this work we utilize an aromatic amino acid (pyrene-appended glutamic acid, PGlu) to prepare nanovesicles that aggregate to form vesicle clusters selectively induced by Fe(3+) or Cu(2+), and the vesicles transform into irregular nano-objects when interacting with Al(3+). Vesicle clusters have better stability than pristine vesicles, which hinders the spontaneous morphological transformation from vesicles into lamellar nanosheets with long incubation period. The difference between complexation of Fe(3+) and Al(3+) with vesicles was studied by various techniques. On the basis of metal ion-vesicle interactions, this self-assembled nanovesicle system also behaves as an effective fluorescent sensor for Fe(3+) and Al(3+), which cause fluorescence quenching and enhanced excimer emission, respectively.

Synaptic vesicle pool‐specific modification of neurotransmitter release by intravesicular free radical generation

PubMed Central

Afuwape, Olusoji A. T.; Wasser, Catherine R.; Schikorski, Thomas

2016-01-01

Key points Synaptic transmission is mediated by the release of neurotransmitters from synaptic vesicles in response to stimulation or through the spontaneous fusion of a synaptic vesicle with the presynaptic plasma membrane.There is growing evidence that synaptic vesicles undergoing spontaneous fusion versus those fusing in response to stimuli are functionally distinct.In this study, we acutely probe the effects of intravesicular free radical generation on synaptic vesicles that fuse spontaneously or in response to stimuli.By targeting vesicles that preferentially release spontaneously, we can dissociate the effects of intravesicular free radical generation on spontaneous neurotransmission from evoked neurotransmission and vice versa.Taken together, these results further advance our knowledge of the synapse and the nature of the different synaptic vesicle pools mediating neurotransmission. Abstract Earlier studies suggest that spontaneous and evoked neurotransmitter release processes are maintained by synaptic vesicles which are segregated into functionally distinct pools. However, direct interrogation of the link between this putative synaptic vesicle pool heterogeneity and neurotransmission has been difficult. To examine this link, we tagged vesicles with horseradish peroxidase (HRP) – a haem‐containing plant enzyme – or antibodies against synaptotagmin‐1 (syt1). Filling recycling vesicles in hippocampal neurons with HRP and subsequent treatment with hydrogen peroxide (H2O2) modified the properties of neurotransmitter release depending on the route of HRP uptake. While strong depolarization‐induced uptake of HRP suppressed evoked release and augmented spontaneous release, HRP uptake during mild activity selectively impaired evoked release, whereas HRP uptake at rest solely potentiated spontaneous release. Expression of a luminal HRP‐tagged syt1 construct and subsequent H2O2 application resulted in a similar increase in spontaneous release and

Vesicle Motion during Sustained Exocytosis in Chromaffin Cells: Numerical Model Based on Amperometric Measurements

PubMed Central

Jarukanont, Daungruthai; Bonifas Arredondo, Imelda; Femat, Ricardo; Garcia, Martin E.

2015-01-01

Chromaffin cells release catecholamines by exocytosis, a process that includes vesicle docking, priming and fusion. Although all these steps have been intensively studied, some aspects of their mechanisms, particularly those regarding vesicle transport to the active sites situated at the membrane, are still unclear. In this work, we show that it is possible to extract information on vesicle motion in Chromaffin cells from the combination of Langevin simulations and amperometric measurements. We developed a numerical model based on Langevin simulations of vesicle motion towards the cell membrane and on the statistical analysis of vesicle arrival times. We also performed amperometric experiments in bovine-adrenal Chromaffin cells under Ba2+ stimulation to capture neurotransmitter releases during sustained exocytosis. In the sustained phase, each amperometric peak can be related to a single release from a new vesicle arriving at the active site. The amperometric signal can then be mapped into a spike-series of release events. We normalized the spike-series resulting from the current peaks using a time-rescaling transformation, thus making signals coming from different cells comparable. We discuss why the obtained spike-series may contain information about the motion of all vesicles leading to release of catecholamines. We show that the release statistics in our experiments considerably deviate from Poisson processes. Moreover, the interspike-time probability is reasonably well described by two-parameter gamma distributions. In order to interpret this result we computed the vesicles’ arrival statistics from our Langevin simulations. As expected, assuming purely diffusive vesicle motion we obtain Poisson statistics. However, if we assume that all vesicles are guided toward the membrane by an attractive harmonic potential, simulations also lead to gamma distributions of the interspike-time probability, in remarkably good agreement with experiment. We also show that

Single-molecule studies of the neuronal SNARE fusion machinery.

PubMed

Brunger, Axel T; Weninger, Keith; Bowen, Mark; Chu, Steven

2009-01-01

SNAREs are essential components of the machinery for Ca(2+)-triggered fusion of synaptic vesicles with the plasma membrane, resulting in neurotransmitter release into the synaptic cleft. Although much is known about their biophysical and structural properties and their interactions with accessory proteins such as the Ca(2+) sensor synaptotagmin, their precise role in membrane fusion remains an enigma. Ensemble studies of liposomes with reconstituted SNAREs have demonstrated that SNAREs and accessory proteins can trigger lipid mixing/fusion, but the inability to study individual fusion events has precluded molecular insights into the fusion process. Thus, this field is ripe for studies with single-molecule methodology. In this review, we discuss applications of single-molecule approaches to observe reconstituted SNAREs, their complexes, associated proteins, and their effect on biological membranes. Some of the findings are provocative, such as the possibility of parallel and antiparallel SNARE complexes or of vesicle docking with only syntaxin and synaptobrevin, but have been confirmed by other experiments.

Nucleation theory with delayed interactions: An application to the early stages of the receptor-mediated adhesion/fusion kinetics of lipid vesicles

NASA Astrophysics Data System (ADS)

Raudino, Antonio; Pannuzzo, Martina

2010-01-01

A semiquantitative theory aimed to describe the adhesion kinetics between soft objects, such as living cells or vesicles, has been developed. When rigid bodies are considered, the adhesion kinetics is successfully described by the classical Derjaguin, Landau, Verwey, and Overbeek (DLVO) picture, where the energy profile of two approaching bodies is given by a two asymmetrical potential wells separated by a barrier. The transition probability from the long-distance to the short-distance minimum defines the adhesion rate. Conversely, soft bodies might follow a different pathway to reach the short-distance minimum: thermally excited fluctuations give rise to local protrusions connecting the approaching bodies. These transient adhesion sites are stabilized by short-range adhesion forces (e.g., ligand-receptor interactions between membranes brought at contact distance), while they are destabilized both by repulsive forces and by the elastic deformation energy. Above a critical area of the contact site, the adhesion forces prevail: the contact site grows in size until the complete adhesion of the two bodies inside a short-distance minimum is attained. This nucleation mechanism has been developed in the framework of a nonequilibrium Fokker-Planck picture by considering both the adhesive patch growth and dissolution processes. In addition, we also investigated the effect of the ligand-receptor pairing kinetics at the adhesion site in the time course of the patch expansion. The ratio between the ligand-receptor pairing kinetics and the expansion rate of the adhesion site is of paramount relevance in determining the overall nucleation rate. The theory enables one to self-consistently include both thermodynamics (energy barrier height) and dynamic (viscosity) parameters, giving rise in some limiting cases to simple analytical formulas. The model could be employed to rationalize fusion kinetics between vesicles, provided the short-range adhesion transition is the rate

Nucleation theory with delayed interactions: an application to the early stages of the receptor-mediated adhesion/fusion kinetics of lipid vesicles.

PubMed

Raudino, Antonio; Pannuzzo, Martina

2010-01-28

A semiquantitative theory aimed to describe the adhesion kinetics between soft objects, such as living cells or vesicles, has been developed. When rigid bodies are considered, the adhesion kinetics is successfully described by the classical Derjaguin, Landau, Verwey, and Overbeek (DLVO) picture, where the energy profile of two approaching bodies is given by a two asymmetrical potential wells separated by a barrier. The transition probability from the long-distance to the short-distance minimum defines the adhesion rate. Conversely, soft bodies might follow a different pathway to reach the short-distance minimum: thermally excited fluctuations give rise to local protrusions connecting the approaching bodies. These transient adhesion sites are stabilized by short-range adhesion forces (e.g., ligand-receptor interactions between membranes brought at contact distance), while they are destabilized both by repulsive forces and by the elastic deformation energy. Above a critical area of the contact site, the adhesion forces prevail: the contact site grows in size until the complete adhesion of the two bodies inside a short-distance minimum is attained. This nucleation mechanism has been developed in the framework of a nonequilibrium Fokker-Planck picture by considering both the adhesive patch growth and dissolution processes. In addition, we also investigated the effect of the ligand-receptor pairing kinetics at the adhesion site in the time course of the patch expansion. The ratio between the ligand-receptor pairing kinetics and the expansion rate of the adhesion site is of paramount relevance in determining the overall nucleation rate. The theory enables one to self-consistently include both thermodynamics (energy barrier height) and dynamic (viscosity) parameters, giving rise in some limiting cases to simple analytical formulas. The model could be employed to rationalize fusion kinetics between vesicles, provided the short-range adhesion transition is the rate

DNA-mediated self-assembly of artificial vesicles.

PubMed

Hadorn, Maik; Eggenberger Hotz, Peter

2010-03-26

Although multicompartment systems made of single unilamellar vesicles offer the potential to outperform single compartment systems widely used in analytic, synthetic, and medical applications, their use has remained marginal to date. On the one hand, this can be attributed to the binary character of the majority of the current tethering protocols that impedes the implementation of real multicomponent or multifunctional systems. On the other hand, the few tethering protocols theoretically providing multicompartment systems composed of several distinct vesicle populations suffer from the readjustment of the vesicle formation procedure as well as from the loss of specificity of the linking mechanism over time. In previous studies, we presented implementations of multicompartment systems and resolved the readjustment of the vesicle formation procedure as well as the loss of specificity by using linkers consisting of biotinylated DNA single strands that were anchored to phospholipid-grafted biotinylated PEG tethers via streptavidin as a connector. The systematic analysis presented herein provides evidences for the incorporation of phospholipid-grafted biotinylated PEG tethers to the vesicle membrane during vesicle formation, providing specific anchoring sites for the streptavidin loading of the vesicle membrane. Furthermore, DNA-mediated vesicle-vesicle self-assembly was found to be sequence-dependent and to depend on the presence of monovalent salts. This study provides a solid basis for the implementation of multi-vesicle assemblies that may affect at least three distinct domains. (i) Analysis. Starting with a minimal system, the complexity of a bottom-up system is increased gradually facilitating the understanding of the components and their interaction. (ii) Synthesis. Consecutive reactions may be implemented in networks of vesicles that outperform current single compartment bioreactors in versatility and productivity. (iii) Personalized medicine. Transport and

Kinetics of DNA-mediated docking reactions between vesicles tethered to supported lipid bilayers

PubMed Central

Chan, Yee-Hung M.; Lenz, Peter; Boxer, Steven G.

2007-01-01

Membrane–membrane recognition and binding are crucial in many biological processes. We report an approach to studying the dynamics of such reactions by using DNA-tethered vesicles as a general scaffold for displaying membrane components. This system was used to characterize the docking reaction between two populations of tethered vesicles that display complementary DNA. Deposition of vesicles onto a supported lipid bilayer was performed by using a microfluidic device to prevent mixing of the vesicles in bulk during sample preparation. Once tethered onto the surface, vesicles mixed via two-dimensional diffusion. DNA-mediated docking of two reacting vesicles results in their colocalization after collision and their subsequent tandem motion. Individual docking events and population kinetics were observed via epifluorescence microscopy. A lattice-diffusion simulation was implemented to extract from experimental data the probability, Pdock, that a collision leads to docking. For individual vesicles displaying small numbers of docking DNA, Pdock shows a first-order relationship with copy number as well as a strong dependence on the DNA sequence. Both trends are explained by a model that includes both tethered vesicle diffusion on the supported bilayer and docking DNA diffusion over each vesicle's surface. These results provide the basis for the application of tethered vesicles to study other membrane reactions including protein-mediated docking and fusion. PMID:18025472

The role of blood cell membrane lipids on the mode of action of HIV-1 fusion inhibitor sifuvirtide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matos, Pedro M.; Freitas, Teresa; Castanho, Miguel A.R.B.

2010-12-17

Research highlights: {yields} Sifuvirtide interacts with erythrocyte and lymphocyte membrane in a concentration dependent manner by decreasing its dipole potential. {yields} Dipole potential variations in lipid vesicles show sifuvirtide's lipid selectivity towards saturated phosphatidylcholines. {yields} This peptide-membrane interaction may direct the drug towards raft-like membrane domains where the receptors used by HIV are located, facilitating its inhibitory action. -- Abstract: Sifuvirtide is a gp41 based peptide that inhibits HIV-1 fusion with the host cells and is currently under clinical trials. Previous studies showed that sifuvirtide partitions preferably to saturated phosphatidylcholine lipid membranes, instead of fluid-phase lipid vesicles. We extended themore » study to the interaction of the peptide with circulating blood cells, by using the dipole potential sensitive probe di-8-ANEPPS. Sifuvirtide decreased the dipole potential of erythrocyte and lymphocyte membranes in a concentration dependent manner, demonstrating its interaction. Also, the lipid selectivity of the peptide towards more rigid phosphatidylcholines was confirmed based on the dipole potential variations. Overall, the interaction of the peptide with the cell membranes is a contribution of different lipid preferences that presumably directs the peptide towards raft-like domains where the receptors are located, facilitating the reach of the peptide to its molecular target, the gp41 in its pre-fusion conformation.« less

Cholesterol-dependent balance between evoked and spontaneous synaptic vesicle recycling

PubMed Central

Wasser, Catherine R; Ertunc, Mert; Liu, Xinran; Kavalali, Ege T

2007-01-01

Cholesterol is a prominent component of nerve terminals. To examine cholesterol's role in central neurotransmission, we treated hippocampal cultures with methyl-β-cyclodextrin, which reversibly binds cholesterol, or mevastatin, an inhibitor of cholesterol biosynthesis, to deplete cholesterol. We also used hippocampal cultures from Niemann-Pick type C1-deficient mice defective in intracellular cholesterol trafficking. These conditions revealed an augmentation in spontaneous neurotransmission detected electrically and an increase in spontaneous vesicle endocytosis judged by horseradish peroxidase uptake after cholesterol depletion by methyl-β-cyclodextrin. In contrast, responses evoked by action potentials and hypertonicity were severely impaired after the same treatments. The increase in spontaneous vesicle recycling and the decrease in evoked neurotransmission were reversible upon cholesterol addition. Cholesterol removal did not impact on the low level of evoked neurotransmission seen in the absence of synaptic vesicle SNARE protein synaptobrevin-2 whereas the increase in spontaneous fusion remained. These results suggest that synaptic cholesterol balances evoked and spontaneous neurotransmission by hindering spontaneous synaptic vesicle turnover and sustaining evoked exo-endocytosis. PMID:17170046

Thin film drainage between pre-inflated capsules or vesicles

NASA Astrophysics Data System (ADS)

Keh, Martin; Walter, Johann; Leal, Gary

2013-11-01

Capsules and vesicles are often used as vehicles to carry active ingredients or fragrance in drug delivery and consumer products and oftentimes in these applications the particles may be pre-inflated due to the existence of a small osmotic pressure difference between the interior and exterior fluid. We study the dynamics of thin film drainage between capsules and vesicles in flow as it is crucial to fusion and deposition of the particles and, therefore, the stability and effectiveness of the products. Simulations are conducted using a numerical model coupling the boundary integral method for the motion of the fluids and a finite element method for the membrane mechanics. For low capillary numbers, the drainage behavior of vesicles and capsules are approximately the same, and also similar to that of drops as the flow-independent and uniform tension due to pre-inflation dominates. The tension due to deformation caused by flow will become more important as the strength of the external flow (i.e. the capillary number) increases. In this case, the shapes of the thin film region are fundamentally different for capsules and vesicles, and the drainage behavior in both cases differs from a drop. Funded by P&G.

Morphological changes in vesicles and release of an encapsulated compound triggered by a photoresponsive Malachite Green leuconitrile derivative.

PubMed

Uda, Ryoko M; Hiraishi, Eri; Ohnishi, Ryo; Nakahara, Yoshio; Kimura, Keiichi

2010-04-20

Photoinduced morphological changes in phosphatidylcholine vesicles are triggered by a Malachite Green leuconitrile derivative dissolved in the lipidic membrane, and are observed at Malachite Green derivative/lipid ratios <5 mol %. This Malachite Green derivative is a photoresponsive compound that undergoes ionization to afford a positive charge on the molecule by UV irradiation. The Malachite Green derivative exhibits amphiphilicity when ionized photochemically, whereas it behaves as a lipophilic compound under dark conditions. Cryo-transmission electron microscopy was used to determine vesicle morphology. The effects of the Malachite Green derivative on vesicles were studied by dynamic light scattering and fluorescence resonance energy transfer. Irradiation of vesicles containing the Malachite Green derivative induces nonspherical vesicle morphology, fusion of vesicles, and membrane solubilization, depending on conditions. Furthermore, irradiation of the Malachite Green derivative induces the release of a vesicle-encapsulated compound.

Surgical planning and manual image fusion based on 3D model facilitate laparoscopic partial nephrectomy for intrarenal tumors.

PubMed

Chen, Yuanbo; Li, Hulin; Wu, Dingtao; Bi, Keming; Liu, Chunxiao

2014-12-01

Construction of three-dimensional (3D) model of renal tumor facilitated surgical planning and imaging guidance of manual image fusion in laparoscopic partial nephrectomy (LPN) for intrarenal tumors. Fifteen patients with intrarenal tumors underwent LPN between January and December 2012. Computed tomography-based reconstruction of the 3D models of renal tumors was performed using Mimics 12.1 software. Surgical planning was performed through morphometry and multi-angle visual views of the tumor model. Two-step manual image fusion superimposed 3D model images onto 2D laparoscopic images. The image fusion was verified by intraoperative ultrasound. Imaging-guided laparoscopic hilar clamping and tumor excision was performed. Manual fusion time, patient demographics, surgical details, and postoperative treatment parameters were analyzed. The reconstructed 3D tumor models accurately represented the patient's physiological anatomical landmarks. The surgical planning markers were marked successfully. Manual image fusion was flexible and feasible with fusion time of 6 min (5-7 min). All surgeries were completed laparoscopically. The median tumor excision time was 5.4 min (3.5-10 min), whereas the median warm ischemia time was 25.5 min (16-32 min). Twelve patients (80 %) demonstrated renal cell carcinoma on final pathology, and all surgical margins were negative. No tumor recurrence was detected after a media follow-up of 1 year (3-15 months). The surgical planning and two-step manual image fusion based on 3D model of renal tumor facilitated visible-imaging-guided tumor resection with negative margin in LPN for intrarenal tumor. It is promising and moves us one step closer to imaging-guided surgery.

Endothelial microparticles: Sophisticated vesicles modulating vascular function

PubMed Central

Curtis, Anne M; Edelberg, Jay; Jonas, Rebecca; Rogers, Wade T; Moore, Jonni S; Syed, Wajihuddin; Mohler, Emile R

2015-01-01

Endothelial microparticles (EMPs) belong to a family of extracellular vesicles that are dynamic, mobile, biological effectors capable of mediating vascular physiology and function. The release of EMPs can impart autocrine and paracrine effects on target cells through surface interaction, cellular fusion, and, possibly, the delivery of intra-vesicular cargo. A greater understanding of the formation, composition, and function of EMPs will broaden our understanding of endothelial communication and may expose new pathways amenable for therapeutic manipulation. PMID:23892447

Nanoparticle orientation to control RNA loading and ligand display on extracellular vesicles for cancer regression

NASA Astrophysics Data System (ADS)

Pi, Fengmei; Binzel, Daniel W.; Lee, Tae Jin; Li, Zhefeng; Sun, Meiyan; Rychahou, Piotr; Li, Hui; Haque, Farzin; Wang, Shaoying; Croce, Carlo M.; Guo, Bin; Evers, B. Mark; Guo, Peixuan

2018-01-01

Nanotechnology offers many benefits, and here we report an advantage of applying RNA nanotechnology for directional control. The orientation of arrow-shaped RNA was altered to control ligand display on extracellular vesicle membranes for specific cell targeting, or to regulate intracellular trafficking of small interfering RNA (siRNA) or microRNA (miRNA). Placing membrane-anchoring cholesterol at the tail of the arrow results in display of RNA aptamer or folate on the outer surface of the extracellular vesicle. In contrast, placing the cholesterol at the arrowhead results in partial loading of RNA nanoparticles into the extracellular vesicles. Taking advantage of the RNA ligand for specific targeting and extracellular vesicles for efficient membrane fusion, the resulting ligand-displaying extracellular vesicles were capable of specific delivery of siRNA to cells, and efficiently blocked tumour growth in three cancer models. Extracellular vesicles displaying an aptamer that binds to prostate-specific membrane antigen, and loaded with survivin siRNA, inhibited prostate cancer xenograft. The same extracellular vesicle instead displaying epidermal growth-factor receptor aptamer inhibited orthotopic breast cancer models. Likewise, survivin siRNA-loaded and folate-displaying extracellular vesicles inhibited patient-derived colorectal cancer xenograft.

Rapid synaptic vesicle endocytosis in cone photoreceptors of salamander retina

PubMed Central

Van Hook, Matthew J.; Thoreson, Wallace B.

2013-01-01

Following synaptic vesicle exocytosis, neurons retrieve the fused membrane by a process of endocytosis in order to provide a supply of vesicles for subsequent release and maintain the presynaptic active zone. Rod and cone photoreceptors use a specialized structure called the synaptic ribbon that enables them to sustain high rates of neurotransmitter release. They must also employ mechanisms of synaptic vesicle endocytosis capable of keeping up with release. While much is known about endocytosis at another retinal ribbon synapse, that of the goldfish Mb1 bipolar cell, less is known about endocytosis in photoreceptors. We used capacitance recording techniques to measure vesicle membrane fusion and retrieval in photoreceptors from salamander retinal slices. We found that application of brief depolarizing steps (<100 ms) to cones evoked exocytosis followed by rapid endocytosis with a time constant ~250 ms. In some cases, the capacitance trace overshot the baseline, indicating excess endocytosis. Calcium had no effect on the time constant, but enhanced excess endocytosis resulting in a faster rate of membrane retrieval. Surprisingly, endocytosis was unaffected by blockers of dynamin, suggesting that cone endocytosis is dynamin-independent. This contrasts with synaptic vesicle endocytosis in rods, which was inhibited by the dynamin inhibitor dynasore and GTPγS introduced through the patch pipette, suggesting that the two photoreceptor types employ distinct pathways for vesicle retrieval. The fast kinetics of synaptic vesicle endocytosis in photoreceptors likely enables these cells to maintain a high rate of transmitter release, allowing them to faithfully signal changes in illumination to second-order neurons. PMID:23238726

Heterotypic endosomal fusion as an initial trigger for insulin-induced glucose transporter 4 (GLUT4) translocation in skeletal muscle.

PubMed

Hatakeyama, Hiroyasu; Kanzaki, Makoto

2017-08-15

Comprehensive imaging analyses of glucose transporter 4 (GLUT4) behaviour in mouse skeletal muscle was conducted. Quantum dot-based single molecule nanometry revealed that GLUT4 molecules in skeletal myofibres are governed by regulatory systems involving 'static retention' and 'stimulus-dependent liberation'. Vital imaging analyses and super-resolution microscopy-based morphometry demonstrated that insulin liberates the GLUT4 molecule from its static state by triggering acute heterotypic endomembrane fusion arising from the very small GLUT4-containing vesicles in skeletal myofibres. Prior exposure to exercise-mimetic stimuli potentiated this insulin-responsive endomembrane fusion event involving GLUT4-containing vesicles, suggesting that this endomembranous regulation process is a potential site related to the effects of exercise. Skeletal muscle is the major systemic glucose disposal site. Both insulin and exercise facilitate translocation of the glucose transporter glucose transporter 4 (GLUT4) via distinct signalling pathways and exercise also enhances insulin sensitivity. However, the trafficking mechanisms controlling GLUT4 mobilization in skeletal muscle remain poorly understood as a resuly of technical limitations. In the present study, which employs various imaging techniques on isolated skeletal myofibres, we show that one of the initial triggers of insulin-induced GLUT4 translocation is heterotypic endomembrane fusion arising from very small static GLUT4-containing vesicles with a subset of transferrin receptor-containing endosomes. Importantly, pretreatment with exercise-mimetic stimuli potentiated the susceptibility to insulin responsiveness, as indicated by these acute endomembranous activities. We also found that AS160 exhibited stripe-like localization close to sarcomeric α-actinin and that insulin induced a reduction of the stripe-like localization accompanying changes in its detergent solubility. The results of the present study thus provide a

Commercial cow milk contains physically stable extracellular vesicles expressing immunoregulatory TGF-β.

PubMed

Pieters, Bartijn C H; Arntz, Onno J; Bennink, Miranda B; Broeren, Mathijs G A; van Caam, Arjan P M; Koenders, Marije I; van Lent, Peter L E M; van den Berg, Wim B; de Vries, Marieke; van der Kraan, Peter M; van de Loo, Fons A J

2015-01-01

Extracellular vesicles, including exosomes, have been identified in all biological fluids and rediscovered as an important part of the intercellular communication. Breast milk also contains extracellular vesicles and the proposed biological function is to enhance the antimicrobial defense in newborns. It is, however, unknown whether extracellular vesicles are still present in commercial milk and, more importantly, whether they retained their bioactivity. Here, we characterize the extracellular vesicles present in semi-skimmed cow milk available for consumers and study their effect on T cells. Extracellular vesicles from commercial milk were isolated and characterized. Milk-derived extracellular vesicles contained several immunomodulating miRNAs and membrane protein CD63, characteristics of exosomes. In contrast to RAW 267.4 derived extracellular vesicles the milk-derived extracellular vesicles were extremely stable under degrading conditions, including low pH, boiling and freezing. Milk-derived extracellular vesicles were easily taken up by murine macrophages in vitro. Furthermore, we found that they can facilitate T cell differentiation towards the pathogenic Th17 lineage. Using a (CAGA)12-luc reporter assay we showed that these extracellular vesicles carried bioactive TGF-β, and that anti-TGF-β antibodies blocked Th17 differentiation. Our findings show that commercial milk contains stable extracellular vesicles, including exosomes, and carry immunoregulatory cargo. These data suggest that the extracellular vesicles present in commercial cow milk remains intact in the gastrointestinal tract and exert an immunoregulatory effect.

A Monte Carlo Simulation of Vesicle Exocytosis in the Buffered Diffusion of Calcium Channel Currents

NASA Astrophysics Data System (ADS)

Dimcovic, Z.; Eagan, T. P.; Brown, R. W.; Petschek, R. G.; Eppell, S. J.; Yunker, A. M. R.; Sharp, A. H.; McEnery, M. W.

2001-04-01

The voltage-dependent opening of calcium channels results in an influx of calcium ions that leads to the fusion of synaptic vesicles with the cell membrane, resulting in the release of neurotransmitters. This allows nerve impulses to be transmitted from one neuron to another. A Monte Carlo model of the three-dimensional diffusion of calcium following a channel opening is employed to estimate the space and time dependence of the calcium density. The effects of fixed and mobile calcium buffers are included, and a tethered nearby vesicle is considered. The importance of the size and location of the vesicle is studied. When the vesicle is ignored, these results are compared with the analytical calculations of Naraghi and Neher and the Monte Carlo calculations of Bennett et al. The finite-vesicle-size analysis offers new insights into the process of neurosecretion. Support: NIH MH55747, AHA 96001250, NSF 0086643, and CWRU Presidential Research Initiative grants.

Splaying of aliphatic tails plays a central role in barrier crossing during liposome fusion.

PubMed

Mirjanian, Dina; Dickey, Allison N; Hoh, Jan H; Woolf, Thomas B; Stevens, Mark J

2010-09-02

The fusion between two lipid bilayers involves crossing a complicated energy landscape. The limiting barrier in the process appears to be between two closely opposed bilayers and the intermediate state where the outer leaflets are fused. We have performed molecular dynamics simulations to characterize the free energy barrier for the fusion of two liposomes and to examine the molecular details of barrier crossing. To capture the slow dynamics of fusion, a model using coarse-grained representations of lipids was used. The fusion between pairs of liposomes was simulated for four systems: DPPC, DOPC, a 3:1 mixture of DPPC/DPPE, and an asymmetric lipid tail system in which one tail of DPPC was reduced to half the length (ASTail). The weighted histogram method was used to compute the free energy as a function of separation distance. The relative barrier heights for these systems was found to be ASTail > DPPC > DPPC/DPPE > DOPC, in agreement with experimental observations. Further, the free energy curves for all four can be overlaid on a single curve by plotting the free energy versus the surface separation (differing only in the point of fusion). These simulations also confirm that the two main contributions to the free energy barrier are the removal of water between the vesicles and the deformation of the vesicle. The most prominent molecular detail of barrier crossing in all cases examined was the splaying of lipid tails, where initially a single splayed lipid formed a bridge between the two outer leaflets that promotes additional lipid mixing between the vesicles and eventually leads to fusion. The tail splay appears to be closely connected to the energetics of the process. For example, the high barrier for the ASTail is the result of a smaller distance between terminal methyl groups in the splayed molecule. The shortening of this distance requires the liposomes to be closer together, which significantly increases the cost of water removal and bilayer deformation

Shrink-wrap Vesicles

PubMed Central

Fujikawa, Shelly M.; Chen, Irene A.; Szostak, Jack W.

2008-01-01

We describe a simple approach to the controlled removal of molecules from the membrane of large unilamellar vesicles made of fatty acids. Such vesicles shrink dramatically upon mixing with micelles composed of a mixture of fatty acid and phospholipid (POPC), as fatty acid molecules leave the vesicle membrane and accumulate within the mixed micelles. Vesicle shrinkage was confirmed by dynamic light scattering, fluorescence recovery after photobleaching of labeled vesicles, and fluorescence resonance energy transfer between lipid dyes incorporated into the vesicle membrane. Most of the encapsulated impermeable solute is retained during shrinkage, becoming concentrated by a factor of at least 50-fold in the final small vesicles. This unprecedented combination of vesicle shrinkage with retention of contents allows for the preparation of small vesicles containing high solute concentrations, and may find applications in liposomal drug delivery. PMID:16342983

Reconstruction of mammalian oocytes by germinal vesicle transfer: A systematic review

PubMed Central

Darbandi, Sara; Darbandi, Mahsa; Khorram Khorshid, Hamid Reza; Shirazi, Abolfazl; Sadeghi, Mohammad Reza; Agarwal, Ashok; Al-Hasani, Safaa; Naderi, Mohammad Mehdi; Ayaz, Ahmet; Akhondi, Mohammad Mehdi

2017-01-01

Nuclear transfer procedures have been recently applied for clinical and research targets as a novel assisted reproductive technique and were used for increasing the oocyte activity during its growth and maturation. In this review, we summarized the nuclear transfer technique for germinal vesicle stage oocytes to reconstruct the maturation of them. Our study covered publications between 1966 and August 2017. In result utilized germinal vesicle transfer techniques, fusion, and fertilization survival rate on five different mammalian species are discussed, regarding their potential clinical application. It seems that with a study on this method, there is real hope for effective treatments of old oocytes or oocytes containing mitochondrial problems in the near future. PMID:29387825

A histological study of the seminal vesicle of the armoured catfish Corydoras aeneus.

PubMed

Franceschini-Vicentini, I B; Papa, L P; Bombonato, M T S; Vicentini, C A; Ribeiro, K; Orsi, A M

2007-04-01

Most species of Corydoras exhibited a reproductive behaviour called 'T-position', and exhibited an accessory gland in the male genital tract, called the seminal vesicle. It appeared that both the structure and the composition of the fluid varied considerably between the species investigated. Consequently, different opinions were proposed regarding the possible role of seminal vesicle on this particular reproductive behaviour. Male adults of Corydoras aeneus were collected, anaesthetized, and samples of seminal vesicle were fixed in Bouin's solution. The sections were stained with haematoxylin-eosin and periodic acid Schiff. The seminal vesicle showed a system of anastomosed secretory tubules, forming a vesicular collective network, which gave rise to the vesicular ducts. The latter fused with the testicular efferent ducts and formed the spermatic ducts. Considering this fusion, when the sperm cells reached the spermatic ducts, the fluid produced at the seminal vesicle covered them. Histochemical studies evidenced the presence of neutral and acid glycosaminoglycans in the seminal fluid. Considering the reproductive behaviour of C. aeneus, it is believed that the protection associated with the immobilization of the sperm cells assures the sperm integrity during the passage through female's intestine until fertilization.

The ancient claudin Dni2 facilitates yeast cell fusion by compartmentalizing Dni1 into a membrane subdomain.

PubMed

Curto, M-Ángeles; Moro, Sandra; Yanguas, Francisco; Gutiérrez-González, Carmen; Valdivieso, M-Henar

2018-05-01

Dni1 and Dni2 facilitate cell fusion during mating. Here, we show that these proteins are interdependent for their localization in a plasma membrane subdomain, which we have termed the mating fusion domain. Dni1 compartmentation in the domain is required for cell fusion. The contribution of actin, sterol-dependent membrane organization, and Dni2 to this compartmentation was analysed, and the results showed that Dni2 plays the most relevant role in the process. In turn, the Dni2 exit from the endoplasmic reticulum depends on Dni1. These proteins share the presence of a cysteine motif in their first extracellular loop related to the claudin GLWxxC(8-10 aa)C signature motif. Structure-function analyses show that mutating each Dni1 conserved cysteine has mild effects, and that only simultaneous elimination of several cysteines leads to a mating defect. On the contrary, eliminating each single cysteine and the C-terminal tail in Dni2 abrogates Dni1 compartmentation and cell fusion. Sequence alignments show that claudin trans-membrane helixes bear small-XXX-small motifs at conserved positions. The fourth Dni2 trans-membrane helix tends to form homo-oligomers in Escherichia plasma membrane, and two concatenated small-XXX-small motifs are required for efficient oligomerization and for Dni2 export from the yeast endoplasmic reticulum. Together, our results strongly suggest that Dni2 is an ancient claudin that blocks Dni1 diffusion from the intercellular region where two plasma membranes are in close proximity, and that this function is required for Dni1 to facilitate cell fusion.

Delivery of RNA and its intracellular translation into protein mediated by SDS-CTAB vesicles: potential use in nanobiotechnology.

PubMed

Russo, Laura; Berardi, Valerio; Tardani, Franco; La Mesa, Camillo; Risuleo, Gianfranco

2013-01-01

Catanionic vesicles are supramolecular aggregates spontaneously forming in water by electrostatic attraction between two surfactants mixed in nonstoichiometric ratios. The outer surface charges allow adsorption to the biomembrane by electrostatic interactions. The lipoplex thus obtained penetrates the cell by endocytosis or membrane fusion. We examined the possible cytotoxic effects and evaluated the transfection efficiency of one vesicle type as compared to known commercial carriers. We show that the individual components of two different vesicles types, CTAB (cetyltrimethylammonium bromide) and DDAB (didodecyldimethylammonium bromide) are detrimental for cell survival. We also assayed the cytotoxicity of SDS-DDAB vesicles and showed dose and time dependency, with the DDAB component being per se extremely cytotoxic. The transfection efficiency of exogenous RNA mediated by SDS-CTAB increases if vesicles assemble in the presence of the reporter RNA; finally, freezing abrogates the transfection ability. The results of our experimental strategy suggest that catanionic vesicles may be adopted in gene therapy and control of antiproliferative diseases.

In vitro assay using engineered yeast vacuoles for neuronal SNARE-mediated membrane fusion

PubMed Central

Ko, Young-Joon; Lee, Miriam; Kang, KyeongJin; Song, Woo Keun; Jun, Youngsoo

2014-01-01

Intracellular membrane fusion requires not only SNARE proteins but also other regulatory proteins such as the Rab and Sec1/Munc18 (SM) family proteins. Although neuronal SNARE proteins alone can drive the fusion between synthetic liposomes, it remains unclear whether they are also sufficient to induce the fusion of biological membranes. Here, through the use of engineered yeast vacuoles bearing neuronal SNARE proteins, we show that neuronal SNAREs can induce membrane fusion between yeast vacuoles and that this fusion does not require the function of the Rab protein Ypt7p or the SM family protein Vps33p, both of which are essential for normal yeast vacuole fusion. Although excess vacuolar SNARE proteins were also shown to mediate Rab-bypass fusion, this fusion required homotypic fusion and vacuole protein sorting complex, which bears Vps33p and was accompanied by extensive membrane lysis. We also show that this neuronal SNARE-driven vacuole fusion can be stimulated by the neuronal SM protein Munc18 and blocked by botulinum neurotoxin serotype E, a well-known inhibitor of synaptic vesicle fusion. Taken together, our results suggest that neuronal SNARE proteins are sufficient to induce biological membrane fusion, and that this new assay can be used as a simple and complementary method for investigating synaptic vesicle fusion mechanisms. PMID:24821814

Study of the Interaction of the HIV-1 Fusion Peptide with Lipid Bilayer Membranes

NASA Astrophysics Data System (ADS)

Heller, William; Rai, Durgesh

HIV-1 undergoes fusion with the cell membrane through interactions between its coat proteins and the target cell. Visualization of fusion with sufficient detail to determine the molecular mechanism remains elusive. Here, the interaction between a synthetic variant of the HIV-1 gp41 fusion peptide with vesicles composed of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS) was studied. The peptide was observed to undergo a concentration-dependent conformational transition between an α-helix and an antiparallel β-sheet that is accompanied by a transition in the structure of the lipid bilayer vesicle. The peptide changes the distribution of lipids between the vesicle leaflets. Further, it creates two regions having different thicknesses. The results shed new light on how the peptide modifies the membrane structure to favor fusion. A portion of this research was sponsored by the Laboratory Directed Research and Development Program of Oak Ridge National Laboratory, managed by UT-Battelle, LLC, for the U. S. Department of Energy. Research at Oak Ridge National Laboratory's Spallation Neutron Source was sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, U. S. Department of Energy.

Novel Three-Dimensional Image Fusion Software to Facilitate Guidance of Complex Cardiac Catheterization : 3D image fusion for interventions in CHD.

PubMed

Goreczny, Sebastian; Dryzek, Pawel; Morgan, Gareth J; Lukaszewski, Maciej; Moll, Jadwiga A; Moszura, Tomasz

2017-08-01

We report initial experience with novel three-dimensional (3D) image fusion software for guidance of transcatheter interventions in congenital heart disease. Developments in fusion imaging have facilitated the integration of 3D roadmaps from computed tomography or magnetic resonance imaging datasets. The latest software allows live fusion of two-dimensional (2D) fluoroscopy with pre-registered 3D roadmaps. We reviewed all cardiac catheterizations guided with this software (Philips VesselNavigator). Pre-catheterization imaging and catheterization data were collected focusing on fusion of 3D roadmap, intervention guidance, contrast and radiation exposure. From 09/2015 until 06/2016, VesselNavigator was applied in 34 patients for guidance (n = 28) or planning (n = 6) of cardiac catheterization. In all 28 patients successful 2D-3D registration was performed. Bony structures combined with the cardiovascular silhouette were used for fusion in 26 patients (93%), calcifications in 9 (32%), previously implanted devices in 8 (29%) and low-volume contrast injection in 7 patients (25%). Accurate initial 3D roadmap alignment was achieved in 25 patients (89%). Six patients (22%) required realignment during the procedure due to distortion of the anatomy after introduction of stiff equipment. Overall, VesselNavigator was applied successfully in 27 patients (96%) without any complications related to 3D image overlay. VesselNavigator was useful in guidance of nearly all of cardiac catheterizations. The combination of anatomical markers and low-volume contrast injections allowed reliable 2D-3D registration in the vast majority of patients.

Synaptotagmin C2B Domain Regulates Ca2+-triggered Fusion in Vitro

PubMed Central

Gaffaney, Jon D.; Dunning, F. Mark; Wang, Zhao; Hui, Enfu; Chapman, Edwin R.

2008-01-01

Synaptotagmin (syt) 1 is localized to synaptic vesicles, binds Ca2+, and regulates neuronal exocytosis. Syt 1 harbors two Ca2+-binding motifs referred to as C2A and C2B. In this study we examine the function of the isolated C2 domains of Syt 1 using a reconstituted, SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor)-mediated, fusion assay. We report that inclusion of phosphatidylethanolamine into reconstituted SNARE vesicles enabled isolated C2B, but not C2A, to regulate Ca2+-triggered fusion. The isolated C2B domain had a 6-fold lower EC for Ca2+ 50-activated fusion than the intact cytosolic domain of Syt 1 (C2AB). Phosphatidylethanolamine increased both the rate and efficiency of C2AB- and C2B-regulated fusion without affecting their abilities to bind membrane-embedded syntaxin-SNAP-25 (t-SNARE) complexes. At equimolar concentrations, the isolated C2A domain was an effective inhibitor of C2B-, but not C2AB-regulated fusion; hence, C2A has markedly different effects in the fusion assay depending on whether it is tethered to C2B. Finally, scanning alanine mutagenesis of C2AB revealed four distinct groups of mutations within the C2B domain that play roles in the regulation of SNARE-mediated fusion. Surprisingly, substitution of Arg-398 with alanine, which lies on the opposite end of C2B from the Ca2+/membrane-binding loops, decreases C2AB t-SNARE binding and Ca2+-triggered fusion in vitro without affecting Ca2+-triggered interactions with phosphatidylserine or vesicle aggregation. In addition, some mutations uncouple the clamping and stimulatory functions of syt 1, suggesting that these two activities are mediated by distinct structural determinants in C2B. PMID:18784080

Gas vesicles.

PubMed Central

Walsby, A E

1994-01-01

The gas vesicle is a hollow structure made of protein. It usually has the form of a cylindrical tube closed by conical end caps. Gas vesicles occur in five phyla of the Bacteria and two groups of the Archaea, but they are mostly restricted to planktonic microorganisms, in which they provide buoyancy. By regulating their relative gas vesicle content aquatic microbes are able to perform vertical migrations. In slowly growing organisms such movements are made more efficiently than by swimming with flagella. The gas vesicle is impermeable to liquid water, but it is highly permeable to gases and is normally filled with air. It is a rigid structure of low compressibility, but it collapses flat under a certain critical pressure and buoyancy is then lost. Gas vesicles in different organisms vary in width, from 45 to > 200 nm; in accordance with engineering principles the narrower ones are stronger (have higher critical pressures) than wide ones, but they contain less gas space per wall volume and are therefore less efficient at providing buoyancy. A survey of gas-vacuolate cyanobacteria reveals that there has been natural selection for gas vesicles of the maximum width permitted by the pressure encountered in the natural environment, which is mainly determined by cell turgor pressure and water depth. Gas vesicle width is genetically determined, perhaps through the amino acid sequence of one of the constituent proteins. Up to 14 genes have been implicated in gas vesicle production, but so far the products of only two have been shown to be present in the gas vesicle: GvpA makes the ribs that form the structure, and GvpC binds to the outside of the ribs and stiffens the structure against collapse. The evolution of the gas vesicle is discussed in relation to the homologies of these proteins. Images PMID:8177173

Inverted micellar intermediates and the transitions between lamellar, cubic, and inverted hexagonal lipid phases. II. Implications for membrane-membrane interactions and membrane fusion.

PubMed Central

Siegel, D P

1986-01-01

Results of a kinetic model of thermotropic L alpha----HII phase transitions are used to predict the types and order-of-magnitude rates of interactions between unilamellar vesicles that can occur by intermediates in the L alpha----HII phase transition. These interactions are: outer monolayer lipid exchange between vesicles; vesicle leakage subsequent to aggregation; and (only in systems with ratios of L alpha and HII phase structural dimensions in a certain range or with unusually large bilayer lateral compressibilities) vesicle fusion with retention of contents. It was previously proposed that inverted micellar structures mediate membrane fusion. These inverted micellar structures are thought to form in all systems with such transitions. However, I show that membrane fusion probably occurs via structures that form from these inverted micellar intermediates, and that fusion should occur in only a sub-set of lipid systems that can adopt the HII phase. For single-component phosphatidylethanolamine (PE) systems with thermotropic L alpha----HII transitions, lipid exchange should be observed starting at temperatures several degrees below TH and at all higher temperatures, where TH is the L alpha----HII transition temperature. At temperatures above TH, the HII phase forms between apposed vesicles, and eventually ruptures them (leakage). In most single-component PE systems, fusion via L alpha----HII transition intermediates should not occur. This is the behavior observed by Bentz, Ellens, Lai, Szoka, et al. in PE vesicle systems. Fusion is likely to occur under circumstances in which multilamellar samples of lipid form the so-called "inverted cubic" or "isotropic" phase. This is as observed in the mono-methyl DOPE system (Ellens, H., J. Bentz, and F. C. Szoka. 1986. Fusion of phosphatidylethanolamine containing liposomes and the mechanism of the L alpha-HII phase transition. Biochemistry. In press.) In lipid systems with L alpha----HII transitions driven by cation binding

The Influence of Vesicle Shape and Medium Conductivity on Possible Electrofusion under a Pulsed Electric Field

PubMed Central

Liu, Linying; Mao, Zheng; Zhang, Jianhua; Liu, Na; Liu, Qing Huo

2016-01-01

The effects of electric field on lipid membrane and cells have been extensively studied in the last decades. The phenomena of electroporation and electrofusion are of particular interest due to their wide use in cell biology and biotechnology. However, numerical studies on the electrofusion of cells (or vesicles) with different deformed shapes are still rare. Vesicle, being of cell size, can be treated as a simple model of cell to investigate the behaviors of cell in electric field. Based on the finite element method, we investigate the effect of vesicle shape on electrofusion of contact vesicles in various medium conditions. The transmembrane voltage (TMV) and pore density induced by a pulsed field are examined to analyze the possibility of vesicle fusion. In two different medium conditions, the prolate shape is observed to have selective electroporation at the contact area of vesicles when the exterior conductivity is smaller than the interior one; selective electroporation is more inclined to be found at the poles of the oblate vesicles when the exterior conductivity is larger than the interior one. Furthermore, we find that when the exterior conductivity is lower than the internal conductivity, the pulse can induce a selective electroporation at the contact area between two vesicles regardless of the vesicle shape. Both of these two findings have important practical applications in guiding electrofusion experiments. PMID:27391692

Passive Diffusion as a Mechanism Underlying Ribbon Synapse Vesicle Release and Resupply

PubMed Central

Graydon, Cole W.; Zhang, Jun; Oesch, Nicholas W.; Sousa, Alioscka A.; Leapman, Richard D.

2014-01-01

Synaptic ribbons are presynaptic protein structures found at many synapses that convey graded, “analog” sensory signals in the visual, auditory, and vestibular pathways. Ribbons, typically anchored to the presynaptic membrane and surrounded by tethered synaptic vesicles, are thought to regulate or facilitate vesicle delivery to the presynaptic membrane. No direct evidence exists, however, to indicate how vesicles interact with the ribbon or, once attached, move along the ribbon's surface to reach the presynaptic release sites at its base. To address these questions, we have created, validated, and tested a passive vesicle diffusion model of retinal rod bipolar cell ribbon synapses. We used axial (bright-field) electron tomography in the scanning transmission electron microscopy to obtain 3D structures of rat rod bipolar cell terminals in 1-μm-thick sections of retinal tissue at an isotropic spatial resolution of ∼3 nm. The resulting structures were then incorporated with previously published estimates of vesicle diffusion dynamics into numerical simulations that accurately reproduced electrophysiologically measured vesicle release/replenishment rates and vesicle pool sizes. The simulations suggest that, under physiologically realistic conditions, diffusion of vesicles crowded on the ribbon surface gives rise to a flow field that enhances delivery of vesicles to the presynaptic membrane without requiring an active transport mechanism. Numerical simulations of ribbon–vesicle interactions predict that transient binding and unbinding of multiple tethers to each synaptic vesicle may achieve sufficiently tight association of vesicles to the ribbon while permitting the fast diffusion along the ribbon that is required to sustain high release rates. PMID:24990916

Analysis of membrane fusion as a two-state sequential process: evaluation of the stalk model.

PubMed

Weinreb, Gabriel; Lentz, Barry R

2007-06-01

We propose a model that accounts for the time courses of PEG-induced fusion of membrane vesicles of varying lipid compositions and sizes. The model assumes that fusion proceeds from an initial, aggregated vesicle state ((A) membrane contact) through two sequential intermediate states (I(1) and I(2)) and then on to a fusion pore state (FP). Using this model, we interpreted data on the fusion of seven different vesicle systems. We found that the initial aggregated state involved no lipid or content mixing but did produce leakage. The final state (FP) was not leaky. Lipid mixing normally dominated the first intermediate state (I(1)), but content mixing signal was also observed in this state for most systems. The second intermediate state (I(2)) exhibited both lipid and content mixing signals and leakage, and was sometimes the only leaky state. In some systems, the first and second intermediates were indistinguishable and converted directly to the FP state. Having also tested a parallel, two-intermediate model subject to different assumptions about the nature of the intermediates, we conclude that a sequential, two-intermediate model is the simplest model sufficient to describe PEG-mediated fusion in all vesicle systems studied. We conclude as well that a fusion intermediate "state" should not be thought of as a fixed structure (e.g., "stalk" or "transmembrane contact") of uniform properties. Rather, a fusion "state" describes an ensemble of similar structures that can have different mechanical properties. Thus, a "state" can have varying probabilities of having a given functional property such as content mixing, lipid mixing, or leakage. Our data show that the content mixing signal may occur through two processes, one correlated and one not correlated with leakage. Finally, we consider the implications of our results in terms of the "modified stalk" hypothesis for the mechanism of lipid pore formation. We conclude that our results not only support this hypothesis but

Munc13 homology domain-1 in CAPS/UNC31 mediates SNARE binding required for priming vesicle exocytosis.

PubMed

Khodthong, Chuenchanok; Kabachinski, Greg; James, Declan J; Martin, Thomas F J

2011-08-03

Neuropeptide and peptide hormone secretion from neural and endocrine cells occurs by Ca(2+)-triggered dense-core vesicle exocytosis. The membrane fusion machinery consisting of vesicle and plasma membrane SNARE proteins needs to be assembled for Ca(2+)-triggered vesicle exocytosis. The related Munc13 and CAPS/UNC31 proteins that prime vesicle exocytosis are proposed to promote SNARE complex assembly. CAPS binds SNARE proteins and stimulates SNARE complex formation on liposomes, but the relevance of SNARE binding to CAPS function in cells had not been determined. Here we identify a core SNARE-binding domain in CAPS as corresponding to Munc13 homology domain-1 (MHD1). CAPS lacking a single helix in MHD1 was unable to bind SNARE proteins or to support the Ca(2+)-triggered exocytosis of either docked or newly arrived dense-core vesicles. The results show that MHD1 is a SNARE-binding domain and that SNARE protein binding is essential for CAPS function in dense-core vesicle exocytosis. Copyright © 2011 Elsevier Inc. All rights reserved.

Fusion of single proteoliposomes with planar, cushioned bilayers in microfluidic flow cells

PubMed Central

Karatekin, Erdem; Rothman, James E.

2013-01-01

Many biological processes rely on membrane fusion, therefore assays to study its mechanisms are necessary. Here we report an assay with sensitivity to single-vesicle, even to single-molecule events using fluorescently labeled vesicle-associated v-SNARE liposomes and target-membrane-associated t-SNARE-reconstituted planar, supported bilayers (SBLs). Docking and fusion events can be detected using conventional far-field epifluorescence or total internal reflection fluorsecence microscopy. Unlike most previous attempts, fusion here is dependent on SNAP25, one of the t-SNARE subunits that is required for fusion in vivo. The success of the assay is due to the use of (i) bilayers covered with a thin layer of poly(ethylene glycol) to control bilayer-bilayer and bilayer-substrate interactions, (ii) microfluidic flow channels which presents many advantages such as the removal of non-specifically bound liposomes by flow. The protocol takes 6–8 days to complete. Analysis can take up to two weeks. PMID:22517259

Differential Regulation of Synaptic Vesicle Tethering and Docking by UNC-18 and TOM-1.

PubMed

Gracheva, Elena O; Maryon, Ed B; Berthelot-Grosjean, Martine; Richmond, Janet E

2010-01-01

The assembly of SNARE complexes between syntaxin, SNAP-25 and synaptobrevin is required to prime synaptic vesicles for fusion. Since Munc18 and tomosyn compete for syntaxin interactions, the interplay between these proteins is predicted to be important in regulating synaptic transmission. We explored this possibility, by examining genetic interactions between C. elegans unc-18(Munc18), unc-64(syntaxin) and tom-1(tomosyn). We have previously demonstrated that unc-18 mutants have reduced synaptic transmission, whereas tom-1 mutants exhibit enhanced release. Here we show that the unc-18 mutant release defect is associated with loss of two morphologically distinct vesicle pools; those tethered within 25 nm of the plasma membrane and those docked with the plasma membrane. In contrast, priming defective unc-13 mutants accumulate tethered vesicles, while docked vesicles are greatly reduced, indicating tethering is UNC-18-dependent and occurs in the absence of priming. C. elegans unc-64 mutants phenocopy unc-18 mutants, losing both tethered and docked vesicles, whereas overexpression of open syntaxin preferentially increases vesicle docking, suggesting UNC-18/closed syntaxin interactions are responsible for vesicle tethering. Given the competition between vertebrate tomosyn and Munc18, for syntaxin binding, we hypothesized that C. elegans TOM-1 may inhibit both UNC-18-dependent vesicle targeting steps. Consistent with this hypothesis, tom-1 mutants exhibit enhanced UNC-18 plasma membrane localization and a concomitant increase in both tethered and docked synaptic vesicles. Furthermore, in tom-1;unc-18 double mutants the docked, primed vesicle pool is preferentially rescued relative to unc-18 single mutants. Together these data provide evidence for the differential regulation of two vesicle targeting steps by UNC-18 and TOM-1 through competitive interactions with syntaxin.

Dilation of fusion pores by crowding of SNARE proteins

PubMed Central

Wu, Zhenyong; Bello, Oscar D; Thiyagarajan, Sathish; Auclair, Sarah Marie; Vennekate, Wensi; Krishnakumar, Shyam S; O'Shaughnessy, Ben; Karatekin, Erdem

2017-01-01

Hormones and neurotransmitters are released through fluctuating exocytotic fusion pores that can flicker open and shut multiple times. Cargo release and vesicle recycling depend on the fate of the pore, which may reseal or dilate irreversibly. Pore nucleation requires zippering between vesicle-associated v-SNAREs and target membrane t-SNAREs, but the mechanisms governing the subsequent pore dilation are not understood. Here, we probed the dilation of single fusion pores using v-SNARE-reconstituted ~23-nm-diameter discoidal nanolipoprotein particles (vNLPs) as fusion partners with cells ectopically expressing cognate, 'flipped' t-SNAREs. Pore nucleation required a minimum of two v-SNAREs per NLP face, and further increases in v-SNARE copy numbers did not affect nucleation rate. By contrast, the probability of pore dilation increased with increasing v-SNARE copies and was far from saturating at 15 v-SNARE copies per face, the NLP capacity. Our experimental and computational results suggest that SNARE availability may be pivotal in determining whether neurotransmitters or hormones are released through a transient ('kiss and run') or an irreversibly dilating pore (full fusion). DOI: http://dx.doi.org/10.7554/eLife.22964.001 PMID:28346138

Synaptotagmin-mediated bending of the target membrane is a critical step in Ca2+-regulated fusion

PubMed Central

Hui, Enfu; Johnson, Colin P.; Yao, Jun; Dunning, F. Mark; Chapman, Edwin R.

2009-01-01

Summary Decades ago it was proposed that exocytosis involves invagination of the target membrane, resulting in a highly localized site of contact between the bilayers destined to fuse. The vesicle protein synaptotagmin-I (syt) bends membranes in response to Ca2+, but whether this drives localized invagination of the target membrane to accelerate fusion has not been determined; previous studies relied on reconstituted vesicles that were already highly curved and used mutations in syt that were not selective for membrane-bending activity. Here, we directly address this question by utilizing vesicles with different degrees of curvature. A tubulation-defective syt mutant was able to promote fusion between highly curved SNARE-bearing liposomes, but exhibited a marked loss of activity when the membranes were relatively flat. Moreover, bending of flat membranes by adding an N-BAR domain rescued the function of the tubulation-deficient syt mutant. Hence, syt-mediated membrane bending is a critical step in membrane fusion. PMID:19703397

Passive diffusion as a mechanism underlying ribbon synapse vesicle release and resupply.

PubMed

Graydon, Cole W; Zhang, Jun; Oesch, Nicholas W; Sousa, Alioscka A; Leapman, Richard D; Diamond, Jeffrey S

2014-07-02

Synaptic ribbons are presynaptic protein structures found at many synapses that convey graded, "analog" sensory signals in the visual, auditory, and vestibular pathways. Ribbons, typically anchored to the presynaptic membrane and surrounded by tethered synaptic vesicles, are thought to regulate or facilitate vesicle delivery to the presynaptic membrane. No direct evidence exists, however, to indicate how vesicles interact with the ribbon or, once attached, move along the ribbon's surface to reach the presynaptic release sites at its base. To address these questions, we have created, validated, and tested a passive vesicle diffusion model of retinal rod bipolar cell ribbon synapses. We used axial (bright-field) electron tomography in the scanning transmission electron microscopy to obtain 3D structures of rat rod bipolar cell terminals in 1-μm-thick sections of retinal tissue at an isotropic spatial resolution of ∼3 nm. The resulting structures were then incorporated with previously published estimates of vesicle diffusion dynamics into numerical simulations that accurately reproduced electrophysiologically measured vesicle release/replenishment rates and vesicle pool sizes. The simulations suggest that, under physiologically realistic conditions, diffusion of vesicles crowded on the ribbon surface gives rise to a flow field that enhances delivery of vesicles to the presynaptic membrane without requiring an active transport mechanism. Numerical simulations of ribbon-vesicle interactions predict that transient binding and unbinding of multiple tethers to each synaptic vesicle may achieve sufficiently tight association of vesicles to the ribbon while permitting the fast diffusion along the ribbon that is required to sustain high release rates. Copyright © 2014 the authors 0270-6474/14/348948-15$15.00/0.

Emergence and stability of intermediate open vesicles in disk-to-vesicle transitions.

PubMed

Li, Jianfeng; Zhang, Hongdong; Qiu, Feng; Shi, An-Chang

2013-07-01

The transition between two basic structures, a disk and an enclosed vesicle, of a finite membrane is studied by examining the minimum energy path (MEP) connecting these two states. The MEP is constructed using the string method applied to continuum elastic membrane models. The results reveal that, besides the commonly observed disk and vesicle, open vesicles (bowl-shaped vesicles or vesicles with a pore) can become stable or metastable shapes. The emergence, stability, and probability distribution of these open vesicles are analyzed. It is demonstrated that open vesicles can be stabilized by higher-order elastic energies. The estimated probability distribution of the different structures is in good agreement with available experiments.

Paramyxovirus F1 protein has two fusion peptides: implications for the mechanism of membrane fusion.

PubMed

Peisajovich, S G; Samuel, O; Shai, Y

2000-03-10

Viral fusion proteins contain a highly hydrophobic segment, named the fusion peptide, which is thought to be responsible for the merging of the cellular and viral membranes. Paramyxoviruses are believed to contain a single fusion peptide at the N terminus of the F1 protein. However, here we identified an additional internal segment in the Sendai virus F1 protein (amino acids 214-226) highly homologous to the fusion peptides of HIV-1 and RSV. A synthetic peptide, which includes this region, was found to induce membrane fusion of large unilamellar vesicles, at concentrations where the known N-terminal fusion peptide is not effective. A scrambled peptide as well as several peptides from other regions of the F1 protein, which strongly bind to membranes, are not fusogenic. The functional and structural characterization of this active segment suggest that the F1 protein has an additional internal fusion peptide that could participate in the actual fusion event. The presence of homologous regions in other members of the same family suggests that the concerted action of two fusion peptides, one N-terminal and the other internal, is a general feature of paramyxoviruses. Copyright 2000 Academic Press.

Interaction of phospholipid vesicles with cells. Endocytosis and fusion as alternate mechanisms for the uptake of lipid-soluble and water-soluble molecules

PubMed Central

1975-01-01

Depending on their phospholipid composition, liposomes are endocytosed by, or fuse with, the plasma membrane, of Acanthamoeba castellanii. Unilamellar egg lecithin vesicles are endocytosed by amoeba at 28 degrees C with equal uptake of the phospholipid bilayer and the contents of the internal aqueous space of the vesicles. Uptake is inhibited almost completely by incubation at 4 degrees C or in the presence of dinitrophenol. After uptake at 28 degrees C, the vesicle phospholipid can be visualized by electron microscope autoradiography within cytoplasmic vacuoles. In contrast, uptake of unilamellar dipalmitoyl lecithin vesicles and multilamellar dipalmitoyl lecithin liposomes is only partially inhibited at 4 degrees C, by dinitrophenol and by prior fixation of the amoebae with glutaraldehyde, each of which inhibits pinocytosis. Vesicle contents are taken up only about 40% as well as the phospholipid bilayer. Electron micrographs are compatible with the interpretation that dipalmitoyl lecithin vesicles fuse with the amoeba plasma membrane, adding their phospholipid to the cell surface, while their contents enter the cell cytoplasm. Dimyristoyl lecithin vesicles behave like egg lecithin vesicles while distearoyl lecithin vesicles behave like dipalmitoyl lecithin vesicles. PMID:1174130

Vesicle-mediated growth of tubular branches and centimeter-long microtubes from a single molecule.

PubMed

Abbas, Abdennour; Brimer, Andrew; Tian, Limei; d'Avignon, D André; Hameed, Abdulrahman Shahul; Vittal, Jagadese J; Singamaneni, Srikanth

2013-08-12

The mechanism by which small molecules assemble into microscale tubular structures in aqueous solution remains poorly understood, particularly when the initial building blocks are non-amphiphilic molecules and no surfactant is used. It is here shown how a subnanometric molecule, namely p-aminothiophenol (p-ATP), prepared in normal water with a small amount of ethanol, spontaneously assembles into a new class of nanovesicle. Due to Brownian motion, these nanostructures rapidly grow into micrometric vesicles and start budding to yield macroscale tubular branches with a remarkable growth rate of ∼20 μm s⁻¹. A real-time visualization by optical microscopy reveals that tubular growth proceeds by vesicle walk and fusion on the apex (growth cone) and sides of the branches and ultimately leads to the generation of centimeter-long microtubes. This unprecedented growth mechanism is triggered by a pH-activated proton switch and maintained by hydrogen bonding. The vesicle fusion-mediated synthesis suggests that functional microtubes with biological properties can be efficiently prepared with a mixture of appropriate diaminophenyl blocks and the desired macromolecule. The reversibility, timescale, and very high yield (90%) of this synthetic approach make it a valuable model for the investigation of hierarchical and structural transition between organized assemblies with different size scales and morphologies. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Loading capacity and interaction of DNA binding on catanionic vesicles with different cationic surfactants.

PubMed

Xu, Lu; Chen, Jingfei; Feng, Lei; Dong, Shuli; Hao, Jingcheng

2014-12-07

Cationic and anionic (catanionic) vesicles were constructed from the mixtures of sodium laurate (SL) and alkyltrimethylammonium bromide (CnTAB, n = 12, 14, and 16) and were used to control the loading capacity of DNA. The binding saturation point (BSP) of DNA to catanionic vesicles increases with the chain length of cationic surfactants, which is at 1.0, 1.3 and 1.5 for CnTAB with n = 12, 14, and 16, respectively. Our measurements showed that the loading capacity and affinity of DNA can be controlled by catanionic vesicles. It increases with the chain length of cationic surfactants. Because of a large reduction in surface charge density, catanionic vesicles are prone to undergo re-aggregation or fusion with the addition of DNA. DNA molecules can still maintain original coil state during the interaction with catanionic CnTAL vesicles. (1)H NMR data reveals that the obvious dissociation of anionic ions, L(-), from catanionic C14TAL vesicles is due to the interaction with DNA; however, this phenomenon cannot be observed in C12TAB-SL vesicles. Agarose gel electrophoresis (AGE) results demonstrate that the electrostatic interaction between the two oppositely charged cationic and anionic surfactants is stronger than that between DNA and cationic surfactant, CnTAB (n = 12, 14, and 16). Not only is the dissociation of L(-) simply determined by the charge competition, but it also depends largely on the variations in the surface charge density as well as the cationic and anionic surfactant competing ability in geometry configuration of catanionic vesicles. The complicated interaction between DNA and catanionic vesicles induces the deformation of cationic vesicles. Our results should provide clear guidance for choosing more proper vectors for DNA delivery and gene therapy in cell experiments.

Entropic forces drive self-organization and membrane fusion by SNARE proteins

PubMed Central

Stratton, Benjamin S.; Warner, Jason M.; Rothman, James E.; O’Shaughnessy, Ben

2017-01-01

SNARE proteins are the core of the cell’s fusion machinery and mediate virtually all known intracellular membrane fusion reactions on which exocytosis and trafficking depend. Fusion is catalyzed when vesicle-associated v-SNAREs form trans-SNARE complexes (“SNAREpins”) with target membrane-associated t-SNAREs, a zippering-like process releasing ∼65 kT per SNAREpin. Fusion requires several SNAREpins, but how they cooperate is unknown and reports of the number required vary widely. To capture the collective behavior on the long timescales of fusion, we developed a highly coarse-grained model that retains key biophysical SNARE properties such as the zippering energy landscape and the surface charge distribution. In simulations the ∼65-kT zippering energy was almost entirely dissipated, with fully assembled SNARE motifs but uncomplexed linker domains. The SNAREpins self-organized into a circular cluster at the fusion site, driven by entropic forces that originate in steric–electrostatic interactions among SNAREpins and membranes. Cooperative entropic forces expanded the cluster and pulled the membranes together at the center point with high force. We find that there is no critical number of SNAREs required for fusion, but instead the fusion rate increases rapidly with the number of SNAREpins due to increasing entropic forces. We hypothesize that this principle finds physiological use to boost fusion rates to meet the demanding timescales of neurotransmission, exploiting the large number of v-SNAREs available in synaptic vesicles. Once in an unfettered cluster, we estimate ≥15 SNAREpins are required for fusion within the ∼1-ms timescale of neurotransmitter release. PMID:28490503

The interaction between Pseudomonas aeruginosa cells and cationic PC:Chol:DOTAP liposomal vesicles versus outer-membrane structure and envelope properties of bacterial cell.

PubMed

Drulis-Kawa, Zuzanna; Dorotkiewicz-Jach, Agata; Gubernator, Jerzy; Gula, Grzegorz; Bocer, Tomasz; Doroszkiewicz, Wlodzimierz

2009-02-09

The interactions between cationic liposomal formulations (PC:Chol:DOTAP 3:4:3) and 23 Pseudomonas aeruginosa strains were tested. The study was undertaken because different antimicrobial results had been obtained by the authors for Pseudomonas aeruginosa strains and liposomal antibiotics (Drulis-Kawa, Z., Gubernator, J., Dorotkiewicz-Jach, A., Doroszkiewicz, W., Kozubek, A., 2006. The comparison of in vitro antimicrobial activity of liposomes containing meropenem and gentamicin. Cell. Mol. Biol. Lett., 11, 360-375; Drulis-Kawa, Z., Gubernator, J., Dorotkiewicz-Jach, A., Doroszkiewicz W., Kozubek, A., 2006. In vitro antimicrobial activity of liposomal meropenem against Pseudomonas aeruginosa strains. Int. J. Pharm., 315, 59-66). The experiments evaluate the roles of the bacterial outer-membrane structure, especially outer-membrane proteins and LPS, and envelope properties (hydrophobicity and electrostatic potential) in the interactions/fusion process between cells and lipid vesicles. The interactions were examined by fluorescent microscopy using PE-rhodamine-labelled liposomes. Some of the strains exhibited red-light emission (fusion with vesicles or vesicles surrounding the cell) and some showed negative reaction (no red-light emission). The main aim of the study was to determine what kinds of bacterial structure or envelope properties have a major influence on the fusion process. Negatively charged cells and hydrophobic properties promote interaction with cationic lipid vesicles, but no specific correlation was noted for the tested strains. A similar situation concerned LPS structure, where parent strains and their mutants possessing identical ladder-like band patterns in SDS-PAGE analysis exhibited totally different results with fluorescent microscopy. Outer-membrane protein analysis showed that an 18-kDA protein occurred in the isolates showing fusion with rhodamine-labelled vesicles and, conversely, strains lacking the 18-kDA protein exhibited no positive

Monitoring and modulating ion traffic in hybrid lipid/polymer vesicles

DOE PAGES

Paxton, Walter F.; McAninch, Patrick T.; Achyuthan, Komandoor E.; ...

2017-08-01

Controlling the traffic of molecules and ions across membranes is a critical feature in a number of biologically relevant processes and highly desirable for the development of technologies based on membrane materials. In this study, ion transport behavior of hybrid lipid/polymer membranes was studied in the absence and presence of ion transfer agents. A pH-sensitive fluorophore was used to investigate ion (H +/OH -) permeability across hybrid lipid/polymer membranes as a function of the fraction of amphiphilic block copolymer. It was observed that vesicles with intermediate lipid/polymer ratios tend to be surprisingly more permeable to ion transport than the puremore » lipid or pure polymer vesicles. Hybrid vesicle permeability could be further modulated with valinomycin, nigericin, or gramicidin A, which significantly expedite the dissipation of externally-imposed pH gradients by facilitating the transport of the rate-limiting co-ions (e.g. K +) ions across the membrane. For gramicidin A, ion permeability decreased with increasing polymer mole fraction, and the method of introduction of gramicidin A into the membrane played an important role. Finally, strategies to incorporate biofunctional molecules and facilitate their activity in synthetic systems are highly desirable for developing artificial organelles or other synthetic compartmentalized structures requiring control over molecular traffic across biomimetic membranes.« less

Fusion pores and their control of neurotransmitter and hormone release

PubMed Central

Chang, Che-Wei; Chiang, Chung-Wei

2017-01-01

Ca2+-triggered exocytosis functions broadly in the secretion of chemical signals, enabling neurons to release neurotransmitters and endocrine cells to release hormones. The biological demands on this process can vary enormously. Although synapses often release neurotransmitter in a small fraction of a millisecond, hormone release can be orders of magnitude slower. Vesicles usually contain multiple signaling molecules that can be released selectively and conditionally. Cells are able to control the speed, concentration profile, and content selectivity of release by tuning and tailoring exocytosis to meet different biological demands. Much of this regulation depends on the fusion pore—the aqueous pathway by which molecules leave a vesicle and move out into the surrounding extracellular space. Studies of fusion pores have illuminated how cells regulate secretion. Furthermore, the formation and growth of fusion pores serve as a readout for the progress of exocytosis, thus revealing key kinetic stages that provide clues about the underlying mechanisms. Herein, we review the structure, composition, and dynamics of fusion pores and discuss the implications for molecular mechanisms as well as for the cellular regulation of neurotransmitter and hormone release. PMID:28167663

Regulated Production of Mineralization-competent Matrix Vesicles in Hypertrophic Chondrocytes

PubMed Central

Kirsch, Thorsten; Nah, Hyun-Duck; Shapiro, Irving M.; Pacifici, Maurizio

1997-01-01

hypertrophic chondrocytes can qualitatively modulate their production of matrix vesicles and only when induced to initiate mineralization, will release mineralization-competent matrix vesicles rich in annexin V and alkaline phosphatase. The occurrence of type I collagen in concert with cartilage matrix calcification suggests that the protein may facilitate crystal growth after rupture of the matrix vesicle membrane; it may also offer a smooth transition from mineralized type II/type X collagen-rich cartilage matrix to type I collagen-rich bone matrix. PMID:9166414

Septin 7 reduces nonmuscle myosin IIA activity in the SNAP23 complex and hinders GLUT4 storage vesicle docking and fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wasik, Anita A.; Dumont, Vincent; Tienari, Jukka

Glomerular epithelial cells, podocytes, are insulin responsive and can develop insulin resistance. Here, we demonstrate that the small GTPase septin 7 forms a complex with nonmuscle myosin heavy chain IIA (NMHC-IIA; encoded by MYH9), a component of the nonmuscle myosin IIA (NM-IIA) hexameric complex. We observed that knockdown of NMHC-IIA decreases insulin-stimulated glucose uptake into podocytes. Both septin 7 and NM-IIA associate with SNAP23, a SNARE protein involved in GLUT4 storage vesicle (GSV) docking and fusion with the plasma membrane. We observed that insulin decreases the level of septin 7 and increases the activity of NM-IIA in the SNAP23 complex,more » as visualized by increased phosphorylation of myosin regulatory light chain. Also knockdown of septin 7 increases the activity of NM-IIA in the complex. The activity of NM-IIA is increased in diabetic rat glomeruli and cultured human podocytes exposed to macroalbuminuric sera from patients with type 1 diabetes. Collectively, the data suggest that the activity of NM-IIA in the SNAP23 complex plays a key role in insulin-stimulated glucose uptake into podocytes. Furthermore, we observed that septin 7 reduces the activity of NM-IIA in the SNAP23 complex and thereby hinders GSV docking and fusion with the plasma membrane. - Highlights: • Septin 7, nonmuscle myosin heavy chain IIA (NMHC-IIA) and SNAP23 form a complex. • Knockdown of septin 7 increases NM-IIA activity in the SNAP23 complex. • Insulin decreases septin 7 level and increases NM-IIA activity in the SNAP23 complex. • Septin 7 hinders GSV docking/fusion by reducing NM-IIA activity in the SNAP23 complex.« less

Changes in lipid bilayer structure caused by the helix-to-sheet transition of an HIV-1 gp41 fusion peptide derivative

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heller, William T.; Rai, Durgesh K.

HIV-1, like other enveloped viruses, undergoes fusion with the cell membrane to infect it. Viral coat proteins are thought to bind the virus to the membrane and actively fuse the viral and cellular membranes together. The actual molecular mechanism of fusion is challenging to visualize, resulting in the use of model systems. In this paper, the bilayer curvature modifying properties of a synthetic variant of the HIV-1 gp41 fusion peptide with lipid bilayer vesicles composed of a mixture of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS) were studied. In 7:3 DMPC:DMPS vesicles made with deuterium-labeled DMPC, the peptide was observedmore » to undergo a concentration-dependent conformational transition between an α-helix and an antiparallel β-sheet. Through the use of small-angle neutron scattering (SANS) and selective deuterium labeling, it was revealed that conformational transition of the peptide is also accompanied by a transition in the structure of the lipid bilayer. In addition to changes in the distribution of the lipid between the leaflets of the vesicle, the SANS data are consistent with two regions having different thicknesses. Finally, of the two different bilayer structures, the one corresponding to the smaller area fraction, being ~8% of the vesicle area, is much thicker than the remainder of the vesicle, which suggests that there are regions of localized negative curvature similar to what takes place at the point of contact between two membranes immediately preceding fusion.« less

Changes in lipid bilayer structure caused by the helix-to-sheet transition of an HIV-1 gp41 fusion peptide derivative

DOE PAGES

Heller, William T.; Rai, Durgesh K.

2017-01-16

HIV-1, like other enveloped viruses, undergoes fusion with the cell membrane to infect it. Viral coat proteins are thought to bind the virus to the membrane and actively fuse the viral and cellular membranes together. The actual molecular mechanism of fusion is challenging to visualize, resulting in the use of model systems. In this paper, the bilayer curvature modifying properties of a synthetic variant of the HIV-1 gp41 fusion peptide with lipid bilayer vesicles composed of a mixture of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS) were studied. In 7:3 DMPC:DMPS vesicles made with deuterium-labeled DMPC, the peptide was observedmore » to undergo a concentration-dependent conformational transition between an α-helix and an antiparallel β-sheet. Through the use of small-angle neutron scattering (SANS) and selective deuterium labeling, it was revealed that conformational transition of the peptide is also accompanied by a transition in the structure of the lipid bilayer. In addition to changes in the distribution of the lipid between the leaflets of the vesicle, the SANS data are consistent with two regions having different thicknesses. Finally, of the two different bilayer structures, the one corresponding to the smaller area fraction, being ~8% of the vesicle area, is much thicker than the remainder of the vesicle, which suggests that there are regions of localized negative curvature similar to what takes place at the point of contact between two membranes immediately preceding fusion.« less

Extracellular Vesicles, Tunneling Nanotubes, and Cellular Interplay: Synergies and Missing Links

PubMed Central

Nawaz, Muhammad; Fatima, Farah

2017-01-01

The process of intercellular communication seems to have been a highly conserved evolutionary process. Higher eukaryotes use several means of intercellular communication to address both the changing physiological demands of the body and to fight against diseases. In recent years, there has been an increasing interest in understanding how cell-derived nanovesicles, known as extracellular vesicles (EVs), can function as normal paracrine mediators of intercellular communication, but can also elicit disease progression and may be used for innovative therapies. Over the last decade, a large body of evidence has accumulated to show that cells use cytoplasmic extensions comprising open-ended channels called tunneling nanotubes (TNTs) to connect cells at a long distance and facilitate the exchange of cytoplasmic material. TNTs are a different means of communication to classical gap junctions or cell fusions; since they are characterized by long distance bridging that transfers cytoplasmic organelles and intracellular vesicles between cells and represent the process of heteroplasmy. The role of EVs in cell communication is relatively well-understood, but how TNTs fit into this process is just emerging. The aim of this review is to describe the relationship between TNTs and EVs, and to discuss the synergies between these two crucial processes in the context of normal cellular cross-talk, physiological roles, modulation of immune responses, development of diseases, and their combinatory effects in tissue repair. At the present time this review appears to be the first summary of the implications of the overlapping roles of TNTs and EVs. We believe that a better appreciation of these parallel processes will improve our understanding on how these nanoscale conduits can be utilized as novel tools for targeted therapies. PMID:28770210

Vesicle electrohydrodynamics.

PubMed

Schwalbe, Jonathan T; Vlahovska, Petia M; Miksis, Michael J

2011-04-01

A small amplitude perturbation analysis is developed to describe the effect of a uniform electric field on the dynamics of a lipid bilayer vesicle in a simple shear flow. All media are treated as leaky dielectrics and fluid motion is described by the Stokes equations. The instantaneous vesicle shape is obtained by balancing electric, hydrodynamic, bending, and tension stresses exerted on the membrane. We find that in the absence of ambient shear flow, it is possible that an applied stepwise uniform dc electric field could cause the vesicle shape to evolve from oblate to prolate over time if the encapsulated fluid is less conducting than the suspending fluid. For a vesicle in ambient shear flow, the electric field damps the tumbling motion, leading to a stable tank-treading state.

Viral Membrane Fusion and Nucleocapsid Delivery into the Cytoplasm are Distinct Events in Some Flaviviruses

PubMed Central

Nour, Adel M.; Li, Yue; Wolenski, Joseph; Modis, Yorgo

2013-01-01

Flaviviruses deliver their genome into the cell by fusing the viral lipid membrane to an endosomal membrane. The sequence and kinetics of the steps required for nucleocapsid delivery into the cytoplasm remain unclear. Here we dissect the cell entry pathway of virions and virus-like particles from two flaviviruses using single-particle tracking in live cells, a biochemical membrane fusion assay and virus infectivity assays. We show that the virus particles fuse with a small endosomal compartment in which the nucleocapsid remains trapped for several minutes. Endosomal maturation inhibitors inhibit infectivity but not membrane fusion. We propose a flavivirus cell entry mechanism in which the virus particles fuse preferentially with small endosomal carrier vesicles and depend on back-fusion of the vesicles with the late endosomal membrane to deliver the nucleocapsid into the cytoplasm. Virus entry modulates intracellular calcium release and phosphatidylinositol-3-phosphate kinase signaling. Moreover, the broadly cross-reactive therapeutic antibody scFv11 binds to virus-like particles and inhibits fusion. PMID:24039574

The Rab3A-22A Chimera Prevents Sperm Exocytosis by Stabilizing Open Fusion Pores*

PubMed Central

Quevedo, María F.; Lucchesi, Ornella; Bustos, Matías A.; Pocognoni, Cristian A.; De la Iglesia, Paola X.

2016-01-01

At the final stage of exocytotis, a fusion pore opens between the plasma and a secretory vesicle membranes; typically, when the pore dilates the vesicle releases its cargo. Sperm contain a large dense-core secretory granule (the acrosome) whose contents are secreted by regulated exocytosis at fertilization. Minutes after the arrival of the triggering signal, the acrosomal and plasma membranes dock at multiple sites and fusion pores open at the contact points. It is believed that immediately afterward, fusion pores dilate spontaneously. Rab3A is an essential component of human sperm exocytotic machinery. Yet, recombinant, persistently active Rab3A halts calcium-triggered secretion when introduced after docking into streptolysin O-permeabilized cells; so does a Rab3A-22A chimera. Here, we applied functional assays, electron and confocal microscopy to show that the secretion blockage is due to the stabilization of open fusion pores. Other novel findings are that sperm SNAREs engage in α-SNAP/NSF-sensitive complexes at a post-fusion stage. Complexes are disentangled by these chaperons to achieve vesiculation and acrosomal contents release. Thus, post-fusion regulation of the pores determines their expansion and the success of the acrosome reaction. PMID:27613869

Multiple roles for the actin cytoskeleton during regulated exocytosis

PubMed Central

Porat-Shliom, Natalie; Milberg, Oleg; Masedunskas, Andrius; Weigert, Roberto

2014-01-01

Regulated exocytosis is the main mechanism utilized by specialized secretory cells to deliver molecules to the cell surface by virtue of membranous containers (i.e. secretory vesicles). The process involves a series of highly coordinated and sequential steps, which include the biogenesis of the vesicles, their delivery to the cell periphery, their fusion with the plasma membrane and the release of their content into the extracellular space. Each of these steps is regulated by the actin cytoskeleton. In this review, we summarize the current knowledge regarding the involvement of actin and its associated molecules during each of the exocytic steps in vertebrates, and suggest that the overall role of the actin cytoskeleton during regulated exocytosis is linked to the architecture and the physiology of the secretory cells under examination. Specifically, in neurons, neuroendocrine, endocrine, and hematopoietic cells, which contain small secretory vesicles that undergo rapid exocytosis (on the order of milliseconds), the actin cytoskeleton plays a role in pre-fusion events, where it acts primarily as a functional barrier and facilitates docking. In exocrine and other secretory cells, which contain large secretory vesicles that undergo slow exocytosis (seconds to minutes), the actin cytoskeleton plays a role in post-fusion events, where it regulates the dynamics of the fusion pore, facilitates the integration of the vesicles into the plasma membrane, provides structural support, and promotes the expulsion of large cargo molecules. PMID:22986507

Visualization of Membrane Pore in Live Cells Reveals a Dynamic-Pore Theory Governing Fusion and Endocytosis.

PubMed

Shin, Wonchul; Ge, Lihao; Arpino, Gianvito; Villarreal, Seth A; Hamid, Edaeni; Liu, Huisheng; Zhao, Wei-Dong; Wen, Peter J; Chiang, Hsueh-Cheng; Wu, Ling-Gang

2018-05-03

Fusion is thought to open a pore to release vesicular cargoes vital for many biological processes, including exocytosis, intracellular trafficking, fertilization, and viral entry. However, fusion pores have not been observed and thus proved in live cells. Its regulatory mechanisms and functions remain poorly understood. With super-resolution STED microscopy, we observed dynamic fusion pore behaviors in live (neuroendocrine) cells, including opening, expansion, constriction, and closure, where pore size may vary between 0 and 490 nm within 26 milliseconds to seconds (vesicle size: 180-720 nm). These pore dynamics crucially determine the efficiency of vesicular cargo release and vesicle retrieval. They are generated by competition between pore expansion and constriction. Pharmacology and mutation experiments suggest that expansion and constriction are mediated by F-actin-dependent membrane tension and calcium/dynamin, respectively. These findings provide the missing live-cell evidence, proving the fusion-pore hypothesis, and establish a live-cell dynamic-pore theory accounting for fusion, fission, and their regulation. Published by Elsevier Inc.

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

Sensor fusion for synthetic vision

NASA Technical Reports Server (NTRS)

Pavel, M.; Larimer, J.; Ahumada, A.

1991-01-01

Display methodologies are explored for fusing images gathered by millimeter wave sensors with images rendered from an on-board terrain data base to facilitate visually guided flight and ground operations in low visibility conditions. An approach to fusion based on multiresolution image representation and processing is described which facilitates fusion of images differing in resolution within and between images. To investigate possible fusion methods, a workstation-based simulation environment is being developed.

A simplified method to recover urinary vesicles for clinical applications, and sample banking.

PubMed

Musante, Luca; Tataruch, Dorota; Gu, Dongfeng; Benito-Martin, Alberto; Calzaferri, Giulio; Aherne, Sinead; Holthofer, Harry

2014-12-23

Urinary extracellular vesicles provide a novel source for valuable biomarkers for kidney and urogenital diseases: Current isolation protocols include laborious, sequential centrifugation steps which hampers their widespread research and clinical use. Furthermore, large individual urine sample volumes or sizable target cohorts are to be processed (e.g. for biobanking), the storage capacity is an additional problem. Thus, alternative methods are necessary to overcome such limitations. We have developed a practical vesicle isolation technique to yield easily manageable sample volumes in an exceptionally cost efficient way to facilitate their full utilization in less privileged environments and maximize the benefit of biobanking. Urinary vesicles were isolated by hydrostatic dialysis with minimal interference of soluble proteins or vesicle loss. Large volumes of urine were concentrated up to 1/100 of original volume and the dialysis step allowed equalization of urine physico-chemical characteristics. Vesicle fractions were found suitable to any applications, including RNA analysis. In the yield, our hydrostatic filtration dialysis system outperforms the conventional ultracentrifugation-based methods and the labour intensive and potentially hazardous step of ultracentrifugations are eliminated. Likewise, the need for trained laboratory personnel and heavy initial investment is avoided. Thus, our method qualifies as a method for laboratories working with urinary vesicles and biobanking.

A Simplified Method to Recover Urinary Vesicles for Clinical Applications, and Sample Banking

PubMed Central

Musante, Luca; Tataruch, Dorota; Gu, Dongfeng; Benito-Martin, Alberto; Calzaferri, Giulio; Aherne, Sinead; Holthofer, Harry

2014-01-01

Urinary extracellular vesicles provide a novel source for valuable biomarkers for kidney and urogenital diseases: Current isolation protocols include laborious, sequential centrifugation steps which hampers their widespread research and clinical use. Furthermore, large individual urine sample volumes or sizable target cohorts are to be processed (e.g. for biobanking), the storage capacity is an additional problem. Thus, alternative methods are necessary to overcome such limitations. We have developed a practical vesicle isolation technique to yield easily manageable sample volumes in an exceptionally cost efficient way to facilitate their full utilization in less privileged environments and maximize the benefit of biobanking. Urinary vesicles were isolated by hydrostatic dialysis with minimal interference of soluble proteins or vesicle loss. Large volumes of urine were concentrated up to 1/100 of original volume and the dialysis step allowed equalization of urine physico-chemical characteristics. Vesicle fractions were found suitable to any applications, including RNA analysis. In the yield, our hydrostatic filtration dialysis system outperforms the conventional ultracentrifugation-based methods and the labour intensive and potentially hazardous step of ultracentrifugations are eliminated. Likewise, the need for trained laboratory personnel and heavy initial investment is avoided. Thus, our method qualifies as a method for laboratories working with urinary vesicles and biobanking. PMID:25532487

ATG14 promotes membrane tethering and fusion of autophagosomes to endolysosomes.

PubMed

Diao, Jiajie; Liu, Rong; Rong, Yueguang; Zhao, Minglei; Zhang, Jing; Lai, Ying; Zhou, Qiangjun; Wilz, Livia M; Li, Jianxu; Vivona, Sandro; Pfuetzner, Richard A; Brunger, Axel T; Zhong, Qing

2015-04-23

Autophagy, an important catabolic pathway implicated in a broad spectrum of human diseases, begins by forming double membrane autophagosomes that engulf cytosolic cargo and ends by fusing autophagosomes with lysosomes for degradation. Membrane fusion activity is required for early biogenesis of autophagosomes and late degradation in lysosomes. However, the key regulatory mechanisms of autophagic membrane tethering and fusion remain largely unknown. Here we report that ATG14 (also known as beclin-1-associated autophagy-related key regulator (Barkor) or ATG14L), an essential autophagy-specific regulator of the class III phosphatidylinositol 3-kinase complex, promotes membrane tethering of protein-free liposomes, and enhances hemifusion and full fusion of proteoliposomes reconstituted with the target (t)-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) syntaxin 17 (STX17) and SNAP29, and the vesicle (v)-SNARE VAMP8 (vesicle-associated membrane protein 8). ATG14 binds to the SNARE core domain of STX17 through its coiled-coil domain, and stabilizes the STX17-SNAP29 binary t-SNARE complex on autophagosomes. The STX17 binding, membrane tethering and fusion-enhancing activities of ATG14 require its homo-oligomerization by cysteine repeats. In ATG14 homo-oligomerization-defective cells, autophagosomes still efficiently form but their fusion with endolysosomes is blocked. Recombinant ATG14 homo-oligomerization mutants also completely lose their ability to promote membrane tethering and to enhance SNARE-mediated fusion in vitro. Taken together, our data suggest an autophagy-specific membrane fusion mechanism in which oligomeric ATG14 directly binds to STX17-SNAP29 binary t-SNARE complex on autophagosomes and primes it for VAMP8 interaction to promote autophagosome-endolysosome fusion.

Rolling blackout is required for synaptic vesicle exocytosis.

PubMed

Huang, Fu-De; Woodruff, Elvin; Mohrmann, Ralf; Broadie, Kendal

2006-03-01

Rolling blackout (RBO) is a putative transmembrane lipase required for phospholipase C-dependent phosphatidylinositol 4,5-bisphosphate-diacylglycerol signaling in Drosophila neurons. Conditional temperature-sensitive (TS) rbo mutants display complete, reversible paralysis within minutes, demonstrating that RBO is acutely required for movement. RBO protein is localized predominantly in presynaptic boutons at neuromuscular junction (NMJ) synapses and throughout central synaptic neuropil, and rbo TS mutants display a complete, reversible block of both central and peripheral synaptic transmission within minutes. This phenotype appears limited to adults, because larval NMJs do not manifest the acute blockade. Electron microscopy of adult rbo TS mutant boutons reveals an increase in total synaptic vesicle (SV) content, with a concomitant shrinkage of presynaptic bouton size and an accumulation of docked SVs at presynaptic active zones within minutes. Genetic tests reveal a synergistic interaction between rbo and syntaxin1A TS mutants, suggesting that RBO is required in the mechanism of N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated SV exocytosis, or in a parallel pathway necessary for SV fusion. The rbo TS mutation does not detectably alter SNARE complex assembly, suggesting a downstream requirement in SV fusion. We conclude that RBO plays an essential role in neurotransmitter release, downstream of SV docking, likely mediating SV fusion.

Extracellular vesicles in Alzheimer's disease: friends or foes? Focus on aβ-vesicle interaction.

PubMed

Joshi, Pooja; Benussi, Luisa; Furlan, Roberto; Ghidoni, Roberta; Verderio, Claudia

2015-03-03

The intercellular transfer of amyloid-β (Aβ) and tau proteins has received increasing attention in Alzheimer's disease (AD). Among other transfer modes, Aβ and tau dissemination has been suggested to occur through release of Extracellular Vesicles (EVs), which may facilitate delivery of pathogenic proteins over large distances. Recent evidence indicates that EVs carry on their surface, specific molecules which bind to extracellular Aβ, opening the possibility that EVs may also influence Aβ assembly and synaptotoxicity. In this review we focus on studies which investigated the impact of EVs in Aβ-mediated neurodegeneration and showed either detrimental or protective role for EVs in the pathology.

Amphiphilic gold nanoparticles as modulators of lipid membrane fusion

NASA Astrophysics Data System (ADS)

Tahir, Mukarram; Alexander-Katz, Alfredo

The fusion of lipid membranes is central to biological functions like inter-cellular transport and signaling and is coordinated by proteins of the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) superfamily. We utilize molecular dynamics simulations to demonstrate that gold nanoparticles functionalized with a mixed-monolayer of hydrophobic and hydrophilic alkanethiol ligands can act as synthetic analogues of these fusion proteins and mediate lipid membrane fusion by catalyzing the formation of a toroidal stalk between adjacent membranes and enabling the formation of a fusion pore upon influx of Ca2+ into the exterior solvent. The fusion pathway enabled by these synthetic nanostructures is analogous to the regulated fast fusion pathway observed during synaptic vesicle fusion; it therefore provides novel physical insights into this important biological process while also being relevant in a number of single-cell therapeutic applications. Computational resources from NSF XSEDE contract TG-DMR130042. Financial support from DOE CSGF fellowship DE-FG02-97ER25308.

Ion transport studies with H+-K+-ATPase-rich vesicles: implications for HCl secretion and parietal cell physiology.

PubMed

Wolosin, J M

1985-06-01

A summary of recent studies on relations between the properties of the membrane incorporating the H+-K+-ATPase, the H+ motive force in gastric acid secretion, and the secretory state of the parietal cell is presented. Depending on tissue secretory state, two distinct H+-K+-ATPase-rich membranes predominate in tissue homogenates, the gastric microsomes derived from the intracellular tubulovesicles of the resting cell and the stimulation-associated (SA) vesicle derived from the apical membrane of the acid-secreting cell. Structural and chemical differences between both vesicular types lend support to the notion that the formation of an expanded, elaborated apical membrane in the secreting parietal cell results from fusion of tubulovesicles containing the H+-K+-ATPase to an apical membrane of different chemical composition. Comparison of polypeptide composition of microsomes and SA membranes provides a way to identify and isolate membrane and cytoskeletal components putatively involved in the membrane interconversion process. Comparison of transport properties between gastric microsomes and SA vesicles demonstrates that stimulation triggers the appearance of rapid K+ and Cl- permeabilities in the H+-K+-ATPase membrane, allowing efficient acid accumulation in SA vesicles by the combination of rapid KCl influx followed by ATPase-driven H+ for K+ exchange, i.e., by K+ recycling. These stimulation-triggered conductances are functionally independent. Nevertheless, their concurrent inhibition by certain divalent cations (Mn2+,Zn2+) suggests their location within a single physical domain. The compatibility of the K+-recycling model for HCl accumulation in SA vesicles with gastric HCl secretion and selected electrophysiological observations and certain implications of the findings for cellular mechanisms of transport regulation in the context of a membrane fusion and recycling model are discussed.

SNX9 - a prelude to vesicle release.

PubMed

Lundmark, Richard; Carlsson, Sven R

2009-01-01

The sorting nexin SNX9 has, in the past few years, been singled out as an important protein that participates in fundamental cellular activities. SNX9 binds strongly to dynamin and is partly responsible for the recruitment of this GTPase to sites of endocytosis. SNX9 also has a high capacity for modulation of the membrane and might therefore participate in the formation of the narrow neck of endocytic vesicles before scission occurs. Once assembled on the membrane, SNX9 stimulates the GTPase activity of dynamin to facilitate the scission reaction. It has also become clear that SNX9 has the ability to activate the actin regulator N-WASP in a membrane-dependent manner to coordinate actin polymerization with vesicle release. In this Commentary, we summarize several aspects of SNX9 structure and function in the context of membrane remodeling, discuss its interplay with various interaction partners and present a model of how SNX9 might work in endocytosis.

The Rab3A-22A Chimera Prevents Sperm Exocytosis by Stabilizing Open Fusion Pores.

PubMed

Quevedo, María F; Lucchesi, Ornella; Bustos, Matías A; Pocognoni, Cristian A; De la Iglesia, Paola X; Tomes, Claudia N

2016-10-28

At the final stage of exocytotis, a fusion pore opens between the plasma and a secretory vesicle membranes; typically, when the pore dilates the vesicle releases its cargo. Sperm contain a large dense-core secretory granule (the acrosome) whose contents are secreted by regulated exocytosis at fertilization. Minutes after the arrival of the triggering signal, the acrosomal and plasma membranes dock at multiple sites and fusion pores open at the contact points. It is believed that immediately afterward, fusion pores dilate spontaneously. Rab3A is an essential component of human sperm exocytotic machinery. Yet, recombinant, persistently active Rab3A halts calcium-triggered secretion when introduced after docking into streptolysin O-permeabilized cells; so does a Rab3A-22A chimera. Here, we applied functional assays, electron and confocal microscopy to show that the secretion blockage is due to the stabilization of open fusion pores. Other novel findings are that sperm SNAREs engage in α-SNAP/NSF-sensitive complexes at a post-fusion stage. Complexes are disentangled by these chaperons to achieve vesiculation and acrosomal contents release. Thus, post-fusion regulation of the pores determines their expansion and the success of the acrosome reaction. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The Habc Domain of the SNARE Vam3 Interacts with the HOPS Tethering Complex to Facilitate Vacuole Fusion*

PubMed Central

Lürick, Anna; Kuhlee, Anne; Bröcker, Cornelia; Kümmel, Daniel; Raunser, Stefan; Ungermann, Christian

2015-01-01

Membrane fusion at vacuoles requires a consecutive action of the HOPS tethering complex, which is recruited by the Rab GTPase Ypt7, and vacuolar SNAREs to drive membrane fusion. It is assumed that the Sec1/Munc18-like Vps33 within the HOPS complex is largely responsible for SNARE chaperoning. Here, we present direct evidence for HOPS binding to SNAREs and the Habc domain of the Vam3 SNARE protein, which may explain its function during fusion. We show that HOPS interacts strongly with the Vam3 Habc domain, assembled Q-SNAREs, and the R-SNARE Ykt6, but not the Q-SNARE Vti1 or the Vam3 SNARE domain. Electron microscopy combined with Nanogold labeling reveals that the binding sites for vacuolar SNAREs and the Habc domain are located in the large head of the HOPS complex, where Vps16 and Vps33 have been identified before. Competition experiments suggest that HOPS bound to the Habc domain can still interact with assembled Q-SNAREs, whereas Q-SNARE binding prevents recognition of the Habc domain. In agreement, membranes carrying Vam3ΔHabc fuse poorly unless an excess of HOPS is provided. These data suggest that the Habc domain of Vam3 facilitates the assembly of the HOPS/SNARE machinery at fusion sites and thus supports efficient membrane fusion. PMID:25564619

A Ratiometric Two-Photon Fluorescent Probe for Tracking the Lysosomal ATP Level: Direct in cellulo Observation of Lysosomal Membrane Fusion Processes.

PubMed

Jun, Yong Woong; Wang, Taejun; Hwang, Sekyu; Kim, Dokyoung; Ma, Donghee; Kim, Ki Hean; Kim, Sungjee; Jung, Junyang; Ahn, Kyo Han

2018-06-05

Vesicles exchange its contents through membrane fusion processes-kiss-and-run and full-collapse fusion. Indirect observation of these fusion processes using artificial vesicles enhanced our understanding on the molecular mechanisms involved. Direct observation of the fusion processes in a real biological system, however, remains a challenge owing to many technical obstacles. We disclose a ratiometric two-photon probe offering real-time tracking of lysosomal ATP with quantitative information for the first time. By applying the probe to two-photon live-cell imaging technique, lysosomal membrane fusion process in cells has been directly observed along with the concentration of its content-lysosomal ATP. Results show that the kiss-and-run process between lysosomes proceeds through repeating transient interactions with gradual content mixing, whereas the full-fusion process occurs at once. Furthermore, it is confirmed that both the fusion processes proceed with conservation of the content. Such a small-molecule probe exerts minimal disturbance and hence has potential for studying various biological processes associated with lysosomal ATP. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pretargeting with bispecific fusion proteins facilitates delivery of nanoparticles to tumor cells with distinct surface antigens.

PubMed

Yang, Qi; Parker, Christina L; Lin, Yukang; Press, Oliver W; Park, Steven I; Lai, Samuel K

2017-06-10

Tumor heterogeneity, which describes the genetically and phenotypically distinct subpopulations of tumor cells present within the same tumor or patient, presents a major challenge to targeted delivery of diagnostic and/or therapeutic agents. An ideal targeting strategy should deliver a given nanocarrier to the full diversity of cancer cells, which is difficult to achieve with conventional ligand-conjugated nanoparticles. We evaluated pretargeting (i.e., multistep targeting) as a strategy to facilitate nanoparticle delivery to multiple target cells by measuring the uptake of biotinylated nanoparticles by lymphoma cells with distinct surface antigens pretreated with different bispecific streptavidin-scFv fusion proteins. Fusion proteins targeting CD20 or tumor-associated glycoprotein 72 (TAG-72) mediated the specific in vitro uptake of 100nm biotin-functionalized nanoparticles by Raji and Jurkat lymphoma cells (CD20-positive and TAG-72-positive cells, respectively). Greater uptake was observed for pretargeted nanoparticles with increasing amounts of surface biotin, with 6- to 18-fold higher uptake vs. non-biotinylated nanoparticle and fusion protein controls. Fully biotin-modified particles remained resistant to cultured macrophage cell uptake, although they were still quickly cleared from systemic circulation in vivo (t 1/2 <1h). For single Raji tumor-bearing mice, pretargeting with CD20-specific FP significantly increased nanoparticle tumor targeting. In mice bearing both Raji and Jurkat tumors, pretargeting with both fusion proteins markedly increased nanoparticle targeting to both tumor types, compared to animals dosed with nanoparticles alone. These in vitro and in vivo observations support further evaluations of pretargeting fusion protein cocktails as a strategy to enhance nanoparticle delivery to a diverse array of molecularly distinct target cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Calcium-Activated Proteases Are Critical for Refilling Depleted Vesicle Stores in Cultured Sensory-Motor Synapses of "Aplysia"

ERIC Educational Resources Information Center

Khoutorsky, Arkady; Spira, Micha E.

2005-01-01

"Aplysia" motoneurons cocultured with a presynaptic sensory neuron exhibit homosynaptic depression when stimulated at low frequencies. A single bath application of serotonin (5HT) leads within seconds to facilitation of the depressed synapse. The facilitation is attributed to mobilization of neurotransmitter-containing vesicles from a…

Activity-Dependence of Synaptic Vesicle Dynamics

PubMed Central

Forte, Luca A.

2017-01-01

The proper function of synapses relies on efficient recycling of synaptic vesicles. The small size of synaptic boutons has hampered efforts to define the dynamical states of vesicles during recycling. Moreover, whether vesicle motion during recycling is regulated by neural activity remains largely unknown. We combined nanoscale-resolution tracking of individual synaptic vesicles in cultured hippocampal neurons from rats of both sexes with advanced motion analyses to demonstrate that the majority of recently endocytosed vesicles undergo sequences of transient dynamical states including epochs of directed, diffusional, and stalled motion. We observed that vesicle motion is modulated in an activity-dependent manner, with dynamical changes apparent in ∼20% of observed boutons. Within this subpopulation of boutons, 35% of observed vesicles exhibited acceleration and 65% exhibited deceleration, accompanied by corresponding changes in directed motion. Individual vesicles observed in the remaining ∼80% of boutons did not exhibit apparent dynamical changes in response to stimulation. More quantitative transient motion analyses revealed that the overall reduction of vesicle mobility, and specifically of the directed motion component, is the predominant activity-evoked change across the entire bouton population. Activity-dependent modulation of vesicle mobility may represent an important mechanism controlling vesicle availability and neurotransmitter release. SIGNIFICANCE STATEMENT Mechanisms governing synaptic vesicle dynamics during recycling remain poorly understood. Using nanoscale resolution tracking of individual synaptic vesicles in hippocampal synapses and advanced motion analysis tools we demonstrate that synaptic vesicles undergo complex sets of dynamical states that include epochs of directed, diffusive, and stalled motion. Most importantly, our analyses revealed that vesicle motion is modulated in an activity-dependent manner apparent as the reduction in

Improving nuclear envelope dynamics by EBV BFRF1 facilitates intranuclear component clearance through autophagy.

PubMed

Liu, Guan-Ting; Kung, Hsiu-Ni; Chen, Chung-Kuan; Huang, Cheng; Wang, Yung-Li; Yu, Cheng-Pu; Lee, Chung-Pei

2018-02-26

Although a vesicular nucleocytoplasmic transport system is believed to exist in eukaryotic cells, the features of this pathway are mostly unknown. Here, we report that the BFRF1 protein of the Epstein-Barr virus improves vesicular transport of nuclear envelope (NE) to facilitate the translocation and clearance of nuclear components. BFRF1 expression induces vesicles that selectively transport nuclear components to the cytoplasm. With the use of aggregation-prone proteins as tools, we found that aggregated nuclear proteins are dispersed when these BFRF1-induced vesicles are formed. BFRF1-containing vesicles engulf the NE-associated aggregates, exit through from the NE, and putatively fuse with autophagic vacuoles. Chemical treatment and genetic ablation of autophagy-related factors indicate that autophagosome formation and autophagy-linked FYVE protein-mediated autophagic proteolysis are involved in this selective clearance of nuclear proteins. Remarkably, vesicular transport, elicited by BFRF1, also attenuated nuclear aggregates accumulated in neuroblastoma cells. Accordingly, induction of NE-derived vesicles by BFRF1 facilitates nuclear protein translocation and clearance, suggesting that autophagy-coupled transport of nucleus-derived vesicles can be elicited for nuclear component catabolism in mammalian cells.-Liu, G.-T., Kung, H.-N., Chen, C.-K., Huang, C., Wang, Y.-L., Yu, C.-P., Lee, C.-P. Improving nuclear envelope dynamics by EBV BFRF1 facilitates intranuclear component clearance through autophagy.

Regulation of Exocytotic Fusion Pores by SNARE Protein Transmembrane Domains

PubMed Central

Wu, Zhenyong; Thiyagarajan, Sathish; O’Shaughnessy, Ben; Karatekin, Erdem

2017-01-01

Calcium-triggered exocytotic release of neurotransmitters and hormones from neurons and neuroendocrine cells underlies neuronal communication, motor activity and endocrine functions. The core of the neuronal exocytotic machinery is composed of soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs). Formation of complexes between vesicle-attached v- and plasma-membrane anchored t-SNAREs in a highly regulated fashion brings the membranes into close apposition. Small, soluble proteins called Complexins (Cpx) and calcium-sensing Synaptotagmins cooperate to block fusion at low resting calcium concentrations, but trigger release upon calcium increase. A growing body of evidence suggests that the transmembrane domains (TMDs) of SNARE proteins play important roles in regulating the processes of fusion and release, but the mechanisms involved are only starting to be uncovered. Here we review recent evidence that SNARE TMDs exert influence by regulating the dynamics of the fusion pore, the initial aqueous connection between the vesicular lumen and the extracellular space. Even after the fusion pore is established, hormone release by neuroendocrine cells is tightly controlled, and the same may be true of neurotransmitter release by neurons. The dynamics of the fusion pore can regulate the kinetics of cargo release and the net amount released, and can determine the mode of vesicle recycling. Manipulations of SNARE TMDs were found to affect fusion pore properties profoundly, both during exocytosis and in biochemical reconstitutions. To explain these effects, TMD flexibility, and interactions among TMDs or between TMDs and lipids have been invoked. Exocytosis has provided the best setting in which to unravel the underlying mechanisms, being unique among membrane fusion reactions in that single fusion pores can be probed using high-resolution methods. An important role will likely be played by methods that can probe single fusion pores in a biochemically

Naked2 Acts as a Cargo Recognition and Targeting Protein to Ensure Proper Delivery and Fusion of TGF-α–containing Exocytic Vesicles at the Lower Lateral Membrane of Polarized MDCK Cells

PubMed Central

Li, Cunxi; Hao, Mingming; Cao, Zheng; Ding, Wei; Graves-Deal, Ramona; Hu, Jianyong; Piston, David W.

2007-01-01

Transforming growth factor-α (TGF-α) is the major autocrine EGF receptor ligand in vivo. In polarized epithelial cells, proTGF-α is synthesized and then delivered to the basolateral cell surface. We previously reported that Naked2 interacts with basolateral sorting determinants in the cytoplasmic tail of a Golgi-processed form of TGF-α and that TGF-α is not detected at the basolateral surface of Madin-Darby canine kidney (MDCK) cells expressing myristoylation-deficient (G2A) Naked2. By high-resolution microscopy, we now show that wild-type, but not G2A, Naked2-associated vesicles fuse at the plasma membrane. We further demonstrate that Naked2-associated vesicles are delivered to the lower lateral membrane of polarized MDCK cells independent of μ1B adaptin. We identify a basolateral targeting segment within Naked2; residues 1-173 redirect NHERF-1 from the apical cytoplasm to the basolateral membrane, and internal deletion of residues 37-104 results in apical mislocalization of Naked2 and TGF-α. Short hairpin RNA knockdown of Naked2 leads to a dramatic reduction in the 16-kDa cell surface isoform of TGF-α and increased cytosolic TGF-α immunoreactivity. We propose that Naked2 acts as a cargo recognition and targeting (CaRT) protein to ensure proper delivery, tethering, and fusion of TGF-α–containing vesicles to a distinct region at the basolateral surface of polarized epithelial cells. PMID:17553928

Fusion activity of influenza virus PR8/34 correlates with a temperature-induced conformational change within the hemagglutinin ectodomain detected by photochemical labeling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brunner, J.; Zugliani, C.; Mischler, R.

1991-03-05

Fusion of influenza viruses with membranes is catalyzed by the viral spike protein hemagglutinin (HA). Under mildly acidic conditions ({approximately}pH 5) this protein undergoes a conformational change that triggers the exposure of the fusion peptide, the hydrophobic N-terminal segment of the HA2 polypeptide chain. Insertion of this segment into the target membrane (or viral membrane ) is likely to represent a key step along the fusion pathway, but the details are far from being clear. The photoreactive phospholipid 1-palmitoyl-2-(11-(4-(3-(trifluoromethyl)diazirinyl)phenyl)(2-{sup 3}H)undecanoyl)-sn-glycero-3-phosphocholine (({sup 3}H)PTPC/11), inserted into the bilayer of large unilamellar vesicles (LUVs), allowed the authors to investigate both the interaction ofmore » viruses with the vesicles under perfusion conditions and the fusion process itself occurring at elevated temperatures only. Despite the observed binding of viruses to LUVs at pH 5 and 0C, labeling of HA2 was very weak. They have studied also the effect of temperature on the acid-induced (pH 5) interaction of bromelain-solubilized HA (BHA) with vesicles.« less

New sterically stabilized vesicles based on nonionic surfactant, cholesterol, and poly(ethylene glycol)-cholesterol conjugates.

PubMed Central

Beugin, S; Edwards, K; Karlsson, G; Ollivon, M; Lesieur, S

1998-01-01

Monomethoxypoly(ethylene glycol) cholesteryl carbonates (M-PEG-Chol) with polymer chain molecular weights of 1000 (M-PEG1000-Chol) and 2000 (M-PEG2000-Chol) have been newly synthesized and characterized. Their aggregation behavior in mixture with diglycerol hexadecyl ether (C16G2) and cholesterol has been examined by cryotransmission electron microscopy, high-performance gel exclusion chromatography, and quasielastic light scattering. Nonaggregated, stable, unilamellar vesicles were obtained at low polymer levels with optimal shape and size homogeneity at cholesteryl conjugate/ lipids ratios of 10 mol% M-PEG1000-Chol or 5 mol% M-PEG2000-Chol, corresponding to the theoretically predicted brush conformational state of the PEG chains. At 20 mol% M-PEG1000-Chol or 10 mol% M-PEG2000-Chol, the saturation threshold of the C16G2/cholesterol membrane in polymer is exceeded, and open disk-shaped aggregates are seen in coexistence with closed vesicles. Higher levels up to 30 mol% lead to the complete solubilization of the vesicles into disk-like structures of decreasing size with increasing PEG content. This study underlines the bivalent role of M-PEG-Chol derivatives: while behaving as solubilizing surfactants, they provide an efficient steric barrier, preventing the vesicles from aggregation and fusion over a period of at least 2 weeks. PMID:9635773

Outer Membrane Vesicles from Neisseria Meningitidis (Proteossome) Used for Nanostructured Zika Virus Vaccine Production.

PubMed

Martins, Paula; Machado, Daisy; Theizen, Thais Holtz; Guarnieri, João Paulo Oliveira; Bernardes, Bruno Gaia; Gomide, Gabriel Piccirillo; Corat, Marcus Alexandre Finzi; Abbehausen, Camilla; Módena, José Luiz Proença; Melo, Carlos Fernando Odir Rodrigues; Morishita, Karen Noda; Catharino, Rodrigo Ramos; Arns, Clarice Weis; Lancellotti, Marcelo

2018-05-29

The increase of Zika virus (ZIKV) infections in Brazil in the last two years leaves a prophylactic measures on alert for this new and emerging pathogen. Concerning of our positive experience, we developed a new prototype using Neisseria meningitidis outer membrane vesicles (OMV) on ZIKV cell growth in a fusion of OMV in the envelope of virus particles. The fusion of nanoparticles resulting from outer membrane vesicles of N. meningitidis with infected C6/36 cells line were analyzed by Nano tracking analysis (NTA), zeta potential, differential light scattering (DLS), scan and scanning transmission eletronic microscopy (SEM and STEM) and high resolution mass spectometry (HRMS) for nanostructure characterization. Also, the vaccination effects were viewed by immune response in mice protocols immunization (ELISA and inflammatory chemokines) confirmed by Zika virus soroneutralization test. The results of immunizations in mice showed that antibody production had a titer greater than 1:160 as compared to unvaccinated mice. The immune response of the adjuvant and non-adjuvant formulation activated the cellular immune response TH1 and TH2. In addition, the serum neutralization was able to prevent infection of virus particles in the glial tumor cell model (M059J). This research shows efficient strategies without recombinant technology or DNA vaccines.

A sequential vesicle pool model with a single release sensor and a Ca(2+)-dependent priming catalyst effectively explains Ca(2+)-dependent properties of neurosecretion.

PubMed

Walter, Alexander M; Pinheiro, Paulo S; Verhage, Matthijs; Sørensen, Jakob B

2013-01-01

Neurotransmitter release depends on the fusion of secretory vesicles with the plasma membrane and the release of their contents. The final fusion step displays higher-order Ca(2+) dependence, but also upstream steps depend on Ca(2+). After deletion of the Ca(2+) sensor for fast release - synaptotagmin-1 - slower Ca(2+)-dependent release components persist. These findings have provoked working models involving parallel releasable vesicle pools (Parallel Pool Models, PPM) driven by alternative Ca(2+) sensors for release, but no slow release sensor acting on a parallel vesicle pool has been identified. We here propose a Sequential Pool Model (SPM), assuming a novel Ca(2+)-dependent action: a Ca(2+)-dependent catalyst that accelerates both forward and reverse priming reactions. While both models account for fast fusion from the Readily-Releasable Pool (RRP) under control of synaptotagmin-1, the origins of slow release differ. In the SPM the slow release component is attributed to the Ca(2+)-dependent refilling of the RRP from a Non-Releasable upstream Pool (NRP), whereas the PPM attributes slow release to a separate slowly-releasable vesicle pool. Using numerical integration we compared model predictions to data from mouse chromaffin cells. Like the PPM, the SPM explains biphasic release, Ca(2+)-dependence and pool sizes in mouse chromaffin cells. In addition, the SPM accounts for the rapid recovery of the fast component after strong stimulation, where the PPM fails. The SPM also predicts the simultaneous changes in release rate and amplitude seen when mutating the SNARE-complex. Finally, it can account for the loss of fast- and the persistence of slow release in the synaptotagmin-1 knockout by assuming that the RRP is depleted, leading to slow and Ca(2+)-dependent fusion from the NRP. We conclude that the elusive 'alternative Ca(2+) sensor' for slow release might be the upstream priming catalyst, and that a sequential model effectively explains Ca(2+)-dependent

A Sequential Vesicle Pool Model with a Single Release Sensor and a Ca2+-Dependent Priming Catalyst Effectively Explains Ca2+-Dependent Properties of Neurosecretion

PubMed Central

Walter, Alexander M.; Pinheiro, Paulo S.; Verhage, Matthijs; Sørensen, Jakob B.

2013-01-01

Neurotransmitter release depends on the fusion of secretory vesicles with the plasma membrane and the release of their contents. The final fusion step displays higher-order Ca2+ dependence, but also upstream steps depend on Ca2+. After deletion of the Ca2+ sensor for fast release – synaptotagmin-1 – slower Ca2+-dependent release components persist. These findings have provoked working models involving parallel releasable vesicle pools (Parallel Pool Models, PPM) driven by alternative Ca2+ sensors for release, but no slow release sensor acting on a parallel vesicle pool has been identified. We here propose a Sequential Pool Model (SPM), assuming a novel Ca2+-dependent action: a Ca2+-dependent catalyst that accelerates both forward and reverse priming reactions. While both models account for fast fusion from the Readily-Releasable Pool (RRP) under control of synaptotagmin-1, the origins of slow release differ. In the SPM the slow release component is attributed to the Ca2+-dependent refilling of the RRP from a Non-Releasable upstream Pool (NRP), whereas the PPM attributes slow release to a separate slowly-releasable vesicle pool. Using numerical integration we compared model predictions to data from mouse chromaffin cells. Like the PPM, the SPM explains biphasic release, Ca2+-dependence and pool sizes in mouse chromaffin cells. In addition, the SPM accounts for the rapid recovery of the fast component after strong stimulation, where the PPM fails. The SPM also predicts the simultaneous changes in release rate and amplitude seen when mutating the SNARE-complex. Finally, it can account for the loss of fast- and the persistence of slow release in the synaptotagmin-1 knockout by assuming that the RRP is depleted, leading to slow and Ca2+-dependent fusion from the NRP. We conclude that the elusive ‘alternative Ca2+ sensor’ for slow release might be the upstream priming catalyst, and that a sequential model effectively explains Ca2+-dependent properties of

EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research.

PubMed

Van Deun, Jan; Mestdagh, Pieter; Agostinis, Patrizia; Akay, Özden; Anand, Sushma; Anckaert, Jasper; Martinez, Zoraida Andreu; Baetens, Tine; Beghein, Els; Bertier, Laurence; Berx, Geert; Boere, Janneke; Boukouris, Stephanie; Bremer, Michel; Buschmann, Dominik; Byrd, James B; Casert, Clara; Cheng, Lesley; Cmoch, Anna; Daveloose, Delphine; De Smedt, Eva; Demirsoy, Seyma; Depoorter, Victoria; Dhondt, Bert; Driedonks, Tom A P; Dudek, Aleksandra; Elsharawy, Abdou; Floris, Ilaria; Foers, Andrew D; Gärtner, Kathrin; Garg, Abhishek D; Geeurickx, Edward; Gettemans, Jan; Ghazavi, Farzaneh; Giebel, Bernd; Kormelink, Tom Groot; Hancock, Grace; Helsmoortel, Hetty; Hill, Andrew F; Hyenne, Vincent; Kalra, Hina; Kim, David; Kowal, Joanna; Kraemer, Sandra; Leidinger, Petra; Leonelli, Carina; Liang, Yaxuan; Lippens, Lien; Liu, Shu; Lo Cicero, Alessandra; Martin, Shaun; Mathivanan, Suresh; Mathiyalagan, Prabhu; Matusek, Támas; Milani, Gloria; Monguió-Tortajada, Marta; Mus, Liselot M; Muth, Dillon C; Németh, Andrea; Nolte-'t Hoen, Esther N M; O'Driscoll, Lorraine; Palmulli, Roberta; Pfaffl, Michael W; Primdal-Bengtson, Bjarke; Romano, Erminia; Rousseau, Quentin; Sahoo, Susmita; Sampaio, Natalia; Samuel, Monisha; Scicluna, Benjamin; Soen, Bieke; Steels, Anneleen; Swinnen, Johannes V; Takatalo, Maarit; Thaminy, Safia; Théry, Clotilde; Tulkens, Joeri; Van Audenhove, Isabel; van der Grein, Susanne; Van Goethem, Alan; van Herwijnen, Martijn J; Van Niel, Guillaume; Van Roy, Nadine; Van Vliet, Alexander R; Vandamme, Niels; Vanhauwaert, Suzanne; Vergauwen, Glenn; Verweij, Frederik; Wallaert, Annelynn; Wauben, Marca; Witwer, Kenneth W; Zonneveld, Marijke I; De Wever, Olivier; Vandesompele, Jo; Hendrix, An

2017-02-28

We argue that the field of extracellular vesicle (EV) biology needs more transparent reporting to facilitate interpretation and replication of experiments. To achieve this, we describe EV-TRACK, a crowdsourcing knowledgebase (http://evtrack.org) that centralizes EV biology and methodology with the goal of stimulating authors, reviewers, editors and funders to put experimental guidelines into practice.

Interactive, computer-assisted tracking of speckle trajectories in fluorescence microscopy: application to actin polymerization and membrane fusion.

PubMed

Smith, Matthew B; Karatekin, Erdem; Gohlke, Andrea; Mizuno, Hiroaki; Watanabe, Naoki; Vavylonis, Dimitrios

2011-10-05

Analysis of particle trajectories in images obtained by fluorescence microscopy reveals biophysical properties such as diffusion coefficient or rates of association and dissociation. Particle tracking and lifetime measurement is often limited by noise, large mobilities, image inhomogeneities, and path crossings. We present Speckle TrackerJ, a tool that addresses some of these challenges using computer-assisted techniques for finding positions and tracking particles in different situations. A dynamic user interface assists in the creation, editing, and refining of particle tracks. The following are results from application of this program: 1), Tracking single molecule diffusion in simulated images. The shape of the diffusing marker on the image changes from speckle to cloud, depending on the relationship of the diffusion coefficient to the camera exposure time. We use these images to illustrate the range of diffusion coefficients that can be measured. 2), We used the program to measure the diffusion coefficient of capping proteins in the lamellipodium. We found values ∼0.5 μm(2)/s, suggesting capping protein association with protein complexes or the membrane. 3), We demonstrate efficient measuring of appearance and disappearance of EGFP-actin speckles within the lamellipodium of motile cells that indicate actin monomer incorporation into the actin filament network. 4), We marked appearance and disappearance events of fluorescently labeled vesicles to supported lipid bilayers and tracked single lipids from the fused vesicle on the bilayer. This is the first time, to our knowledge, that vesicle fusion has been detected with single molecule sensitivity and the program allowed us to perform a quantitative analysis. 5), By discriminating between undocking and fusion events, dwell times for vesicle fusion after vesicle docking to membranes can be measured. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Towards reconstitution of membrane fusion mediated by SNAREs and other synaptic proteins

PubMed Central

Brunger, Axel T.; Cipriano, Daniel J.; Diao, Jiajie

2015-01-01

Abstract Proteoliposomes have been widely used for in vitro studies of membrane fusion mediated by synaptic proteins. Initially, such studies were made with large unsynchronized ensembles of vesicles. Such ensemble assays limited the insights into the SNARE-mediated fusion mechanism that could be obtained from them. Single particle microscopy experiments can alleviate many of these limitations but they pose significant technical challenges. Here we summarize various approaches that have enabled studies of fusion mediated by SNAREs and other synaptic proteins at a single-particle level. Currently available methods are described and their advantages and limitations are discussed. PMID:25788028

Membrane Fusion Induced by Small Molecules and Ions

PubMed Central

Mondal Roy, Sutapa; Sarkar, Munna

2011-01-01

Membrane fusion is a key event in many biological processes. These processes are controlled by various fusogenic agents of which proteins and peptides from the principal group. The fusion process is characterized by three major steps, namely, inter membrane contact, lipid mixing forming the intermediate step, pore opening and finally mixing of inner contents of the cells/vesicles. These steps are governed by energy barriers, which need to be overcome to complete fusion. Structural reorganization of big molecules like proteins/peptides, supplies the required driving force to overcome the energy barrier of the different intermediate steps. Small molecules/ions do not share this advantage. Hence fusion induced by small molecules/ions is expected to be different from that induced by proteins/peptides. Although several reviews exist on membrane fusion, no recent review is devoted solely to small moleculs/ions induced membrane fusion. Here we intend to present, how a variety of small molecules/ions act as independent fusogens. The detailed mechanism of some are well understood but for many it is still an unanswered question. Clearer understanding of how a particular small molecule can control fusion will open up a vista to use these moleucles instead of proteins/peptides to induce fusion both in vivo and in vitro fusion processes. PMID:21660306

Vesicle-MaNiA: extracellular vesicles in liquid biopsy and cancer

PubMed Central

Torrano, Veronica; Royo, Felix; Peinado, Héctor; Loizaga-Iriarte, Ana; Unda, Miguel; Falcón-Perez, Juan M.; Carracedo, Arkaitz

2016-01-01

Normal and tumor cells shed vesicles to the environment. Within the large family of extracellular vesicles, exosomes and microvesicles have attracted much attention in the recent years. Their interest ranges from mediators of cancer progression, inflammation, immune regulation and metastatic niche regulation, to non-invasive biomarkers of disease. In this respect, the procedures to purify and analyze extracellular vesicles have quickly evolved and represent a source of variability for data integration in the field. In this review, we provide an updated view of the potential of exosomes and microvesicles as biomarkers and the available technologies for their isolation. PMID:27366992

Single calcium channel domain gating of synaptic vesicle fusion at fast synapses; analysis by graphic modeling

PubMed Central

Stanley, Elise F

2015-01-01

At fast-transmitting presynaptic terminals Ca2+ enter through voltage gated calcium channels (CaVs) and bind to a synaptic vesicle (SV) -associated calcium sensor (SV-sensor) to gate fusion and discharge. An open CaV generates a high-concentration plume, or nanodomain of Ca2+ that dissipates precipitously with distance from the pore. At most fast synapses, such as the frog neuromuscular junction (NMJ), the SV sensors are located sufficiently close to individual CaVs to be gated by single nanodomains. However, at others, such as the mature rodent calyx of Held (calyx of Held), the physiology is more complex with evidence that CaVs that are both close and distant from the SV sensor and it is argued that release is gated primarily by the overlapping Ca2+ nanodomains from many CaVs. We devised a 'graphic modeling' method to sum Ca2+ from individual CaVs located at varying distances from the SV-sensor to determine the SV release probability and also the fraction of that probability that can be attributed to single domain gating. This method was applied first to simplified, low and high CaV density model release sites and then to published data on the contrasting frog NMJ and the rodent calyx of Held native synapses. We report 3 main predictions: the SV-sensor is positioned very close to the point at which the SV fuses with the membrane; single domain-release gating predominates even at synapses where the SV abuts a large cluster of CaVs, and even relatively remote CaVs can contribute significantly to single domain-based gating. PMID:26457441

Vibrio effector protein VopQ inhibits fusion of V-ATPase–containing membranes

PubMed Central

Sreelatha, Anju; Bennett, Terry L.; Carpinone, Emily M.; O’Brien, Kevin M.; Jordan, Kamyron D.; Burdette, Dara L.; Orth, Kim; Starai, Vincent J.

2015-01-01

Vesicle fusion governs many important biological processes, and imbalances in the regulation of membrane fusion can lead to a variety of diseases such as diabetes and neurological disorders. Here we show that the Vibrio parahaemolyticus effector protein VopQ is a potent inhibitor of membrane fusion based on an in vitro yeast vacuole fusion model. Previously, we demonstrated that VopQ binds to the Vo domain of the conserved V-type H+-ATPase (V-ATPase) found on acidic compartments such as the yeast vacuole. VopQ forms a nonspecific, voltage-gated membrane channel of 18 Å resulting in neutralization of these compartments. We now present data showing that VopQ inhibits yeast vacuole fusion. Furthermore, we identified a unique mutation in VopQ that delineates its two functions, deacidification and inhibition of membrane fusion. The use of VopQ as a membrane fusion inhibitor in this manner now provides convincing evidence that vacuole fusion occurs independently of luminal acidification in vitro. PMID:25453092

Vibrio effector protein VopQ inhibits fusion of V-ATPase-containing membranes.

PubMed

Sreelatha, Anju; Bennett, Terry L; Carpinone, Emily M; O'Brien, Kevin M; Jordan, Kamyron D; Burdette, Dara L; Orth, Kim; Starai, Vincent J

2015-01-06

Vesicle fusion governs many important biological processes, and imbalances in the regulation of membrane fusion can lead to a variety of diseases such as diabetes and neurological disorders. Here we show that the Vibrio parahaemolyticus effector protein VopQ is a potent inhibitor of membrane fusion based on an in vitro yeast vacuole fusion model. Previously, we demonstrated that VopQ binds to the V(o) domain of the conserved V-type H(+)-ATPase (V-ATPase) found on acidic compartments such as the yeast vacuole. VopQ forms a nonspecific, voltage-gated membrane channel of 18 Å resulting in neutralization of these compartments. We now present data showing that VopQ inhibits yeast vacuole fusion. Furthermore, we identified a unique mutation in VopQ that delineates its two functions, deacidification and inhibition of membrane fusion. The use of VopQ as a membrane fusion inhibitor in this manner now provides convincing evidence that vacuole fusion occurs independently of luminal acidification in vitro.

Posing for a picture: vesicle immobilization in agarose gel

NASA Astrophysics Data System (ADS)

Lira, Rafael B.; Steinkühler, Jan; Knorr, Roland L.; Dimova, Rumiana; Riske, Karin A.

2016-05-01

Taking a photo typically requires the object of interest to stand still. In science, imaging is potentiated by optical and electron microscopy. However, living and soft matter are not still. Thus, biological preparations for microscopy usually include a fixation step. Similarly, immobilization strategies are required for or substantially facilitate imaging of cells or lipid vesicles, and even more so for acquiring high-quality data via fluorescence-based techniques. Here, we describe a simple yet efficient method to immobilize objects such as lipid vesicles with sizes between 0.1 and 100 μm using agarose gel. We show that while large and giant unilamellar vesicles (LUVs and GUVs) can be caged in the pockets of the gel meshwork, small molecules, proteins and micelles remain free to diffuse through the gel and interact with membranes as in agarose-free solutions, and complex biochemical reactions involving several proteins can proceed in the gel. At the same time, immobilization in agarose has no adverse effect on the GUV size and stability. By applying techniques such as FRAP and FCS, we show that the lateral diffusion of lipids is not affected by the gel. Finally, our immobilization strategy allows capturing high-resolution 3D images of GUVs.

Single-vesicle imaging reveals different transport mechanisms between glutamatergic and GABAergic vesicles.

PubMed

Farsi, Zohreh; Preobraschenski, Julia; van den Bogaart, Geert; Riedel, Dietmar; Jahn, Reinhard; Woehler, Andrew

2016-02-26

Synaptic transmission is mediated by the release of neurotransmitters, which involves exo-endocytotic cycling of synaptic vesicles. To maintain synaptic function, synaptic vesicles are refilled with thousands of neurotransmitter molecules within seconds after endocytosis, using the energy provided by an electrochemical proton gradient. However, it is unclear how transmitter molecules carrying different net charges can be efficiently sequestered while maintaining charge neutrality and osmotic balance. We used single-vesicle imaging to monitor pH and electrical gradients and directly showed different uptake mechanisms for glutamate and γ-aminobutyric acid (GABA) operating in parallel. In contrast to glutamate, GABA was exchanged for protons, with no other ions participating in the transport cycle. Thus, only a few components are needed to guarantee reliable vesicle filling with different neurotransmitters. Copyright © 2016, American Association for the Advancement of Science.

Vesicle-MaNiA: extracellular vesicles in liquid biopsy and cancer.

PubMed

Torrano, Veronica; Royo, Felix; Peinado, Héctor; Loizaga-Iriarte, Ana; Unda, Miguel; Falcón-Perez, Juan M; Carracedo, Arkaitz

2016-08-01

Normal and tumor cells shed vesicles to the environment. Within the large family of extracellular vesicles, exosomes and microvesicles have attracted much attention in the recent years. Their interest ranges from mediators of cancer progression, inflammation, immune regulation and metastatic niche regulation, to non-invasive biomarkers of disease. In this respect, the procedures to purify and analyze extracellular vesicles have quickly evolved and represent a source of variability for data integration in the field. In this review, we provide an updated view of the potential of exosomes and microvesicles as biomarkers and the available technologies for their isolation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Vesicle Docking Is a Key Target of Local PI(4,5)P2 Metabolism in the Secretory Pathway of INS-1 Cells.

PubMed

Ji, Chen; Fan, Fan; Lou, Xuelin

2017-08-08

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) signaling is transient and spatially confined in live cells. How this pattern of signaling regulates transmitter release and hormone secretion has not been addressed. We devised an optogenetic approach to control PI(4,5)P 2 levels in time and space in insulin-secreting cells. Combining this approach with total internal reflection fluorescence microscopy, we examined individual vesicle-trafficking steps. Unlike long-term PI(4,5)P 2 perturbations, rapid and cell-wide PI(4,5)P 2 reduction in the plasma membrane (PM) strongly inhibits secretion and intracellular Ca 2+ concentration ([Ca 2+ ] i ) responses, but not sytaxin1a clustering. Interestingly, local PI(4,5)P 2 reduction selectively at vesicle docking sites causes remarkable vesicle undocking from the PM without affecting [Ca 2+ ] i . These results highlight a key role of local PI(4,5)P 2 in vesicle tethering and docking, coordinated with its role in priming and fusion. Thus, different spatiotemporal PI(4,5)P 2 signaling regulates distinct steps of vesicle trafficking, and vesicle docking may be a key target of local PI(4,5)P 2 signaling in vivo. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Activation of Rab GTPase Sec4 by its GEF Sec2 is required for prospore membrane formation during sporulation in yeast Saccharomyces cerevisiae.

PubMed

Suda, Yasuyuki; Tachikawa, Hiroyuki; Inoue, Ichiro; Kurita, Tomokazu; Saito, Chieko; Kurokawa, Kazuo; Nakano, Akihiko; Irie, Kenji

2018-02-01

Sec2 activates Sec4 Rab GTPase as a guanine nucleotide exchange factor for the recruitment of downstream effectors to facilitate tethering and fusion of post-Golgi vesicles at the plasma membrane. During the meiosis and sporulation of budding yeast, post-Golgi vesicles are transported to and fused at the spindle pole body (SPB) to form a de novo membrane, called the prospore membrane. Previous studies have revealed the role of the SPB outer surface called the meiotic outer plaque (MOP) in docking and fusion of post-Golgi vesicles. However, the upstream molecular machinery for post-Golgi vesicular fusion that facilitates prospore membrane formation remains enigmatic. Here, we demonstrate that the GTP exchange factor for Sec4, Sec2, participates in the formation of the prospore membrane. A conditional mutant in which the SEC2 expression is shut off during sporulation showed sporulation defects. Inactivation of Sec2 caused Sec4 targeting defects along the prospore membranes, thereby causing insufficient targeting of downstream effectors and cargo proteins to the prospore membrane. These results suggest that the activation of Sec4 by Sec2 is required for the efficient supply of post-Golgi vesicles to the prospore membrane and thus for prospore membrane formation/extension and subsequent deposition of spore wall materials. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The Vici Syndrome Protein EPG5 Is a Rab7 Effector that Determines the Fusion Specificity of Autophagosomes with Late Endosomes/Lysosomes.

PubMed

Wang, Zheng; Miao, Guangyan; Xue, Xue; Guo, Xiangyang; Yuan, Chongzhen; Wang, Zhaoyu; Zhang, Gangming; Chen, Yingyu; Feng, Du; Hu, Junjie; Zhang, Hong

2016-09-01

Mutations in the human autophagy gene EPG5 cause the multisystem disorder Vici syndrome. Here we demonstrated that EPG5 is a Rab7 effector that determines the fusion specificity of autophagosomes with late endosomes/lysosomes. EPG5 is recruited to late endosomes/lysosomes by direct interaction with Rab7 and the late endosomal/lysosomal R-SNARE VAMP7/8. EPG5 also binds to LC3/LGG-1 (mammalian and C. elegans Atg8 homolog, respectively) and to assembled STX17-SNAP29 Qabc SNARE complexes on autophagosomes. EPG5 stabilizes and facilitates the assembly of STX17-SNAP29-VAMP7/8 trans-SNARE complexes, and promotes STX17-SNAP29-VAMP7-mediated fusion of reconstituted proteoliposomes. Loss of EPG5 activity causes abnormal fusion of autophagosomes with various endocytic vesicles, in part due to elevated assembly of STX17-SNAP25-VAMP8 complexes. SNAP25 knockdown partially suppresses the autophagy defect caused by EPG5 depletion. Our study reveals that EPG5 is a Rab7 effector involved in autophagosome maturation, providing insight into the molecular mechanism underlying Vici syndrome. Copyright © 2016 Elsevier Inc. All rights reserved.

Dynamics of small unilamellar vesicles

NASA Astrophysics Data System (ADS)

Hoffmann, Ingo; Hoffmann, Claudia; Farago, Bela; Prévost, Sylvain; Gradzielski, Michael

2018-03-01

In this paper, we investigate the dynamics of small unilamellar vesicles with the aid of neutron spin-echo spectroscopy. The purpose of this investigation is twofold. On the one hand, we investigate the influence of solubilised cosurfactant on the dynamics of the vesicle's surfactant bilayer. On the other hand, the small unilamellar vesicles used here have a size between larger vesicles, with dynamics being well described by the Zilman-Granek model and smaller microemulsion droplets which can be described by the Milner-Safran model. Therefore, we want to elucidate the question, which model is more suitable for the description of the membrane dynamics of small vesicles, where the finite curvature of the bilayer is felt by the contained amphiphilic molecules. This question is of substantial relevance for our understanding of membranes and how their dynamics is affected by curvature, a problem that is also of key importance in a number of biological questions. Our results indicate the even down to vesicle radii of 20 nm the Zilman-Granek model appears to be the more suitable one.

Identification of a human synaptotagmin-1 mutation that perturbs synaptic vesicle cycling

PubMed Central

Baker, Kate; Gordon, Sarah L.; Grozeva, Detelina; van Kogelenberg, Margriet; Roberts, Nicola Y.; Pike, Michael; Blair, Edward; Hurles, Matthew E.; Chong, W. Kling; Baldeweg, Torsten; Kurian, Manju A.; Boyd, Stewart G.; Cousin, Michael A.; Raymond, F. Lucy

2015-01-01

Synaptotagmin-1 (SYT1) is a calcium-binding synaptic vesicle protein that is required for both exocytosis and endocytosis. Here, we describe a human condition associated with a rare variant in SYT1. The individual harboring this variant presented with an early onset dyskinetic movement disorder, severe motor delay, and profound cognitive impairment. Structural MRI was normal, but EEG showed extensive neurophysiological disturbances that included the unusual features of low-frequency oscillatory bursts and enhanced paired-pulse depression of visual evoked potentials. Trio analysis of whole-exome sequence identified a de novo SYT1 missense variant (I368T). Expression of rat SYT1 containing the equivalent human variant in WT mouse primary hippocampal cultures revealed that the mutant form of SYT1 correctly localizes to nerve terminals and is expressed at levels that are approximately equal to levels of endogenous WT protein. The presence of the mutant SYT1 slowed synaptic vesicle fusion kinetics, a finding that agrees with the previously demonstrated role for I368 in calcium-dependent membrane penetration. Expression of the I368T variant also altered the kinetics of synaptic vesicle endocytosis. Together, the clinical features, electrophysiological phenotype, and in vitro neuronal phenotype associated with this dominant negative SYT1 mutation highlight presynaptic mechanisms that mediate human motor control and cognitive development. PMID:25705886

Molecular scaffold reorganization at the transmitter release site with vesicle exocytosis or botulinum toxin C1.

PubMed

Stanley, Elise F; Reese, Tom S; Wang, Gary Z

2003-10-01

Neurotransmitter release sites at the freeze-fractured frog neuromuscular junction are composed of inner and outer paired rows of large membrane particles, the putative calcium channels, anchored by the ribs of an underlying protein scaffold. We analysed the locations of the release site particles as a reflection of the scaffold structure, comparing particle distributions in secreting terminals with those where secretion was blocked with botulinum toxin A, which cleaves a small segment off SNAP-25, or botulinum toxin C1, which cleaves the cytoplasmic domain of syntaxin. In the idle terminal the inner and outer paired rows were located approximately 25 and approximately 44 nm, respectively, from the release site midline. However, adjacent to vesicular fusion sites both particle rows were displaced towards the midline by approximately 25%. The intervals between the particles along each row were examined by a nearest-neighbour approach. In control terminals the peak interval along the inner row was approximately 17 nm, consistent with previous reports and the spacing of the scaffold ribs. While the average distance between particles in the outer row was also approximately 17 nm, a detailed analysis revealed short 'linear clusters' with a approximately 14 nm interval. These clusters were enriched at vesicle fusion sites, suggesting an association with the docking sites, and were eliminated by botulinum C1, but not A. Our findings suggest, first, that the release site scaffold ribs undergo a predictable, and possibly active, shortening during exocytosis and, second, that at the vesicle docking site syntaxin plays a role in the cross-linking of the rib tips to form the vesicle docking sites.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hasan, Nazarul; Hu, Chuan, E-mail: chuan.hu@louisville.edu

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 cellmore » 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.« less

NO and CO binding profiles of hemoglobin vesicles as artificial oxygen carriers.

PubMed

Sakai, Hiromi; Sato, Atsushi; Sobolewski, Peter; Takeoka, Shinji; Frangos, John A; Kobayashi, Koichi; Intaglietta, Marcos; Tsuchida, Eishun

2008-10-01

Hemoglobin vesicles (HbVs) are artificial oxygen carriers encapsulating purified and concentrated Hb solution in phospholipid vesicles (liposomes). We examined in-vitro reaction profiles of a formulation of HbV with NO and CO in anaerobic and aerobic conditions using stopped-flow spectrophotometry and a NO electrode. Reaction rate constants of NO to deoxygenated and oxygenated HbV were considerably smaller than those of cell-free Hb because of the intracellular NO-diffusion barrier. The reaction of CO with deoxygenated HbV was slightly slower than that of cell-free Hb solely because of the co-encapsulated allosteric effector, pyridoxal 5'-phosphate. The NO depletion in an aerobic condition in the presence of empty vesicles was monitored using a NO electrode, showing that the hydrophobic bilayer membrane of HbV, which might have higher gas solubility, does not markedly facilitate the O(2) and NO reaction, and that the intracellular Hb is the major component of NO depletion. In conclusion, HbV shows retarded gas reactions, providing some useful information to explain the absence of vasoconstriction and hypertension when they are intravenously injected.

Coupling Between Metabolism and Compartmentalization: Vesicle Growth in the Presence of Dipeptides

NASA Technical Reports Server (NTRS)

Wei, C.; Pohorille, A.

2017-01-01

A fundamental unresolved question in studies on the origin of life is: how different, ubiquitous protocellular functions begun to work in concert setting the stage for Darwinian evolution of nascent life? From this perspective, of particular significance is coupling between growth of protocellular compartments and the encapsulated, primordial metabolism, which is one of the focal topics of the current ISSOL meeting. Specifically, growth and division of cells facilitated by the products of a metabolic reaction would confer an evolutionary advantage on protocells encapsulating this reaction, as their population would increase at the expense of other protocells. Along these lines, Adamala and Szostak have recently demonstrated that a dipeptide captured inside fatty acid vesicles catalyzes the formation of other dipeptides from activated monomers. Some of the newly synthesized dipeptides, in turn, are capable to promote competitive growth of vesicles in the presence of fatty acid micelles. As vesicles become larger, they adapt filamentous shape, which has been shown to promote their division. On the basis of computer simulations, we provide a molecularly detailed explanation of this process and draw conclusions about its generality.

GABARAP-mediated targeting of PI4K2A/PI4KIIα to autophagosomes regulates PtdIns4P-dependent autophagosome-lysosome fusion.

PubMed

Albanesi, Joseph; Wang, Hanzhi; Sun, Hui-Qiao; Levine, Beth; Yin, Helen

2015-11-02

For decades, phosphatidylinositol 4-phosphate (PtdIns4P) was considered primarily as a precursor in the synthesis of phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P 2 ). More recently, specific functions for PtdIns4P itself have been identified, particularly in the regulation of intracellular membrane trafficking. PI4K2A/PI4KIIα (phosphatidylinositol 4-kinase type 2 α), one of the 4 enzymes that catalyze PtdIns4P production in mammalian cells, promotes vesicle formation from the trans-Golgi network (TGN) and endosomes. We recently identified a novel function for PI4K2A-derived PtdIns4P, as a facilitator of autophagosome-lysosome (A-L) fusion. We further showed that that this function requires the presence of the autophagic adaptor protein GABARAP (GABA[A] receptor-associated protein), which binds to PI4K2A and recruits it to autophagosomes. The mechanism whereby GABARAP-PI4K2A-PtdIns4P promotes A-L fusion remains to be defined. Based on other examples of phosphoinositide involvement in membrane trafficking, we speculate that it acts by recruiting elements of the membrane docking and fusion machinery.

Chemokine Signaling during Midline Epithelial Seam Disintegration Facilitates Palatal Fusion

PubMed Central

Suttorp, Christiaan M.; Cremers, Niels A.; van Rheden, René; Regan, Raymond F.; Helmich, Pia; van Kempen, Sven; Kuijpers-Jagtman, Anne M.; Wagener, Frank A.D.T.G.

2017-01-01

Disintegration of the midline epithelial seam (MES) is crucial for palatal fusion, and failure results in cleft palate. Palatal fusion and wound repair share many common signaling pathways related to epithelial-mesenchymal cross-talk. We postulate that chemokine CXCL11, its receptor CXCR3, and the cytoprotective enzyme heme oxygenase (HO), which are crucial during wound repair, also play a decisive role in MES disintegration. Fetal growth restriction and craniofacial abnormalities were present in HO-2 knockout (KO) mice without effects on palatal fusion. CXCL11 and CXCR3 were highly expressed in the disintegrating MES in both wild-type and HO-2 KO animals. Multiple apoptotic DNA fragments were present within the disintegrating MES and phagocytized by recruited CXCR3-positive wt and HO-2 KO macrophages. Macrophages located near the MES were HO-1-positive, and more HO-1-positive cells were present in HO-2 KO mice compared to wild-type. This study of embryonic and palatal development provided evidence that supports the hypothesis that the MES itself plays a prominent role in palatal fusion by orchestrating epithelial apoptosis and macrophage recruitment via CXCL11-CXCR3 signaling. PMID:29164113

Gene Fusion Markup Language: a prototype for exchanging gene fusion data.

PubMed

Kalyana-Sundaram, Shanker; Shanmugam, Achiraman; Chinnaiyan, Arul M

2012-10-16

An avalanche of next generation sequencing (NGS) studies has generated an unprecedented amount of genomic structural variation data. These studies have also identified many novel gene fusion candidates with more detailed resolution than previously achieved. However, in the excitement and necessity of publishing the observations from this recently developed cutting-edge technology, no community standardization approach has arisen to organize and represent the data with the essential attributes in an interchangeable manner. As transcriptome studies have been widely used for gene fusion discoveries, the current non-standard mode of data representation could potentially impede data accessibility, critical analyses, and further discoveries in the near future. Here we propose a prototype, Gene Fusion Markup Language (GFML) as an initiative to provide a standard format for organizing and representing the significant features of gene fusion data. GFML will offer the advantage of representing the data in a machine-readable format to enable data exchange, automated analysis interpretation, and independent verification. As this database-independent exchange initiative evolves it will further facilitate the formation of related databases, repositories, and analysis tools. The GFML prototype is made available at http://code.google.com/p/gfml-prototype/. The Gene Fusion Markup Language (GFML) presented here could facilitate the development of a standard format for organizing, integrating and representing the significant features of gene fusion data in an inter-operable and query-able fashion that will enable biologically intuitive access to gene fusion findings and expedite functional characterization. A similar model is envisaged for other NGS data analyses.

Gene Fusion Markup Language: a prototype for exchanging gene fusion data

PubMed Central

2012-01-01

Background An avalanche of next generation sequencing (NGS) studies has generated an unprecedented amount of genomic structural variation data. These studies have also identified many novel gene fusion candidates with more detailed resolution than previously achieved. However, in the excitement and necessity of publishing the observations from this recently developed cutting-edge technology, no community standardization approach has arisen to organize and represent the data with the essential attributes in an interchangeable manner. As transcriptome studies have been widely used for gene fusion discoveries, the current non-standard mode of data representation could potentially impede data accessibility, critical analyses, and further discoveries in the near future. Results Here we propose a prototype, Gene Fusion Markup Language (GFML) as an initiative to provide a standard format for organizing and representing the significant features of gene fusion data. GFML will offer the advantage of representing the data in a machine-readable format to enable data exchange, automated analysis interpretation, and independent verification. As this database-independent exchange initiative evolves it will further facilitate the formation of related databases, repositories, and analysis tools. The GFML prototype is made available at http://code.google.com/p/gfml-prototype/. Conclusion The Gene Fusion Markup Language (GFML) presented here could facilitate the development of a standard format for organizing, integrating and representing the significant features of gene fusion data in an inter-operable and query-able fashion that will enable biologically intuitive access to gene fusion findings and expedite functional characterization. A similar model is envisaged for other NGS data analyses. PMID:23072312

Overproduction of individual gas vesicle proteins perturbs flotation, antibiotic production and cell division in the enterobacterium Serratia sp. ATCC 39006.

PubMed

Monson, Rita E; Tashiro, Yosuke; Salmond, George P C

2016-09-01

Gas vesicles are intracellular proteinaceous organelles that facilitate bacterial colonization of static water columns. In the enterobacterium Serratia sp. ATCC 39006, gas vesicle formation requires the proteins GvpA1, GvpF1, GvpG, GvpA2, GvpK, GvpA3, GvpF2 and GvpF3 and the three gas vesicle regulatory proteins GvrA, GvrB and GvrC. Deletion of gvpC alters gas vesicle robustness and deletion of gvpN or gvpV results in small bicone vesicles. In this work, we assessed the impacts on gas vesicle formation when each of these 14 essential proteins was overexpressed. Overproduction of GvpF1, GvpF2, GvrA, GvrB or GvrC all resulted in significantly reduced gas vesicle synthesis. Perturbations in gas vesicle formation were also observed when GvpV and GvpA3 were in excess. In addition to impacts on gas vesicle formation, overproduction of GvrA or GvrB led to elevated biosynthesis of the tripyrrole pigment, prodigiosin, a secondary metabolite of increasing medical interest due to its antimalarial and anticancer properties. Finally, when GvpG was overexpressed, gas vesicles were still produced, but the cells exhibited a growth defect. Further analysis showed that induction of GvpG arrested cell growth and caused a drop in viable count, suggesting a possible physiological role for this protein linking gas vesicle biogenesis and binary fission. These combined results demonstrate that the stoichiometry of individual gas vesicle proteins is crucially important for controlled organelle morphogenesis and flotation and provides evidence for the first link between gas vesicle assembly and cell division, to our knowledge.

Viral membrane fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harrison, Stephen C., E-mail: harrison@crystal.harvard.edu

2015-05-15

Membrane fusion is an essential step when enveloped viruses enter cells. Lipid bilayer fusion requires catalysis to overcome a high kinetic barrier; viral fusion proteins are the agents that fulfill this catalytic function. Despite a variety of molecular architectures, these proteins facilitate fusion by essentially the same generic mechanism. Stimulated by a signal associated with arrival at the cell to be infected (e.g., receptor or co-receptor binding, proton binding in an endosome), they undergo a series of conformational changes. A hydrophobic segment (a “fusion loop” or “fusion peptide”) engages the target-cell membrane and collapse of the bridging intermediate thus formedmore » draws the two membranes (virus and cell) together. We know of three structural classes for viral fusion proteins. Structures for both pre- and postfusion conformations of illustrate the beginning and end points of a process that can be probed by single-virion measurements of fusion kinetics. - Highlights: • Viral fusion proteins overcome the high energy barrier to lipid bilayer merger. • Different molecular structures but the same catalytic mechanism. • Review describes properties of three known fusion-protein structural classes. • Single-virion fusion experiments elucidate mechanism.« less

Mathematical modeling of vesicle drug delivery systems 2: targeted vesicle interactions with cells, tumors, and the body.

PubMed

Ying, Chong T; Wang, Juntian; Lamm, Robert J; Kamei, Daniel T

2013-02-01

Vesicles have been studied for several years in their ability to deliver drugs. Mathematical models have much potential in reducing time and resources required to engineer optimal vesicles, and this review article summarizes these models that aid in understanding the ability of targeted vesicles to bind and internalize into cancer cells, diffuse into tumors, and distribute in the body. With regard to binding and internalization, radiolabeling and surface plasmon resonance experiments can be performed to determine optimal vesicle size and the number and type of ligands conjugated. Binding and internalization properties are also inputs into a mathematical model of vesicle diffusion into tumor spheroids, which highlights the importance of the vesicle diffusion coefficient and the binding affinity of the targeting ligand. Biodistribution of vesicles in the body, along with their half-life, can be predicted with compartmental models for pharmacokinetics that include the effect of targeting ligands, and these predictions can be used in conjunction with in vivo models to aid in the design of drug carriers. Mathematical models can prove to be very useful in drug carrier design, and our hope is that this review will encourage more investigators to combine modeling with quantitative experimentation in the field of vesicle-based drug delivery.

Sensing Exocytosis and Triggering Endocytosis at Synapses: Synaptic Vesicle Exocytosis–Endocytosis Coupling

PubMed Central

Lou, Xuelin

2018-01-01

The intact synaptic structure is critical for information processing in neural circuits. During synaptic transmission, rapid vesicle exocytosis increases the size of never terminals and endocytosis counteracts the increase. Accumulating evidence suggests that SV exocytosis and endocytosis are tightly connected in time and space during SV recycling, and this process is essential for synaptic function and structural stability. Research in the past has illustrated the molecular details of synaptic vesicle (SV) exocytosis and endocytosis; however, the mechanisms that timely connect these two fundamental events are poorly understood at central synapses. Here we discuss recent progress in SV recycling and summarize several emerging mechanisms by which synapses can “sense” the occurrence of exocytosis and timely initiate compensatory endocytosis. They include Ca2+ sensing, SV proteins sensing, and local membrane stress sensing. In addition, the spatial organization of endocytic zones adjacent to active zones provides a structural basis for efficient coupling between SV exocytosis and endocytosis. Through linking different endocytosis pathways with SV fusion, these mechanisms ensure necessary plasticity and robustness of nerve terminals to meet diverse physiological needs. PMID:29593500

Spontaneous charged lipid transfer between lipid vesicles.

PubMed

Richens, Joanna L; Tyler, Arwen I I; Barriga, Hanna M G; Bramble, Jonathan P; Law, Robert V; Brooks, Nicholas J; Seddon, John M; Ces, Oscar; O'Shea, Paul

2017-10-03

An assay to study the spontaneous charged lipid transfer between lipid vesicles is described. A donor/acceptor vesicle system is employed, where neutrally charged acceptor vesicles are fluorescently labelled with the electrostatic membrane probe Fluoresceinphosphatidylethanolamine (FPE). Upon addition of charged donor vesicles, transfer of negatively charged lipid occurs, resulting in a fluorescently detectable change in the membrane potential of the acceptor vesicles. Using this approach we have studied the transfer properties of a range of lipids, varying both the headgroup and the chain length. At the low vesicle concentrations chosen, the transfer follows a first-order process where lipid monomers are transferred presumably through the aqueous solution phase from donor to acceptor vesicle. The rate of transfer decreases with increasing chain length which is consistent with energy models previously reported for lipid monomer vesicle interactions. Our assay improves on existing methods allowing the study of a range of unmodified lipids, continuous monitoring of transfer and simplified experimental procedures.

Vesicles

MedlinePlus

... herpetiformis Chickenpox Contact dermatitis (may be caused by poison ivy) Herpes simplex (cold sores, genital herpes ) Herpes zoster ( ... available for certain conditions that cause vesicles, including poison ivy and cold sores. When to Contact a Medical ...

Signal transduction meets vesicle traffic: the software and hardware of GLUT4 translocation.

PubMed

Klip, Amira; Sun, Yi; Chiu, Tim Ting; Foley, Kevin P

2014-05-15

Skeletal muscle is the major tissue disposing of dietary glucose, a function regulated by insulin-elicited signals that impart mobilization of GLUT4 glucose transporters to the plasma membrane. This phenomenon, also central to adipocyte biology, has been the subject of intense and productive research for decades. We focus on muscle cell studies scrutinizing insulin signals and vesicle traffic in a spatiotemporal manner. Using the analogy of an integrated circuit to approach the intersection between signal transduction and vesicle mobilization, we identify signaling relays ("software") that engage structural/mechanical elements ("hardware") to enact the rapid mobilization and incorporation of GLUT4 into the cell surface. We emphasize how insulin signal transduction switches from tyrosine through lipid and serine phosphorylation down to activation of small G proteins of the Rab and Rho families, describe key negative regulation step of Rab GTPases through the GTPase-activating protein activity of the Akt substrate of 160 kDa (AS160), and focus on the mechanical effectors engaged by Rabs 8A and 10 (the molecular motor myosin Va), and the Rho GTPase Rac1 (actin filament branching and severing through Arp2/3 and cofilin). Finally, we illustrate how actin filaments interact with myosin 1c and α-Actinin4 to promote vesicle tethering as preamble to fusion with the membrane. Copyright © 2014 the American Physiological Society.

A molecular network for the transport of the TI-VAMP/VAMP7 vesicles from cell center to periphery.

PubMed

Burgo, Andrea; Proux-Gillardeaux, Véronique; Sotirakis, Emmanuel; Bun, Philippe; Casano, Alessandra; Verraes, Agathe; Liem, Ronald K H; Formstecher, Etienne; Coppey-Moisan, Maïté; Galli, Thierry

2012-07-17

The compartmental organization of eukaryotic cells is maintained dynamically by vesicular trafficking. SNARE proteins play a crucial role in intracellular membrane fusion and need to be targeted to their proper donor or acceptor membrane. The molecular mechanisms that allow for the secretory vesicles carrying the v-SNARE TI-VAMP/VAMP7 to leave the cell center, load onto microtubules, and reach the periphery to mediate exocytosis are largely unknown. Here, we show that the TI-VAMP/VAMP7 partner Varp, a Rab21 guanine nucleotide exchange factor, interacts with GolginA4 and the kinesin 1 Kif5A. Activated Rab21-GTP in turn binds to MACF1, an actin and microtubule regulator, which is itself a partner of GolginA4. These components are required for directed movement of TI-VAMP/VAMP7 vesicles from the cell center to the cell periphery. The molecular mechanisms uncovered here suggest an integrated view of the transport of vesicles carrying a specific v-SNARE toward the cell surface. Copyright © 2012 Elsevier Inc. All rights reserved.

Amyloglucosidase enzymatic reactivity inside lipid vesicles

PubMed Central

Li, Mian; Hanford, Michael J; Kim, Jin-Woo; Peeples, Tonya L

2007-01-01

Efficient functioning of enzymes inside liposomes would open new avenues for applications in biocatalysis and bioanalytical tools. In this study, the entrapment of amyloglucosidase (AMG) (EC 3.2.1.3) from Aspergillus niger into dipalmitoylphosphatidylcholine (DPPC) multilamellar vesicles (MLVs) and large unilamellar vesicles (LUVs) was investigated. Negative-stain, freeze-fracture, and cryo-transmission electron microscopy images verified vesicle formation in the presence of AMG. Vesicles with entrapped AMG were isolated from the solution by centrifugation, and vesicle lamellarity was identified using fluorescence laser confocal microscopy. The kinetics of starch hydrolysis by AMG was modeled for two different systems, free enzyme in aqueous solution and entrapped enzyme within vesicles in aqueous suspension. For the free enzyme system, intrinsic kinetics were described by a Michaelis-Menten kinetic model with product inhibition. The kinetic constants, Vmax and Km, were determined by initial velocity measurements, and Ki was obtained by fitting the model to experimental data of glucose concentration-time curves. Predicted concentration-time curves using these kinetic constants were in good agreement with experimental measurements. In the case of the vesicles, the time-dependence of product (glucose) formation was experimentally determined and simulated by considering the kinetic behavior of the enzyme and the permeation of substrate into the vesicle. Experimental results demonstrated that entrapped enzymes were much more stable than free enyzme. The entrapped enzyme could be recycled with retention of 60% activity after 3 cycles. These methodologies can be useful in evaluating other liposomal catalysis operations. PMID:18271982

Superresolution and pulse-chase imaging reveal the role of vesicle transport in polar growth of fungal cells

PubMed Central

Zhou, Lu; Evangelinos, Minoas; Wernet, Valentin; Eckert, Antonia F.; Ishitsuka, Yuji; Fischer, Reinhard; Nienhaus, G. Ulrich; Takeshita, Norio

2018-01-01

Polarized growth of filamentous fungi requires continuous transport of biomolecules to the hyphal tip. To this end, construction materials are packaged in vesicles and transported by motor proteins along microtubules and actin filaments. We have studied these processes with quantitative superresolution localization microscopy of live Aspergillus nidulans cells expressing the photoconvertible protein mEosFPthermo fused to the chitin synthase ChsB. ChsB is mainly located at the Spitzenkörper near the hyphal tip and produces chitin, a key component of the cell wall. We have visualized the pulsatory dynamics of the Spitzenkörper, reflecting vesicle accumulation before exocytosis and their subsequent fusion with the apical plasma membrane. Furthermore, high-speed pulse-chase imaging after photoconversion of mEosFPthermo in a tightly focused spot revealed that ChsB is transported with two different speeds from the cell body to the hyphal tip and vice versa. Comparative analysis using motor protein deletion mutants allowed us to assign the fast movements (7 to 10 μm s−1) to transport of secretory vesicles by kinesin-1, and the slower ones (2 to 7 μm s−1) to transport by kinesin-3 on early endosomes. Our results show how motor proteins ensure the supply of vesicles to the hyphal tip, where temporally regulated exocytosis results in stepwise tip extension. PMID:29387789

Structure-directing star-shaped block copolymers: supramolecular vesicles for the delivery of anticancer drugs.

PubMed

Yang, Chuan; Liu, Shao Qiong; Venkataraman, Shrinivas; Gao, Shu Jun; Ke, Xiyu; Chia, Xin Tian; Hedrick, James L; Yang, Yi Yan

2015-06-28

Amphiphilic polycarbonate/PEG copolymer with a star-like architecture was designed to facilitate a unique supramolecular transformation of micelles to vesicles in aqueous solution for the efficient delivery of anticancer drugs. The star-shaped amphipilic block copolymer was synthesized by initiating the ring-opening polymerization of trimethylene carbonate (TMC) from methyl cholate through a combination of metal-free organo-catalytic living ring-opening polymerization and post-polymerization chain-end derivatization strategies. Subsequently, the self-assembly of the star-like polymer in aqueous solution into nanosized vesicles for anti-cancer drug delivery was studied. DOX was physically encapsulated into vesicles by dialysis and drug loading level was significant (22.5% in weight) for DOX. Importantly, DOX-loaded nanoparticles self-assembled from the star-like copolymer exhibited greater kinetic stability and higher DOX loading capacity than micelles prepared from cholesterol-initiated diblock analogue. The advantageous disparity is believed to be due to the transformation of micelles (diblock copolymer) to vesicles (star-like block copolymer) that possess greater core space for drug loading as well as the ability of such supramolecular structures to encapsulate DOX. DOX-loaded vesicles effectively inhibited the proliferation of 4T1, MDA-MB-231 and BT-474 cells, with IC50 values of 10, 1.5 and 1.0mg/L, respectively. DOX-loaded vesicles injected into 4T1 tumor-bearing mice exhibited enhanced accumulation in tumor tissue due to the enhanced permeation and retention (EPR) effect. Importantly, DOX-loaded vesicles demonstrated greater tumor growth inhibition than free DOX without causing significant body weight loss or cardiotoxicity. The unique ability of the star-like copolymer emanating from the methyl cholate core provided the requisite modification in the block copolymer interfacial curvature to generate vesicles of high loading capacity for DOX with significant

LRRK2 phosphorylates pre-synaptic N-ethylmaleimide sensitive fusion (NSF) protein enhancing its ATPase activity and SNARE complex disassembling rate.

PubMed

Belluzzi, Elisa; Gonnelli, Adriano; Cirnaru, Maria-Daniela; Marte, Antonella; Plotegher, Nicoletta; Russo, Isabella; Civiero, Laura; Cogo, Susanna; Carrion, Maria Perèz; Franchin, Cinzia; Arrigoni, Giorgio; Beltramini, Mariano; Bubacco, Luigi; Onofri, Franco; Piccoli, Giovanni; Greggio, Elisa

2016-01-13

Lrrk2, a gene linked to Parkinson's disease, encodes a large scaffolding protein with kinase and GTPase activities implicated in vesicle and cytoskeletal-related processes. At the presynaptic site, LRRK2 associates with synaptic vesicles through interaction with a panel of presynaptic proteins. Here, we show that LRRK2 kinase activity influences the dynamics of synaptic vesicle fusion. We therefore investigated whether LRRK2 phosphorylates component(s) of the exo/endocytosis machinery. We have previously observed that LRRK2 interacts with NSF, a hexameric AAA+ ATPase that couples ATP hydrolysis to the disassembling of SNARE proteins allowing them to enter another fusion cycle during synaptic exocytosis. Here, we demonstrate that NSF is a substrate of LRRK2 kinase activity. LRRK2 phosphorylates full-length NSF at threonine 645 in the ATP binding pocket of D2 domain. Functionally, NSF phosphorylated by LRRK2 displays enhanced ATPase activity and increased rate of SNARE complex disassembling. Substitution of threonine 645 with alanine abrogates LRRK2-mediated increased ATPase activity. Given that the most common Parkinson's disease LRRK2 G2019S mutation displays increased kinase activity, our results suggest that mutant LRRK2 may impair synaptic vesicle dynamics via aberrant phosphorylation of NSF.

Self-recognition in corals facilitates deep-sea habitat engineering

USGS Publications Warehouse

Hennige, Sebastian J; Morrison, Cheryl L.; Form, Armin U.; Buscher, Janina; Kamenos, Nicholas A.; Roberts, J. Murray

2014-01-01

The ability of coral reefs to engineer complex three-dimensional habitats is central to their success and the rich biodiversity they support. In tropical reefs, encrusting coralline algae bind together substrates and dead coral framework to make continuous reef structures, but beyond the photic zone, the cold-water coral Lophelia pertusa also forms large biogenic reefs, facilitated by skeletal fusion. Skeletal fusion in tropical corals can occur in closely related or juvenile individuals as a result of non-aggressive skeletal overgrowth or allogeneic tissue fusion, but contact reactions in many species result in mortality if there is no ‘self-recognition’ on a broad species level. This study reveals areas of ‘flawless’ skeletal fusion in Lophelia pertusa, potentially facilitated by allogeneic tissue fusion, are identified as having small aragonitic crystals or low levels of crystal organisation, and strong molecular bonding. Regardless of the mechanism, the recognition of ‘self’ between adjacent L. pertusa colonies leads to no observable mortality, facilitates ecosystem engineering and reduces aggression-related energetic expenditure in an environment where energy conservation is crucial. The potential for self-recognition at a species level, and subsequent skeletal fusion in framework-forming cold-water corals is an important first step in understanding their significance as ecological engineers in deep-seas worldwide.

Functionalized Vesicles by Microfluidic Device.

PubMed

Vallejo, Derek; Lee, Shih-Hui; Lee, Abraham

2017-01-01

In recent years, lipid vesicles have become popular vehicles for the creation of biosensors. Vesicles can hold reaction components within a selective permeable membrane that provides an ideal environment for membrane protein biosensing elements. The lipid bilayer allows a protein to retain its native structure and function, and the membrane fluidity can allow for conformational changes and physiological interactions with target analytes. Here, we present two methods for the production of giant unilamellar vesicles (GUVs) within a microfluidic device that can be used as the basis for a biosensor. The vesicles are produced from water-in-oil-in-water (W/O/W) double emulsion templates using a nonvolatile oil phase. To create the GUVs, the oil can be removed via extraction with ethanol, or by altering the interfacial tension between the oil and carrier solution causing the oil to retract into a cap on one side of the structure, leaving behind an exposed lipid bilayer. Methods to integrate sensing elements and membrane protein pores onto the vesicles are also introduced in this work.

Multiple effects of sibutramine on ejaculation and on vas deferens and seminal vesicle contractility.

PubMed

Nojimoto, Fernanda D; Piffer, Renata C; Kiguti, Luiz Ricardo de A; Lameu, Claudiana; de Camargo, Antônio C M; Pereira, Oduvaldo C M; Pupo, André S

2009-09-15

Sibutramine is an inhibitor of norepinephrine and 5-HT reuptake largely used in the management of obesity. Although a fairly safe drug, postmarketing adverse effects of sibutramine were reported including abnormal ejaculation in men. This study investigates the effects of sibutramine on ejaculation and vas deferens and seminal vesicle contractility. Adult male rats received sibutramine (5; 20; or 50 mg kg(-1), ip) and after 60 min were exposed to receptive females for determination of ejaculation parameters. The vasa deferentia and seminal vesicles of untreated rats were mounted in isolated organ baths for recording of isometric contractions and HEK293 cells loaded with fluorescent calcium indicator were used to measure intracellular Ca(2+) transients. Sibutramine 5 and 20 mg kg(-1) reduced ejaculation latency whereas 50 mg kg(-1) increased ejaculation latency. Sibutramine 3 to 30 microM greatly increased the sensitivity of the seminal vesicle and vas deferens to norepinephrine, but at concentrations higher than 10 microM there were striking depressions of maximal contractions induced by norepinephrine, carbachol and CaCl(2). In HEK293 cells, sibutramine 10 to 100 microM inhibited intracellular Ca(2+) transients induced by carbachol. Depending on the doses, sibutramine either facilitates or inhibits ejaculation. Apart from its actions in the central nervous system, facilitation of ejaculation may result from augmented sensitivity of smooth muscles to norepinephrine while reductions of intracellular Ca(2+) may be involved in the delayed ejaculation observed with high doses of sibutramine.

Optogenetic Acidification of Synaptic Vesicles and Lysosomes

PubMed Central

Grauel, M. Katharina; Wozny, Christian; Bentz, Claudia; Blessing, Anja; Rosenmund, Tanja; Jentsch, Thomas J.; Schmitz, Dietmar; Hegemann, Peter; Rosenmund, Christian

2016-01-01

Acidification is required for the function of many intracellular organelles, but methods to acutely manipulate their intraluminal pH have not been available. Here we present a targeting strategy to selectively express the light-driven proton pump Arch3 on synaptic vesicles. Our new tool, pHoenix, can functionally replace endogenous proton pumps, enabling optogenetic control of vesicular acidification and neurotransmitter accumulation. Under physiological conditions, glutamatergic vesicles are nearly full, as additional vesicle acidification with pHoenix only slightly increased the quantal size. By contrast, we found that incompletely filled vesicles exhibited a lower release probability than full vesicles, suggesting preferential exocytosis of vesicles with high transmitter content. Our subcellular targeting approach can be transferred to other organelles, as demonstrated for a pHoenix variant that allows light-activated acidification of lysosomes. PMID:26551543

Optogenetic acidification of synaptic vesicles and lysosomes.

PubMed

Rost, Benjamin R; Schneider, Franziska; Grauel, M Katharina; Wozny, Christian; Bentz, Claudia; Blessing, Anja; Rosenmund, Tanja; Jentsch, Thomas J; Schmitz, Dietmar; Hegemann, Peter; Rosenmund, Christian

2015-12-01

Acidification is required for the function of many intracellular organelles, but methods to acutely manipulate their intraluminal pH have not been available. Here we present a targeting strategy to selectively express the light-driven proton pump Arch3 on synaptic vesicles. Our new tool, pHoenix, can functionally replace endogenous proton pumps, enabling optogenetic control of vesicular acidification and neurotransmitter accumulation. Under physiological conditions, glutamatergic vesicles are nearly full, as additional vesicle acidification with pHoenix only slightly increased the quantal size. By contrast, we found that incompletely filled vesicles exhibited a lower release probability than full vesicles, suggesting preferential exocytosis of vesicles with high transmitter content. Our subcellular targeting approach can be transferred to other organelles, as demonstrated for a pHoenix variant that allows light-activated acidification of lysosomes.

On the entry of an emerging arbovirus into host cells: Mayaro virus takes the highway to the cytoplasm through fusion with early endosomes and caveolae-derived vesicles

PubMed Central

Carvalho, Carlos A.M.; Silva, Jerson L.; Oliveira, Andréa C.

2017-01-01

Mayaro virus (MAYV) is an emergent sylvatic alphavirus in South America, related to sporadic outbreaks of a chikungunya-like human febrile illness accompanied by severe arthralgia. Despite its high potential for urban emergence, MAYV is still an obscure virus with scarce information about its infection cycle, including the corresponding early events. Even for prototypical alphaviruses, the cell entry mechanism still has some rough edges to trim: although clathrin-mediated endocytosis is quoted as the putative route, alternative paths as distinct as direct virus genome injection through the cell plasma membrane seems to be possible. Our aim was to clarify crucial details on the entry route exploited by MAYV to gain access into the host cell. Tracking the virus since its first contact with the surface of Vero cells by fluorescence microscopy, we show that its entry occurs by a fast endocytic process and relies on fusion with acidic endosomal compartments. Moreover, blocking clathrin-mediated endocytosis or depleting cholesterol from the cell membrane leads to a strong inhibition of viral infection, as assessed by plaque assays. Following this clue, we found that early endosomes and caveolae-derived vesicles are both implicated as target membranes for MAYV fusion. Our findings unravel the very first events that culminate in a productive infection by MAYV and shed light on potential targets for a rational antiviral therapy, besides providing a better comprehension of the entry routes exploited by alphaviruses to get into the cell. PMID:28462045

Rab proteins: The key regulators of intracellular vesicle transport

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhuin, Tanmay; Roy, Jagat Kumar, E-mail: jkroy@bhu.ac.in

2014-10-15

Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied bymore » cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. - Highlights: • Rab proteins regulate different signalling pathways. • Deregulation of Rabs is the fundamental causes of a variety of human diseases. • This paper gives potential directions in developing therapeutic targets. • This paper also gives ample directions for modulating pathways central to normal physiology. • These are the huge challenges for drug discovery and delivery in near future.« less

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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Insulin Recruits GLUT4 from Specialized VAMP2-carrying Vesicles as well as from the Dynamic Endosomal/Trans-Golgi Network in Rat Adipocytes.

PubMed Central

Ramm, Georg; Slot, Jan Willem; James, David E.; Stoorvogel, Willem

2000-01-01

Insulin treatment of fat cells results in the translocation of the insulin-responsive glucose transporter type 4, GLUT4, from intracellular compartments to the plasma membrane. However, the precise nature of these intracellular GLUT4-carrying compartments is debated. To resolve the nature of these compartments, we have performed an extensive morphological analysis of GLUT4-containing compartments, using a novel immunocytochemical technique enabling high labeling efficiency and 3-d resolution of cytoplasmic rims isolated from rat epididymal adipocytes. In basal cells, GLUT4 was localized to three morphologically distinct intracellular structures: small vesicles, tubules, and vacuoles. In response to insulin the increase of GLUT4 at the cell surface was compensated by a decrease in small vesicles, whereas the amount in tubules and vacuoles was unchanged. Under basal conditions, many small GLUT4 positive vesicles also contained IRAP (88%) and the v-SNARE, VAMP2 (57%) but not markers of sorting endosomes (EEA1), late endosomes, or lysosomes (lgp120). A largely distinct population of GLUT4 vesicles (56%) contained the cation-dependent mannose 6-phosphate receptor (CD-MPR), a marker protein that shuttles between endosomes and the trans-Golgi network (TGN). In response to insulin, GLUT4 was recruited both from VAMP2 and CD-MPR positive vesicles. However, while the concentration of GLUT4 in the remaining VAMP2-positive vesicles was unchanged, the concentration of GLUT4 in CD-MPR-positive vesicles decreased. Taken together, we provide morphological evidence indicating that, in response to insulin, GLUT4 is recruited to the plasma membrane by fusion of preexisting VAMP2-carrying vesicles as well as by sorting from the dynamic endosomal-TGN system. PMID:11102509

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.

Exosome-like vesicles in Gloydius blomhoffii blomhoffii venom.

PubMed

Ogawa, Yuko; Kanai-Azuma, Masami; Akimoto, Yoshihiro; Kawakami, Hayato; Yanoshita, Ryohei

2008-05-01

Exosomes are small membrane vesicles (30-100 nm) with an endosome-derived limiting membrane that are secreted by a diverse range of cell types. We provide here the first evidence for the presence of exosome-like vesicles in snake venom. We isolated vesicles from fresh venom from Gloydius blomhoffii blomhoffii by gel-filtration. We found that the vesicles showed a typical exosome-like size and morphology as analyzed by electron microscopy. We observed that the vesicles contained dipeptidyl peptidase IV, aminopeptidase A, ecto-5'-nucleotidase and actin. Vesicle preparations truncated bioactive peptides such as angiotensin II, substance P, cholecystokinin-octapeptide, glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1. The role of these vesicles is still unknown, but they may affect blood pressure and glucose homeostasis following envenomation.

In vivo processing and release into the circulation of GFP fusion protein in arginine vasopressin enhanced GFP transgenic rats: response to osmotic stimulation.

PubMed

Satoh, Keita; Oti, Takumi; Katoh, Akiko; Ueta, Yoichi; Morris, John F; Sakamoto, Tatsuya; Sakamoto, Hirotaka

2015-07-01

Arginine vasopressin (AVP) is a neurohypophysial hormone synthesized as a part of a prepropeptide precursor containing the signal peptide, AVP hormone, AVP-associated neurophysin II and copeptin in the hypothalamic neurosecretory neurons. A transgenic (Tg) rat line expressing the AVP-eGFP fusion gene has been generated. To establish the AVP-eGFP Tg rat as a unique model for an analysis of AVP dynamics in vivo, we first examined the in vivo molecular dynamics of the AVP-eGFP fusion gene, and then the release of GFP in response to physiological stimuli. Double immunoelectron microscopy demonstrated that GFP was specifically localized in neurosecretory vesicles of AVP neurons in this Tg rat. After stimulation of the posterior pituitary with high potassium we demonstrated the exocytosis of AVP neurosecretory vesicles containing GFP at the ultrastructural level. Biochemical analyses indicated that the AVP-eGFP fusion gene is subjected to in vivo post-translational modifications like the native AVP gene, and is packaged into neurosecretory vesicles as a fusion protein: copeptin1-14 -GFP. Moreover, GFP release into the circulating blood appeared to be augmented after osmotic stimulation, like native AVP. Thus, here we show for the first time the in vivo molecular processing of the AVP-eGFP fusion gene and stimulated secretion after osmotic stimulation in rats. Because GFP behaved like native AVP in the hypothalamo-pituitary axis, and in particular was released into the circulation in response to a physiological stimulus, the AVP-eGFP Tg rat model appears to be a powerful tool for analyzing neuroendocrine systems at the organismal level. © 2015 FEBS.

Thermodynamics and kinetics of vesicles formation processes.

PubMed

Guida, Vincenzo

2010-12-15

Vesicles are hollow aggregates, composed of bilayers of amphiphilic molecules, dispersed into and filled with a liquid solvent. These aggregates can be formed either as equilibrium or as out of equilibrium meta-stable structures and they exhibit a rich variety of different morphologies. The surprising richness of structures, the vast range of industrial applications and the presence of vesicles in a number of biological systems have attracted the interest of numerous researchers and scientists. In this article, we review both the thermodynamics and the kinetics aspects of the phenomena of formation of vesicles. We start presenting the thermodynamics of bilayer membranes formation and deformation, with the aim of deriving the conditions for the existence of equilibrium vesicles. Specifically, we use the results from continuum thermodynamics to discuss the possibility of formation of stable equilibrium vesicles, from both mixed amphiphiles and single component systems. We also link the bilayer membrane properties to the molecular structure of the starting amphiphiles. In the second part of this article, we focus on the dynamics and kinetics of vesiculation. We review the process of vesicles formation both from planar lamellar phase under shear and from isotropic micelles. In order to clarify the physical mechanisms of vesicles formation, we continuously draw a parallel between emulsification and vesiculation processes. Specifically, we compare the experimental results, the driving forces and the relative scaling laws identified for the two processes. Describing the dynamics of vesicles formation, we also discuss why non equilibrium vesicles can be formed by kinetics control and why they are meta-stable. Understanding how to control the properties, the stability and the formation process of vesicles is of fundamental importance for a vast number of industrial applications. Copyright © 2009. Published by Elsevier B.V.

Bioengineering anembryonic human trophoblast vesicles.

PubMed

Robins, Jared C; Morgan, Jeffrey R; Krueger, Paula; Carson, Sandra A

2011-02-01

Trophoblast cells in vivo form a 3-dimensional structure that promotes complex cell-to-cell interactions that cannot be studied with traditional monolayer culture. We describe a 3-dimensional trophoblast bioreactor to study cellular interactions. Nonadhesive agarose hydrogels were cast from molds using computer-assisted prototyping. Trophoblast cells were seeded into the gels for 10 days. Morphology, viability, and vesicle behavior were assessed. Trophoblast cells formed uniform spheroids. Serial sectioning on days 3, 7, and 10 revealed central vacuolization with a consistent outer rim 12.3-μ thick. The vesicle configuration has been confirmed with confocal imaging. Electron Microscopic (EM) imaging revealed its ultrastructure. The vesicles migrate across a fibronectin-coated surface and invaded basement membrane. Trophoblast cells cultured in a novel substrate-free 3-dimensional system form trophoblast vesicles. This new cell culture technique allows us to better study placental cell-to-cell interactions with the potential of forming microtissues.

Multiple effects of sibutramine on ejaculation and on vas deferens and seminal vesicle contractility

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nojimoto, Fernanda D.; Piffer, Renata C.; Kiguti, Luiz Ricardo de A.

Sibutramine is an inhibitor of norepinephrine and 5-HT reuptake largely used in the management of obesity. Although a fairly safe drug, postmarketing adverse effects of sibutramine were reported including abnormal ejaculation in men. This study investigates the effects of sibutramine on ejaculation and vas deferens and seminal vesicle contractility. Adult male rats received sibutramine (5; 20; or 50 mg kg{sup -1}, ip) and after 60 min were exposed to receptive females for determination of ejaculation parameters. The vasa deferentia and seminal vesicles of untreated rats were mounted in isolated organ baths for recording of isometric contractions and HEK293 cells loadedmore » with fluorescent calcium indicator were used to measure intracellular Ca{sup 2+} transients. Sibutramine 5 and 20 mg kg{sup -1} reduced ejaculation latency whereas 50 mg kg{sup -1} increased ejaculation latency. Sibutramine 3 to 30 {mu}M greatly increased the sensitivity of the seminal vesicle and vas deferens to norepinephrine, but at concentrations higher than 10 {mu}M there were striking depressions of maximal contractions induced by norepinephrine, carbachol and CaCl{sub 2}. In HEK293 cells, sibutramine 10 to 100 {mu}M inhibited intracellular Ca{sup 2+} transients induced by carbachol. Depending on the doses, sibutramine either facilitates or inhibits ejaculation. Apart from its actions in the central nervous system, facilitation of ejaculation may result from augmented sensitivity of smooth muscles to norepinephrine while reductions of intracellular Ca{sup 2+} may be involved in the delayed ejaculation observed with high doses of sibutramine.« less

Yarrowia lipolytica vesicle-mediated protein transport pathways

PubMed Central

Swennen, Dominique; Beckerich, Jean-Marie

2007-01-01

Background Protein secretion is a universal cellular process involving vesicles which bud and fuse between organelles to bring proteins to their final destination. Vesicle budding is mediated by protein coats; vesicle targeting and fusion depend on Rab GTPase, tethering factors and SNARE complexes. The Génolevures II sequencing project made available entire genome sequences of four hemiascomycetous yeasts, Yarrowia lipolytica, Debaryomyces hansenii, Kluyveromyces lactis and Candida glabrata. Y. lipolytica is a dimorphic yeast and has good capacities to secrete proteins. The translocation of nascent protein through the endoplasmic reticulum membrane was well studied in Y. lipolytica and is largely co-translational as in the mammalian protein secretion pathway. Results We identified S. cerevisiae proteins involved in vesicular secretion and these protein sequences were used for the BLAST searches against Génolevures protein database (Y. lipolytica, C. glabrata, K. lactis and D. hansenii). These proteins are well conserved between these yeasts and Saccharomyces cerevisiae. We note several specificities of Y. lipolytica which may be related to its good protein secretion capacities and to its dimorphic aspect. An expansion of the Y. lipolytica Rab protein family was observed with autoBLAST and the Rab2- and Rab4-related members were identified with BLAST against NCBI protein database. An expansion of this family is also found in filamentous fungi and may reflect the greater complexity of the Y. lipolytica secretion pathway. The Rab4p-related protein may play a role in membrane recycling as rab4 deleted strain shows a modification of colony morphology, dimorphic transition and permeability. Similarly, we find three copies of the gene (SSO) encoding the plasma membrane SNARE protein. Quantification of the percentages of proteins with the greatest homology between S. cerevisiae, Y. lipolytica and animal homologues involved in vesicular transport shows that 40% of Y

Extravasation of adhering vesicles

NASA Astrophysics Data System (ADS)

Tordeux, C.; Fournier, J.-B.

2002-12-01

We study how the passage of lipid vesicles through a small pore can be induced by the difference in non-specific adhesion energy between the two sides of the substrate bearing the pore. This process is inspired from the extravasation of cells or liposomes from blood vessels, which involves adhesion binders. We study the adhesion-dominated regime and we show that the passage of a vesicle of volume V and area A is selective in terms of the reduced volume v ~ V/A3/2. Extravasation occurs for adhesion ratios of order unity. We also consider the possibility of pressure-induced extravasation in the presence of adhesion. Finally, we propose a micro-device based on adhesion-induced extravasation, which is designed to sort vesicles according to their deflatedness.

Elastic energy of polyhedral bilayer vesicles

PubMed Central

Haselwandter, Christoph A.; Phillips, Rob

2011-01-01

In recent experiments the spontaneous formation of hollow bilayer vesicles with polyhedral symmetry has been observed. On the basis of the experimental phenomenology it was suggested that the mechanism for the formation of bilayer polyhedra is minimization of elastic bending energy. Motivated by these experiments, we study the elastic bending energy of polyhedral bilayer vesicles. In agreement with experiments, and provided that excess amphiphiles exhibiting spontaneous curvature are present in sufficient quantity, we find that polyhedral bilayer vesicles can indeed be energetically favorable compared to spherical bilayer vesicles. Consistent with experimental observations we also find that the bending energy associated with the vertices of bilayer polyhedra can be locally reduced through the formation of pores. However, the stabilization of polyhedral bilayer vesicles over spherical bilayer vesicles relies crucially on molecular segregation of excess amphiphiles along the ridges rather than the vertices of bilayer polyhedra. Furthermore, our analysis implies that, contrary to what has been suggested on the basis of experiments, the icosahedron does not minimize elastic bending energy among arbitrary polyhedral shapes and sizes. Instead, we find that, for large polyhedron sizes, the snub dodecahedron and the snub cube both have lower total bending energies than the icosahedron. PMID:21797397

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

PubMed Central

Nikolaus, Joerg; Karatekin, Erdem

2016-01-01

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, "kiss-and-run" fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore "openness", the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo. PMID:27585113

Targeted Cell Fusion Facilitates Stable Heterokaryon Generation In Vitro and In Vivo

PubMed Central

Long, Michael A.; Rossi, Fabio M. V.

2011-01-01

Induced cell fusion has enabled several important discoveries, including the phenomenon of nuclear reprogramming and may yet be applied as a novel therapy for degenerative diseases. However, existing fusogens lack the efficiency required to enable investigation of the epigenetic modifications underlying nuclear reprogramming and the specificity required for clinical application. Here we present a chimeric measles hemagglutinin, Hα7, which specifically and efficiently mediates the fusion of diverse cell types with skeletal muscle both in vitro and in vivo. When compared directly to polyethylene glycol, Hα7 consistently generated a substantial increase in heterokaryon yield and exhibited insignificant levels of toxicity. Moreover, this increased fusion efficiency enabled detection of chromatin modifications associated with nuclear reprogramming following Hα7-mediated fusion of human fibroblasts and mouse myotubes. Finally, Hα7 was also capable of increasing the contribution of transplanted fibroblasts to skeletal muscle repair in vivo, suggesting that this strategy could be used for therapeutic gene delivery. PMID:22039476

Spatial attention facilitates assembly of the briefest percepts: Electrophysiological evidence from color fusion.

PubMed

Akyürek, Elkan G; van Asselt, E Manon

2015-12-01

When two different color stimuli are presented in rapid succession, the resulting percept is sometimes that of a mixture of both colors, due to a perceptual process called color fusion. Although color fusion might seem to occur very early in the visual pathway, and only happens across the briefest of stimulus presentation intervals (< 50 ms), the present study showed that spatial attention can alter the fusion process. In a series of experiments, spatial cues were presented that either validly indicated the location of a pair of (different) color stimuli in successive stimulus arrays, or did not, pointing toward isoluminant gray distractors in the other visual hemifield. Increased color fusion was observed for valid cues across a range of stimulus durations, at the expense of individual color reports. By contrast, perception of repeated, same-color stimulus pairs did not change, suggesting that the enhancement was specific to fusion, not color discrimination per se. Electrophysiological measures furthermore showed that the amplitude of the N1, N2pc, and P3 components of the ERP were differentially modulated during the perception of individual and fused colors, as a function of cueing and stimulus duration. Fusion itself, collapsed across cueing conditions, was reflected uniquely in N1 amplitude. Overall, the results suggest that spatial attention enhances color fusion and decreases competition between stimuli, constituting an adaptive slowdown in service of temporal integration. © 2015 Society for Psychophysiological Research.

A Role for an Hsp70 Nucleotide Exchange Factor in the Regulation of Synaptic Vesicle Endocytosis

PubMed Central

Morgan, Jennifer R.; Jiang, Jianwen; Oliphint, Paul A.; Jin, Suping; Gimenez, Luis E.; Busch, David J.; Foldes, Andrea E.; Zhuo, Yue; Sousa, Rui; Lafer, Eileen M.

2013-01-01

Neurotransmission requires a continuously available pool of synaptic vesicles (SVs) that can fuse with the plasma membrane and release their neurotransmitter contents upon stimulation. After fusion, SV membranes and membrane proteins are retrieved from the presynaptic plasma membrane by clathrin-mediated endocytosis. Following the internalization of a clathrin coated vesicle (CCV), the vesicle must uncoat to replenish the pool of SVs. CCV uncoating requires ATP and is mediated by the ubiquitous molecular chaperone Hsc70. In vitro, depolymerized clathrin forms a stable complex with Hsc70*ADP. This complex can be dissociated by nucleotide exchange factors (NEFs) that release ADP from Hsc70, allowing ATP to bind and induce disruption of the clathrin:Hsc70 association. Whether NEFs generally play similar roles in vesicle trafficking in vivo, and whether they play such roles in SV endocytosis in particular is unknown. To address this question we used information from recent structural and mechanistic studies of Hsp70:NEF and Hsp70:cochaperone interactions to design a NEF inhibitor. Using acute perturbations at giant reticulospinal synapses of the sea lamprey (Petromyzon marinus), we found that this NEF inhibitor inhibited SV endocytosis. When this inhibitor was mutated so it could no longer bind and inhibit Hsp110--a NEF that we find to be highly abundant in brain cytosol--its ability to inhibit SV endocytosis was eliminated. These observations indicate that the action of a NEF, most likely Hsp110, is normally required during SV trafficking to release clathrin from Hsc70 and make it available for additional rounds of endocytosis. PMID:23637191

Are calcifying matrix vesicles in atherosclerotic lesions of cellular origin?

PubMed

Bobryshev, Yuri V; Killingsworth, Murray C; Huynh, Thuan G; Lord, Reginald S A; Grabs, Anthony J; Valenzuela, Stella M

2007-03-01

Over recent years, the role of matrix vesicles in the initial stages of arterial calcification has been recognized. Matrix calcifying vesicles have been isolated from atherosclerotic arteries and the biochemical composition of calcified vesicles has been studied. No studies have yet been carried out to examine the fine structure of matrix vesicles in order to visualize the features of the consequent stages of their calcification in arteries. In the present work, a high resolution ultrastructural analysis has been employed and the study revealed that matrix vesicles in human atherosclerotic lesions are heterogeneous with two main types which we classified. Type I calcified vesicles were presented by vesicles surrounded by two electron-dense layers and these vesicles were found to be resistant to the calcification process in atherosclerotic lesions in situ. Type II matrix vesicles were presented by vesicles surrounded by several electron-dense layers and these vesicles were found to represent calcifying vesicles in atherosclerotic lesions. To test the hypothesis that calcification of matrix vesicles surrounded by multilayer sheets may occur simply as a physicochemical process, independently from the cell regulation, we produced multilamellar liposomes and induced their calcification in vitro in a manner similar to that occurring in matrix vesicles in atherosclerotic lesions in situ.

A new level of plasticity: Drosophila smooth-like testes muscles compensate failure of myoblast fusion

PubMed Central

Kuckwa, Jessica; Fritzen, Katharina; Buttgereit, Detlev; Rothenbusch-Fender, Silke; Renkawitz-Pohl, Renate

2016-01-01

The testis of Drosophila resembles an individual testis tubule of mammals. Both are surrounded by a sheath of smooth muscles, which in Drosophila are multinuclear and originate from a pool of myoblasts that are set aside in the embryo and accumulate on the genital disc later in development. These muscle stem cells start to differentiate early during metamorphosis and give rise to all muscles of the inner male reproductive system. Shortly before the genital disc and the developing testes connect, multinuclear nascent myotubes appear on the anterior tips of the seminal vesicles. Here, we show that adhesion molecules are distinctly localized on the seminal vesicles; founder cell (FC)-like myoblasts express Dumbfounded (Duf) and Roughest (Rst), and fusion-competent myoblast (FCM)-like cells mainly express Sticks and stones (Sns). The smooth but multinuclear myotubes of the testes arose by myoblast fusion. RNAi-mediated attenuation of Sns or both Duf and Rst severely reduced the number of nuclei in the testes muscles. Duf and Rst probably act independently in this context. Despite reduced fusion in all of these RNAi-treated animals, myotubes migrated onto the testes, testes were shaped and coiled, muscle filaments were arranged as in the wild type and spermatogenesis proceeded normally. Hence, the testes muscles compensate for fusion defects so that the myofibres encircling the adult testes are indistinguishable from those of the wild type and male fertility is guaranteed. PMID:26657767

Spontaneous vesicle formation at lipid bilayer membranes.

PubMed

Edwards, D A; Schneck, F; Zhang, I; Davis, A M; Chen, H; Langer, R

1996-09-01

Unilamellar vesicles are observed to form spontaneously at planar lipid bilayers agitated by exothermic chemical reactions. The membrane-binding reaction between biotin and streptavidin, two strong transmembrane neutralization reactions, and a weak neutralization reaction involving an "antacid" buffer, all lead to spontaneous vesicle formation. This formation is most dramatic when a viscosity differential exists between the two phases bounding the membrane, in which case vesicles appear exclusively in the more viscous phase. A hydrodynamic analysis explains the phenomenon in terms of a membrane flow driven by liberated reaction energy, leading to vesicle formation. These results suggest that energy liberated by intra- and extracellular chemical reactions near or at cell and internal organelle membranes can play an important role in vesicle formation, membrane agitation, or enhanced transmembrane mass transfer.

SARS-CoV fusion peptides induce membrane surface ordering and curvature

PubMed Central

Basso, Luis G. M.; Vicente, Eduardo F.; Crusca Jr., Edson; Cilli, Eduardo M.; Costa-Filho, Antonio J.

2016-01-01

Viral membrane fusion is an orchestrated process triggered by membrane-anchored viral fusion glycoproteins. The S2 subunit of the spike glycoprotein from severe acute respiratory syndrome (SARS) coronavirus (CoV) contains internal domains called fusion peptides (FP) that play essential roles in virus entry. Although membrane fusion has been broadly studied, there are still major gaps in the molecular details of lipid rearrangements in the bilayer during fusion peptide-membrane interactions. Here we employed differential scanning calorimetry (DSC) and electron spin resonance (ESR) to gather information on the membrane fusion mechanism promoted by two putative SARS FPs. DSC data showed the peptides strongly perturb the structural integrity of anionic vesicles and support the hypothesis that the peptides generate opposing curvature stresses on phosphatidylethanolamine membranes. ESR showed that both FPs increase lipid packing and head group ordering as well as reduce the intramembrane water content for anionic membranes. Therefore, bending moment in the bilayer could be generated, promoting negative curvature. The significance of the ordering effect, membrane dehydration, changes in the curvature properties and the possible role of negatively charged phospholipids in helping to overcome the high kinetic barrier involved in the different stages of the SARS-CoV-mediated membrane fusion are discussed. PMID:27892522

SARS-CoV fusion peptides induce membrane surface ordering and curvature.

PubMed

Basso, Luis G M; Vicente, Eduardo F; Crusca, Edson; Cilli, Eduardo M; Costa-Filho, Antonio J

2016-11-28

Viral membrane fusion is an orchestrated process triggered by membrane-anchored viral fusion glycoproteins. The S2 subunit of the spike glycoprotein from severe acute respiratory syndrome (SARS) coronavirus (CoV) contains internal domains called fusion peptides (FP) that play essential roles in virus entry. Although membrane fusion has been broadly studied, there are still major gaps in the molecular details of lipid rearrangements in the bilayer during fusion peptide-membrane interactions. Here we employed differential scanning calorimetry (DSC) and electron spin resonance (ESR) to gather information on the membrane fusion mechanism promoted by two putative SARS FPs. DSC data showed the peptides strongly perturb the structural integrity of anionic vesicles and support the hypothesis that the peptides generate opposing curvature stresses on phosphatidylethanolamine membranes. ESR showed that both FPs increase lipid packing and head group ordering as well as reduce the intramembrane water content for anionic membranes. Therefore, bending moment in the bilayer could be generated, promoting negative curvature. The significance of the ordering effect, membrane dehydration, changes in the curvature properties and the possible role of negatively charged phospholipids in helping to overcome the high kinetic barrier involved in the different stages of the SARS-CoV-mediated membrane fusion are discussed.

Membrane vesicles shed by oligodendroglioma cells induce neuronal apoptosis.

PubMed

D'Agostino, Stefania; Salamone, Monica; Di Liegro, Italia; Vittorelli, M Letizia

2006-11-01

In order to investigate the mechanism by which oligodendrogliomas cause neuronal damage, media conditioned by G26/24 oligodendroglioma cells, were fractionated into shed vesicles and vesicle-free supernatants, and added to primary cultures of rat fetal cortical neurons. After one night treatment with vesicles, a reproducible, dose-dependent, inhibitory effect on neurite outgrowth was already induced and, after 48-72 h of incubation, neuronal apoptosis was evident. Vesicle-free supernatants and vesicles shed by NIH-3T3 cells had no inhibitory effects on neurons. Western blot analyses showed that treated neurons expressed a decreased amount of neurofilament (NF), growth-associated protein (GAP-43) and microtubule-associated protein (MAP-2). Moreover procaspase-3 and -8 were activated while Bcl-2 expression was reduced. Vesicles were found positive for the proapoptotic molecule, Fas-ligand (Fas-L), and for the B isoform of Nogo protein, a myelin component with inhibitory effects on neurons. Nogo B involvement in the vesicle effects was analyzed both by testing the neutralizing capability of anti-Nogo antibodies and by removing the Nogo receptor from neurons by phospholipase C digestion. These treatments did not revert the vesicle effects. To test the role of Fas-L, vesicles were treated with functional anti-Fas-L monoclonals. Vesicle inhibitory and proapoptotic effects were reduced. Vesicles shed by ovarian carcinoma cells (OvCa), which are known to vehicle biologically active Fas-L, had similar effects on neurons to those of oligodendroglioma vesicles, and their inhibitory effects were also reduced by anti Fas-L antibodies. We therefore conclude that vesicles shed by G26/24 cells induce neuronal apoptosis at least partially by a Fas-L mediated mechanism.

Kinetic regulation of coated vesicle secretion

PubMed Central

Foret, Lionel; Sens, Pierre

2008-01-01

The secretion of vesicles for intracellular transport often relies on the aggregation of specialized membrane-bound proteins into a coat able to curve cell membranes. The nucleation and growth of a protein coat is a kinetic process that competes with the energy-consuming turnover of coat components between the membrane and the cytosol. We propose a generic kinetic description of coat assembly and the formation of coated vesicles and discuss its implication to the dynamics of COP vesicles that traffic within the Golgi and with the endoplasmic reticulum. We show that stationary coats of fixed area emerge from the competition between coat growth and the recycling of coat components, in a fashion resembling the treadmilling of cytoskeletal filaments. We further show that the turnover of coat components allows for a highly sensitive switching mechanism between a quiescent and a vesicle producing membrane, upon a slowing down of the exchange kinetics. We claim that the existence of this switching behavior, also triggered by factors, such as the presence of cargo and variation of the membrane mechanical tension, allows for efficient regulation of vesicle secretion. We propose a model, supported by different experimental observations, in which vesiculation of secretory membranes is impaired by the energy-consuming desorption of coat proteins, until the presence of cargo or other factors triggers a dynamical switch into a vesicle producing state. PMID:18824695

Rings of membrane sterols surround the openings of vesicles and fenestrae, in capillary endothelium.

PubMed

Simionescu, N; Lupu, F; Simionescu, M

1983-11-01

We investigated the distribution of sterols in the cell membrane of microvascular endothelium (mouse pancreas, diaphragm, brain, heart, lung, kidney, thyroid, adrenal, and liver) with the polyene antibiotic filipin, which reportedly has binding specificity for free 3-beta-hydroxysterols. In some experiments, concomitantly, cell-surface anionic sites were detected with cationized ferritin. Vessels were perfused in situ with PBS, followed by light fixation and filipin administration for 10 to 60 min. Tissues were further processed for thin-section and freeze-fracture electron microscopy. Short exposure (10 min) to filipin-glutaraldehyde solution resulted in the initial appearance, on many areas, of rings of characteristic filipin-sterol complexes within the rim surrounding stomata of most plasmalemmal vesicles, transendothelial channels, and fenestrae. Such rings were absent from the rims of the large openings of the sinusoid endothelium (liver, adrenal), coated pits and phagocytic vacuoles. After longer exposure (30-60 min), filipin-sterol complexes labeled randomly the rest of plasma membrane (except for coated pits, and partially the interstrand areas of junctions), and also marked most plasmalemmal vesicles. These peristomal rings of sterols were displayed mostly on the P face, and, at their full development, consisted of 6-8 units around a vesicle stoma, and 10-12 units around a fenestra. At their level, the intramembranous particles and the cell surface anionic sites were virtually excluded. Peristomal rings of sterols were also detected on the plasma membrane of pericytes and smooth muscle cells of the microvascular wall, which otherwise were poorly labeled with filipin-sterol complexes as compared to endothelial plasmalemma. It is presumed that the peristomal rings of cholesterol may represent important contributors to the local transient stabilization of plasma membrane and to the phase separation between cell membrane and vesicle membrane at a certain stage

Zwitterionic lipid assemblies: Molecular dynamics studies of monolayers, bilayers, and vesicles using a new coarse grain force field

PubMed Central

Shinoda, Wataru; DeVane, Russell; Klein, Michael L.

2010-01-01

A new coarse-grained (CG) intermolecular force field is presented for a series of zwitterionic lipids. The model is an extension of our previous work on nonionic surfactants and is designed to reproduce experimental surface/interfacial properties as well as distribution functions from all-atom molecular dynamics (MD) simulations. Using simple functional forms, the force field parameters are optimized for multiple lipid molecules, simultaneously. The resulting CG lipid bilayers have reasonable molecular areas, chain order parameters, and elastic properties. The computed surface pressure vs. area (π-A) curve for a DPPC monolayer demonstrates a significant improvement over the previous CG models. The DPPC monolayer has a longer persistence length than a PEG lipid monolayer, exhibiting a long-lived curved monolayer surface under negative tension. The bud ejected from an oversaturated DPPC monolayer has a large bicelle-like structure, which is different from the micellar bud formed from an oversaturated PEG lipid monolayer. We have successfully observed vesicle formation during CG-MD simulations, starting from an aggregate of DMPC molecules. Depending on the aggregate size, the lipid assembly spontaneously transforms into a closed vesicle or a bicelle. None of the various intermediate structures between these extremes seem to be stable. An attempt to observe fusion of two vesicles through the application of an external adhesion force was not successful. The present CG force field also supports stable multi-lamellar DMPC vesicles. PMID:20438090

Visualization of Synchronous or Asynchronous Release of Single Synaptic Vesicle in Active-Zone-Like Membrane Formed on Neuroligin-Coated Glass Surface.

PubMed

Funahashi, Junichiro; Tanaka, Hiromitsu; Hirano, Tomoo

2018-01-01

Fast repetitive synaptic transmission depends on efficient exocytosis and retrieval of synaptic vesicles around a presynaptic active zone. However, the functional organization of an active zone and regulatory mechanisms of exocytosis, endocytosis and reconstruction of release-competent synaptic vesicles have not been fully elucidated. By developing a novel visualization method, we attempted to identify the location of exocytosis of a single synaptic vesicle within an active zone and examined movement of synaptic vesicle protein synaptophysin (Syp) after exocytosis. Using cultured hippocampal neurons, we induced formation of active-zone-like membranes (AZLMs) directly adjacent and parallel to a glass surface coated with neuroligin, and imaged Syp fused to super-ecliptic pHluorin (Syp-SEP) after its translocation to the plasma membrane from a synaptic vesicle using total internal reflection fluorescence microscopy (TIRFM). An AZLM showed characteristic molecular and functional properties of a presynaptic active zone. It contained active zone proteins, cytomatrix at the active zone-associated structural protein (CAST), Bassoon, Piccolo, Munc13 and RIM, and showed an increase in intracellular Ca 2+ concentration upon electrical stimulation. In addition, single-pulse stimulation sometimes induced a transient increase of Syp-SEP signal followed by lateral spread in an AZLM, which was considered to reflect an exocytosis event of a single synaptic vesicle. The diffusion coefficient of Syp-SEP on the presynaptic plasma membrane after the membrane fusion was estimated to be 0.17-0.19 μm 2 /s, suggesting that Syp-SEP diffused without significant obstruction. Synchronous exocytosis just after the electrical stimulation tended to occur at multiple restricted sites within an AZLM, whereas locations of asynchronous release occurring later after the stimulation tended to be more scattered.

Structure and Function Study of HIV and Influenza Fusion Proteins

NASA Astrophysics Data System (ADS)

Liang, Shuang

Human immunodeficiency virus (HIV) and influenza virus are membrane-enveloped viruses causing acquired immunodeficiency syndrome (AIDS) and flu. The initial step of HIV and influenza virus infection is fusion between viral and host cell membrane catalyzed by the viral fusion protein gp41 and hemagglutinin (HA) respectively. However, the structure of gp41 and HA as well as the infection mechanism are still not fully understood. This work addresses (1) full length gp41 ectodomain and TM domain structure and function and (2) IFP membrane location and IFP-membrane interaction. My studies of gp41 protein and IFP can provide better understanding of the membrane fusion mechanism and may aid development of anti-viral therapeutics and vaccine. The full length ectodomain and transmembrane domain of gp41 and shorter constructs were expressed, purified and solubilized at physiology conditions. The constructs adopt overall alpha helical structure in SDS and DPC detergents, and showed hyperthermostability with Tm > 90 °C. The oligomeric states of these proteins vary in different detergent buffer: predominant trimer for all constructs and some hexamer fraction for HM and HM_TM protein in SDS at pH 7.4; and mixtures of monomer, trimer, and higher-order oligomer protein in DPC at pH 4.0 and 7.4. Substantial protein-induced vesicle fusion was observed, including fusion of neutral vesicles at neutral pH, which are the conditions similar HIV/cell fusion. Vesicle fusion by a gp41 ectodomain construct has rarely been observed under these conditions, and is aided by inclusion of both the FP and TM, and by protein which is predominantly trimer rather than monomer. Current data was integrated with existing data, and a structural model was proposed. Secondary structure and conformation of IFP is a helix-turn-helix structure in membrane. However, there has been arguments about the IFP membrane location. 13C-2H REDOR solid-state NMR is used to solve this problem. The IFP adopts major alpha

The pressure-dependence of the size of extruded vesicles.

PubMed

Patty, Philipus J; Frisken, Barbara J

2003-08-01

Variations in the size of vesicles formed by extrusion through small pores are discussed in terms of a simple model. Our model predicts that the radius should decrease as the square root of the applied pressure, consistent with data for vesicles extruded under various conditions. The model also predicts dependencies on the pore size used and on the lysis tension of the vesicles being extruded that are consistent with our data. The pore size was varied by using track-etched polycarbonate membranes with average pore diameters ranging from 50 to 200 nm. To vary the lysis tension, vesicles made from POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine), mixtures of POPC and cholesterol, and mixtures of POPC and C(16)-ceramide were studied. The lysis tension, as measured by an extrusion-based technique, of POPC:cholesterol vesicles is higher than that of pure POPC vesicles whereas POPC:ceramide vesicles have lower lysis tensions than POPC vesicles.

The Pressure-Dependence of the Size of Extruded Vesicles

PubMed Central

Patty, Philipus J.; Frisken, Barbara J.

2003-01-01

Variations in the size of vesicles formed by extrusion through small pores are discussed in terms of a simple model. Our model predicts that the radius should decrease as the square root of the applied pressure, consistent with data for vesicles extruded under various conditions. The model also predicts dependencies on the pore size used and on the lysis tension of the vesicles being extruded that are consistent with our data. The pore size was varied by using track-etched polycarbonate membranes with average pore diameters ranging from 50 to 200 nm. To vary the lysis tension, vesicles made from POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine), mixtures of POPC and cholesterol, and mixtures of POPC and C16-ceramide were studied. The lysis tension, as measured by an extrusion-based technique, of POPC:cholesterol vesicles is higher than that of pure POPC vesicles whereas POPC:ceramide vesicles have lower lysis tensions than POPC vesicles. PMID:12885646

Electrohydrodynamics of a compound vesicle under an AC electric field

NASA Astrophysics Data System (ADS)

Priti Sinha, Kumari; Thaokar, Rochish M.

2017-07-01

Compound vesicles are relevant as simplified models for biological cells as well as in technological applications such as drug delivery. Characterization of these compound vesicles, especially the inner vesicle, remains a challenge. Similarly their response to electric field assumes importance in light of biomedical applications such as electroporation. Fields lower than that required for electroporation cause electrodeformation in vesicles and can be used to characterize their mechanical and electrical properties. A theoretical analysis of the electrohydrodynamics of a compound vesicle with outer vesicle of radius R o and an inner vesicle of radius λ {{R}o} , is presented. A phase diagram for the compound vesicle is presented and elucidated using detailed plots of electric fields, free charges and electric stresses. The electrohydrodynamics of the outer vesicle in a compound vesicle shows a prolate-sphere and prolate-oblate-sphere shape transitions when the conductivity of the annular fluid is greater than the outer fluid, and vice-versa respectively, akin to single vesicle electrohydrodynamics reported in the literature. The inner vesicle in contrast shows sphere-prolate-sphere and sphere-prolate-oblate-sphere transitions when the inner fluid conductivity is greater and smaller than the annular fluid, respectively. Equations and methodology are provided to determine the bending modulus and capacitance of the outer as well as the inner membrane, thereby providing an easy way to characterize compound vesicles and possibly biological cells.

Distinct features of multivesicular body-lysosome fusion revealed by a new cell-free content-mixing assay.

PubMed

Karim, Mahmoud Abdul; Samyn, Dieter Ronny; Mattie, Sevan; Brett, Christopher Leonard

2018-02-01

When marked for degradation, surface receptor and transporter proteins are internalized and delivered to endosomes where they are packaged into intralumenal vesicles (ILVs). Many rounds of ILV formation create multivesicular bodies (MVBs) that fuse with lysosomes exposing ILVs to hydrolases for catabolism. Despite being critical for protein degradation, the molecular underpinnings of MVB-lysosome fusion remain unclear, although machinery underlying other lysosome fusion events is implicated. But how then is specificity conferred? And how is MVB maturation and fusion coordinated for efficient protein degradation? To address these questions, we developed a cell-free MVB-lysosome fusion assay using Saccharomyces cerevisiae as a model. After confirming that the Rab7 ortholog Ypt7 and the multisubunit tethering complex HOPS (homotypic fusion and vacuole protein sorting complex) are required, we found that the Qa-SNARE Pep12 distinguishes this event from homotypic lysosome fusion. Mutations that impair MVB maturation block fusion by preventing Ypt7 activation, confirming that a Rab-cascade mechanism harmonizes MVB maturation with lysosome fusion. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

SNARE-mediated rapid lysosome fusion in membrane raft clustering and dysfunction of bovine coronary arterial endothelium

PubMed Central

Han, Wei-Qing; Xia, Min; Zhang, Chun; Zhang, Fan; Xu, Ming; Li, Ning-Jun

2011-01-01

The present study attempted to evaluate whether soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate lysosome fusion in response to death receptor activation and contribute to membrane raft (MR) clustering and consequent endothelial dysfunction in coronary arterial endothelial cells. By immunohistochemical analysis, vesicle-associated membrane proteins 2 (VAMP-2, vesicle-SNAREs) were found to be abundantly expressed in the endothelium of bovine coronary arteries. Direct lysosome fusion monitoring by N-(3-triethylammoniumpropyl)-4-[4-(dibutylamino)styryl]pyridinium dibromide (FM1-43) quenching demonstrated that the inhibition of VAMP-2 with tetanus toxin or specific small interfering ribonucleic acid (siRNA) almost completely blocked lysosome fusion to plasma membrane induced by Fas ligand (FasL), a well-known MR clustering stimulator. The involvement of SNAREs was further confirmed by an increased interaction of VAMP-2 with a target-SNARE protein syntaxin-4 after FasL stimulation in coimmunoprecipitation analysis. Also, the inhibition of VAMP-2 with tetanus toxin or VAMP-2 siRNA abolished FasL-induced MR clustering, its colocalization with a NADPH oxidase unit gp91phox, and increased superoxide production. Finally, FasL-induced impairment of endothelium-dependent vasodilation was reversed by the treatment of bovine coronary arteries with tetanus toxin or VAMP-2 siRNA. VAMP-2 is critical to lysosome fusion in MR clustering, and this VAMP-2-mediated lysosome-MR signalosomes contribute to redox regulation of coronary endothelial function. PMID:21926345

Studies of matrix vesicle-induced mineralization in a gelatin gel

NASA Technical Reports Server (NTRS)

Boskey, A. L.; Boyan, B. D.; Doty, S. B.; Feliciano, A.; Greer, K.; Weiland, D.; Swain, L. D.; Schwartz, Z.

1992-01-01

Matrix vesicles isolated from fourth-passage cultures of chondrocytes were tested for their ability to induce hydroxyapatite formation in a gelatin gel in order to gain insight into the function of matrix vesicles in in situ mineralization. These matrix vesicles did not appear to be hydroxyapatite nucleators per se since the extent of mineral accumulation in the gel diffusion system was not altered by the presence of matrix vesicles alone, and in the vesicle containing gels, mineral crystals were formed whether associated with vesicles or not. In gels with these matrix vesicles and beta-glycerophosphate, despite the presence of alkaline phosphatase activity, there was no increase in mineral deposition. This suggested that in the gel system these culture-derived vesicles did not increase local phosphate concentrations. However, when known inhibitors of mineral crystal formation and growth (proteoglycan aggregates [4 mg/ml], or ATP [1 mM], or both proteoglycan and ATP) were included in the gel, more mineral was deposited in gels with the vesicles than in comparable gels without vesicles, indicating that enzymes within these vesicles were functioning to remove the inhibition. These data support the suggestion that one function of the extracellular matrix vesicles is to transport enzymes for matrix modification.

Synaptic vesicle recycling: steps and principles.

PubMed

Rizzoli, Silvio O

2014-04-16

Synaptic vesicle recycling is one of the best-studied cellular pathways. Many of the proteins involved are known, and their interactions are becoming increasingly clear. However, as for many other pathways, it is still difficult to understand synaptic vesicle recycling as a whole. While it is generally possible to point out how synaptic reactions take place, it is not always easy to understand what triggers or controls them. Also, it is often difficult to understand how the availability of the reaction partners is controlled: how the reaction partners manage to find each other in the right place, at the right time. I present here an overview of synaptic vesicle recycling, discussing the mechanisms that trigger different reactions, and those that ensure the availability of reaction partners. A central argument is that synaptic vesicles bind soluble cofactor proteins, with low affinity, and thus control their availability in the synapse, forming a buffer for cofactor proteins. The availability of cofactor proteins, in turn, regulates the different synaptic reactions. Similar mechanisms, in which one of the reaction partners buffers another, may apply to many other processes, from the biogenesis to the degradation of the synaptic vesicle.

Synaptic vesicle recycling: steps and principles

PubMed Central

Rizzoli, Silvio O

2014-01-01

Synaptic vesicle recycling is one of the best-studied cellular pathways. Many of the proteins involved are known, and their interactions are becoming increasingly clear. However, as for many other pathways, it is still difficult to understand synaptic vesicle recycling as a whole. While it is generally possible to point out how synaptic reactions take place, it is not always easy to understand what triggers or controls them. Also, it is often difficult to understand how the availability of the reaction partners is controlled: how the reaction partners manage to find each other in the right place, at the right time. I present here an overview of synaptic vesicle recycling, discussing the mechanisms that trigger different reactions, and those that ensure the availability of reaction partners. A central argument is that synaptic vesicles bind soluble cofactor proteins, with low affinity, and thus control their availability in the synapse, forming a buffer for cofactor proteins. The availability of cofactor proteins, in turn, regulates the different synaptic reactions. Similar mechanisms, in which one of the reaction partners buffers another, may apply to many other processes, from the biogenesis to the degradation of the synaptic vesicle. PMID:24596248

Osmotic shrinkage of giant egg-lecithin vesicles.

PubMed Central

Boroske, E; Elwenspoek, M; Helfrich, W

1981-01-01

Osmotic shrinkage of giant egg-lecithin vesicles was observed by phase-contrast microscopy. The vesicles remained or became spherical when shrinking. Small and thick-walled vesicles formed visible fingers attached to the sphere. The water permeability of the single bilayer was found to be 41 micrometers/s. A variety of observations indicate that osmosis induces a parallel lipid flow between the monolayers of the bilayer, leading to a strong positive spontaneous curvature. They also suggest the formation of mostly submicroscopic daughter vesicles. The estimated coupling constant, 2 . 10(-6) mol/mol, is large enough to be biologically significant. Images FIGURE 1 FIGURE 3 FIGURE 4 PMID:7213933

UNC-18 and Tomosyn Antagonistically Control Synaptic Vesicle Priming Downstream of UNC-13 in Caenorhabditis elegans

PubMed Central

Park, Seungmee; Bin, Na-Ryum; Wong, Raymond; Sitarska, Ewa; Sugita, Kyoko; Ma, Ke; Algouneh, Arash; Turlova, Ekaterina; Wang, Siyan; Siriya, Pranay; Kalia, Lorraine; Feng, Zhong-Ping; Monnier, Philippe P.; Zhen, Mei; Gao, Shangbang

2017-01-01

Munc18-1/UNC-18 is believed to prime SNARE-mediated membrane fusion, yet the underlying mechanisms remain enigmatic. Here, we examine how potential gain-of-function mutations of Munc18-1/UNC-18 affect locomotory behavior and synaptic transmission, and how Munc18-1-mediated priming is related to Munc13-1/UNC-13 and Tomosyn/TOM-1, positive and negative SNARE regulators, respectively. We show that a Munc18-1(P335A)/UNC-18(P334A) mutation leads to significantly increased locomotory activity and acetylcholine release in Caenorhabditis elegans, as well as enhanced synaptic neurotransmission in cultured mammalian neurons. Importantly, similar to tom-1 null mutants, unc-18(P334A) mutants partially bypass the requirement of UNC-13. Moreover, unc-18(P334A) and tom-1 null mutations confer a strong synergy in suppressing the phenotypes of unc-13 mutants. Through biochemical experiments, we demonstrate that Munc18-1(P335A) exhibits enhanced activity in SNARE complex formation as well as in binding to the preformed SNARE complex, and partially bypasses the Munc13-1 requirement in liposome fusion assays. Our results indicate that Munc18-1/UNC-18 primes vesicle fusion downstream of Munc13-1/UNC-13 by templating SNARE complex assembly and acts antagonistically with Tomosyn/TOM-1. SIGNIFICANCE STATEMENT At presynaptic sites, SNARE-mediated membrane fusion is tightly regulated by several key proteins including Munc18/UNC-18, Munc13/UNC-13, and Tomosyn/TOM-1. However, how these proteins interact with each other to achieve the precise regulation of neurotransmitter release remains largely unclear. Using Caenorhabditis elegans as an in vivo model, we found that a gain-of-function mutant of UNC-18 increases locomotory activity and synaptic acetylcholine release, that it partially bypasses the requirement of UNC-13 for release, and that this bypass is synergistically augmented by the lack of TOM-1. We also elucidated the biochemical basis for the gain-of-function caused by this mutation

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

Disrupted apical exocytosis of cargo vesicles causes enteropathy in FHL5 patients with Munc18-2 mutations.

PubMed

Vogel, Georg F; van Rijn, Jorik M; Krainer, Iris M; Janecke, Andreas R; Posovszky, Carsten; Cohen, Marta; Searle, Claire; Jantchou, Prevost; Escher, Johanna C; Patey, Natalie; Cutz, Ernest; Müller, Thomas; Middendorp, Sabine; Hess, Michael W; Huber, Lukas A

2017-07-20

Familial hemophagocytic lymphohistiocytosis 5 (FHL5) is an autosomal recessive disease caused by mutations in STXBP2, coding for Munc18-2, which is required for SNARE-mediated membrane fusion. FHL5 causes hematologic and gastrointestinal symptoms characterized by chronic enteropathy that is reminiscent of microvillus inclusion disease (MVID). However, the molecular pathophysiology of FHL5-associated diarrhea is poorly understood. Five FHL5 patients, including four previously unreported patients, were studied. Morphology of duodenal sections was analyzed by electron and fluorescence microscopy. Small intestinal enterocytes and organoid-derived monolayers displayed the subcellular characteristics of MVID. For the analyses of Munc18-2-dependent SNARE-protein interactions, a Munc18-2 CaCo2-KO model cell line was generated by applying CRISPR/Cas9 technology. Munc18-2 is required for Slp4a/Stx3 interaction in fusion of cargo vesicles with the apical plasma membrane. Cargo trafficking was investigated in patient biopsies, patient-derived organoids, and the genome-edited model cell line. Loss of Munc18-2 selectively disrupts trafficking of certain apical brush-border proteins (NHE3 and GLUT5), while transport of DPPIV remained unaffected. Here, we describe the molecular mechanism how the loss of function of Munc18-2 leads to cargo-selective mislocalization of brush-border components and a subapical accumulation of cargo vesicles, as it is known from the loss of polarity phenotype in MVID.

Synapsin-dependent development of glutamatergic synaptic vesicles and presynaptic plasticity in postnatal mouse brain.

PubMed

Bogen, I L; Jensen, V; Hvalby, O; Walaas, S I

2009-01-12

Inactivation of the genes encoding the neuronal, synaptic vesicle-associated proteins synapsin I and II leads to severe reductions in the number of synaptic vesicles in the CNS. We here define the postnatal developmental period during which the synapsin I and/or II proteins modulate synaptic vesicle number and function in excitatory glutamatergic synapses in mouse brain. In wild-type mice, brain levels of both synapsin I and synapsin IIb showed developmental increases during synaptogenesis from postnatal days 5-20, while synapsin IIa showed a protracted increase during postnatal days 20-30. The vesicular glutamate transporters (VGLUT) 1 and VGLUT2 showed synapsin-independent development during postnatal days 5-10, following which significant reductions were seen when synapsin-deficient brains were compared with wild-type brains following postnatal day 20. A similar, synapsin-dependent developmental profile of vesicular glutamate uptake occurred during the same age periods. Physiological analysis of the development of excitatory glutamatergic synapses, performed in the CA1 stratum radiatum of the hippocampus from the two genotypes, showed that both the synapsin-dependent part of the frequency facilitation and the synapsin-dependent delayed response enhancement were restricted to the period after postnatal day 10. Our data demonstrate that while both synaptic vesicle number and presynaptic short-term plasticity are essentially independent of synapsin I and II prior to postnatal day 10, maturation and function of excitatory synapses appear to be strongly dependent on synapsin I and II from postnatal day 20.

Light-dependent cation gradients and electrical potential in Halobacterium halobium cell envelope vesicles

NASA Technical Reports Server (NTRS)

Lanyi, J. K.; Macdonald, R. E.

1977-01-01

Vesicles can be prepared from Halobacterium halobium cell envelopes, which contain properly oriented bacteriorhodopsin and which extrude H(+) during illumination. The pH difference that is generated across the membranes is accompanied by an electrical potential of 90 to 100 mV (interior negative) and the movements of other cations. Among these is the efflux of Na(+), which proceeds against its electrochemical potential. The relationship between the size and direction of the light-induced pH gradient and the rate of depletion of Na(+) from the vesicles, as well as other evidence, suggest that the active Na(+) extrusion is facilitated by a membrane component that exchanges H(+) for Na(+) with a stoichiometry greater than 1. The gradients of H(+) and Na(+) are thus coupled to one another. The Na(+) gradient (efflux much larger than influx), which arises during illumination, plays a major role in energizing the active transport of amino acids.

Putative terminator and/or effector functions of Arf GAPs in the trafficking of clathrin-coated vesicles.

PubMed

Kon, Shunsuke; Funaki, Tomo; Satake, Masanobu

2011-05-01

The role of ArfGAP1 as a terminator or effector in COPi-vesicle formation has been the subject of ongoing discussions. Here, the discussion on the putative terminator/effector functions has been enlarged to include Arf GAP members involved in the formation of clathrin-coated vesicles. ACAP1, whose role has been studied extensively, enhances the recycling of endocytosed proteins to the plasma membrane. Importantly, this positive role appears to be an overall reflection of both the terminator and effector activities attributed to ACAP1. Other Arf GAP subtypes have also been suggested to possess both terminator and effector activities. Interestingly, while most Arf GAP proteins regulate membrane trafficking by acting as facilitators, a few Arf GAP subtypes act as inhibitors.

Bubble-induced microstreaming: guiding and destroying lipid vesicles

NASA Astrophysics Data System (ADS)

Marmottant, Philippe; Hilgenfeldt, Sascha

2002-11-01

Micron-sized bubbles respond with strong oscillations when submitted to ultrasound. This has led to their use as echographic contrast enhancers. The large energy and force densities generated by the collapsing bubbles also make them non-invasive mechanical tools: Recently, it has been reported that the interaction of cavitating bubbles with nearby cells can render the latter permeable to large molecules (sonoporation), suggesting prospects for drug delivery and gene transfection. We have developed a laboratory setup that allows for a controlled study of the interaction of single microbubbles with single lipid bilayer vesicles. Substituting vesicles for cell membranes is advantageous because the mechanical properties of vesicles are well-known. Microscopic observations reveal that vesicles near a bubble follow the vivid streaming motion set up by the bubble. The vesicles "bounce" off the bubble, being periodically accelerated towards and away from it, and undergo well-defined shape deformations along their trajectory in accordance with fluid-dynamical theory. Break-up of vesicles could also be observed.

α-SNAP Interferes with the Zippering of the SNARE Protein Membrane Fusion Machinery

PubMed Central

Park, Yongsoo; Vennekate, Wensi; Yavuz, Halenur; Preobraschenski, Julia; Hernandez, Javier M.; Riedel, Dietmar; Walla, Peter Jomo; Jahn, Reinhard

2014-01-01

Neuronal exocytosis is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Before fusion, SNARE proteins form complexes bridging the membrane followed by assembly toward the C-terminal membrane anchors, thus initiating membrane fusion. After fusion, the SNARE complex is disassembled by the AAA-ATPase N-ethylmaleimide-sensitive factor that requires the cofactor α-SNAP to first bind to the assembled SNARE complex. Using chromaffin granules and liposomes we now show that α-SNAP on its own interferes with the zippering of membrane-anchored SNARE complexes midway through the zippering reaction, arresting SNAREs in a partially assembled trans-complex and preventing fusion. Intriguingly, the interference does not result in an inhibitory effect on synaptic vesicles, suggesting that membrane properties also influence the final outcome of α-SNAP interference with SNARE zippering. We suggest that binding of α-SNAP to the SNARE complex affects the ability of the SNARE complex to harness energy or transmit force to the membrane. PMID:24778182

Biomimetic proteolipid vesicles for targeting inflamed tissues

NASA Astrophysics Data System (ADS)

Molinaro, R.; Corbo, C.; Martinez, J. O.; Taraballi, F.; Evangelopoulos, M.; Minardi, S.; Yazdi, I. K.; Zhao, P.; De Rosa, E.; Sherman, M. B.; de Vita, A.; Toledano Furman, N. E.; Wang, X.; Parodi, A.; Tasciotti, E.

2016-09-01

A multitude of micro- and nanoparticles have been developed to improve the delivery of systemically administered pharmaceuticals, which are subject to a number of biological barriers that limit their optimal biodistribution. Bioinspired drug-delivery carriers formulated by bottom-up or top-down strategies have emerged as an alternative approach to evade the mononuclear phagocytic system and facilitate transport across the endothelial vessel wall. Here, we describe a method that leverages the advantages of bottom-up and top-down strategies to incorporate proteins derived from the leukocyte plasma membrane into lipid nanoparticles. The resulting proteolipid vesicles--which we refer to as leukosomes--retained the versatility and physicochemical properties typical of liposomal formulations, preferentially targeted inflamed vasculature, enabled the selective and effective delivery of dexamethasone to inflamed tissues, and reduced phlogosis in a localized model of inflammation.

Membrane-shed vesicles from the parasite Trichomonas vaginalis: characterization and their association with cell interaction.

PubMed

Nievas, Yesica R; Coceres, Veronica M; Midlej, Victor; de Souza, Wanderley; Benchimol, Marlene; Pereira-Neves, Antonio; Vashisht, Ajay A; Wohlschlegel, James A; Johnson, Patricia J; de Miguel, Natalia

2018-06-01

Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract, where it remains extracellular and adheres to epithelial cells. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Despite the serious consequences associated with trichomoniasis disease, little is known about parasite or host factors involved in attachment of the parasite-to-host epithelial cells. Here, we report the identification of microvesicle-like structures (MVs) released by T. vaginalis. MVs are considered universal transport vehicles for intercellular communication as they can incorporate peptides, proteins, lipids, miRNA, and mRNA, all of which can be transferred to target cells through receptor-ligand interactions, fusion with the cell membrane, and delivery of a functional cargo to the cytoplasm of the target cell. In the present study, we demonstrated that T. vaginalis release MVs from the plasma and the flagellar membranes of the parasite. We performed proteomic profiling of these structures demonstrating that they possess physical characteristics similar to mammalian extracellular vesicles and might be selectively charged with specific protein content. In addition, we demonstrated that viable T. vaginalis parasites release large vesicles (LVs), membrane structures larger than 1 µm that are able to interact with other parasites and with the host cell. Finally, we show that both populations of vesicles present on the surface of T vaginalis are induced in the presence of host cells, consistent with a role in modulating cell interactions.

ABC Triblock Copolymer Vesicles with Mesh-like Morphology

NASA Astrophysics Data System (ADS)

Zhao, Wei; Russell, Thomas; Grason, Gregory

2010-03-01

Polymer vesicles can be made from poly(isoprene-b-styrene-b-2-vinylpyridene) (PI-b-PS-b-P2VP) triblock copolymer under the confinement of anodic aluminum oxide (AAO) membrane. It was found that these vesicles have well-defined, nanoscopic size and a microphase-separated hydrophobic core, comprised of PS and PI blocks. Vesicle formation was tracked using both transmission and scanning electron microscopy. A mesh-like morphology formed in the core at a well-defined composition of three blocks. Confinement played an important role in generating these vesicles with such an unusual morphology.

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

PubMed

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

2015-05-01

The formation and release of outer membrane vesicles (OMVs) is a phenomenon observed in many bacteria, including Legionella pneumophila. During infection, this human pathogen primarily invades alveolar macrophages and replicates within a unique membrane-bound compartment termed Legionella-containing vacuole. In the current study, we analysed the membrane architecture of L. pneumophila OMVs by small-angle X-ray scattering and biophysically characterized OMV membranes. We investigated the interaction of L. pneumophila OMVs with model membranes by Förster resonance energy transfer and Fourier transform infrared spectroscopy. These experiments demonstrated the incorporation of OMV membrane material into liposomes composed of different eukaryotic phospholipids, revealing an endogenous property of OMVs to fuse with eukaryotic membranes. Cellular co-incubation experiments showed a dose- and time-dependent binding of fluorophore-labelled OMVs to macrophages. Trypan blue quenching experiments disclosed a rapid internalization of OMVs into macrophages at 37 and 4 °C. Purified OMVs induced tumour necrosis factor-α production in human macrophages at concentrations starting at 300 ng ml(-1). Experiments on HEK293-TLR2 and TLR4/MD-2 cell lines demonstrated a dominance of TLR2-dependent signalling pathways. In summary, we demonstrate binding, internalization and biological activity of L. pneumophila OMVs on human macrophages. Our data support OMV membrane fusion as a mechanism for the remote delivery of virulence factors to host cells. © 2014 John Wiley & Sons Ltd.

AC-electric field dependent electroformation of giant lipid vesicles.

PubMed

Politano, Timothy J; Froude, Victoria E; Jing, Benxin; Zhu, Yingxi

2010-08-01

Giant vesicles of larger than 5 microm, which have been of intense interest for their potential as drug delivery vehicles and as a model system for cell membranes, can be rapidly formed from a spin-coated lipid thin film under an electric field. In this work, we explore the AC-field dependent electroformation of giant lipid vesicles in aqueous media over a wide range of AC-frequency from 1 Hz to 1 MHz and peak-to-peak field strength from 0.212 V/mm to 40 V/mm between two parallel conducting electrode surfaces. By using fluorescence microscopy, we perform in-situ microscopic observations of the structural evolution of giant vesicles formed from spin-coated lipid films under varied uniform AC-electric fields. The real-time observation of bilayer bulging from the lipid film, vesicle growth and fusing further examine the critical role of AC-induced electroosmotic flow of surrounding fluids for giant vesicle formation. A rich AC-frequency and field strength phase diagram is obtained experimentally to predict the AC-electroformation of giant unilamellar vesicles (GUVs) of l-alpha-phosphatidylcholine, where a weak dependence of vesicle size on AC-frequency is observed at low AC-field voltages, showing decreased vesicle size with a narrowed size distribution with increased AC-frequency. Formation of vesicles was shown to be constrained by an upper field strength of 10 V/mm and an upper AC-frequency of 10 kHz. Within these parameters, giant lipid vesicles were formed predominantly unilamellar and prevalent across the entire electrode surfaces. Copyright 2010 Elsevier B.V. All rights reserved.

Dynamics of vesicles in electric fields

NASA Astrophysics Data System (ADS)

Vlahovska, Petia; Gracia, Ruben

2007-11-01

Electromechanical forces are widely used for cell manipulation. Knowledge of the physical mechanisms underlying the interaction of cells and external fields is essential for practical applications. Vesicles are model cells made of a lipid bilayer membrane. They are examples of ``soft'' particles, i.e., their shape when subjected to flow or electric field is not given a priori but it is governed by the balance of membrane, fluid and electrical stresses. This generic ``softness'' gives rise to a very complex vesicle dynamics in external fields. In an AC electric field, as the frequency is increased, vesicles filled with a fluid less conducting than the surrounding fluid undergo shape transition from prolate to oblate ellipsoids. The opposite effect is observed with drops. We present an electro- hydrodynamic theory based on the leaky dielectric model that quantitatively describes experimental observations. We compare drops and vesicles, and show how their distinct behavior stems from different interfacial properties.

Ultrastructure and biological function of matrix vesicles in bone mineralization.

PubMed

Hasegawa, Tomoka

2018-04-01

Bone mineralization is initiated by matrix vesicles, small extracellular vesicles secreted by osteoblasts, inducing the nucleation and subsequent growth of calcium phosphate crystals inside. Although calcium ions (Ca 2+ ) are abundant throughout the tissue fluid close to the matrix vesicles, the influx of phosphate ions (PO4 3- ) into matrix vesicles is a critical process mediated by several enzymes and transporters such as ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), ankylosis (ANK), and tissue nonspecific alkaline phosphatase (TNSALP). The catalytic activity of ENPP1 in osteoblasts generates inorganic pyrophosphate (PPi) intracellularly and extracellularly, and ANK may allow the intracellular PPi to pass through the plasma membrane to the outside of the osteoblasts. Although the extracellular PPi binds to growing hydroxyapatite crystals to prevent crystal overgrowth, TNSALP on the osteoblasts and matrix vesicles hydrolyzes PPi into PO4 3- monomers: the prevention of crystal growth is blocked, and PO4 3- monomers are supplied to matrix vesicles. In addition, PHOSPHO1 is thought to function inside matrix vesicles to catalyze phosphocoline, a constituent of the plasma membrane, consequently increasing PO4 3- in the vesicles. Accumulation of Ca 2+ and PO4 3- inside the matrix vesicles then initiates crystalline nucleation associated with the inner leaflet of the matrix vesicles. Calcium phosphate crystals elongate radially, penetrate the matrix vesicle's membrane, and finally grow out of the vesicles to form calcifying nodules, globular assemblies of needle-shaped mineral crystals retaining some of those transporters and enzymes. The subsequent growth of calcifying nodules appears to be regulated by surrounding organic compounds, finally leading to collagen mineralization.

Extracellular Vesicle (EV) Array: microarray capturing of exosomes and other extracellular vesicles for multiplexed phenotyping.

PubMed

Jørgensen, Malene; Bæk, Rikke; Pedersen, Shona; Søndergaard, Evo K L; Kristensen, Søren R; Varming, Kim

2013-01-01

Exosomes are one of the several types of cell-derived vesicles with a diameter of 30-100 nm. These extracellular vesicles are recognized as potential markers of human diseases such as cancer. However, their use in diagnostic tests requires an objective and high-throughput method to define their phenotype and determine their concentration in biological fluids. To identify circulating as well as cell culture-derived vesicles, the current standard is immunoblotting or a flow cytometrical analysis for specific proteins, both of which requires large amounts of purified vesicles. Based on the technology of protein microarray, we hereby present a highly sensitive Extracellular Vesicle (EV) Array capable of detecting and phenotyping exosomes and other extracellular vesicles from unpurified starting material in a high-throughput manner. To only detect the exosomes captured on the EV Array, a cocktail of antibodies against the tetraspanins CD9, CD63 and CD81 was used. These antibodies were selected to ensure that all exosomes captured are detected, and concomitantly excluding the detection of other types of microvesicles. The limit of detection (LOD) was determined on exosomes derived from the colon cancer cell line LS180. It clarified that supernatant from only approximately 10(4) cells was needed to obtain signals or that only 2.5×10(4) exosomes were required for each microarray spot (~1 nL). Phenotyping was performed on plasma (1-10 µL) from 7 healthy donors, which were applied to the EV Array with a panel of antibodies against 21 different cellular surface antigens and cancer antigens. For each donor, there was considerable heterogeneity in the expression levels of individual markers. The protein profiles of the exosomes (defined as positive for CD9, CD63 and CD81) revealed that only the expression level of CD9 and CD81 was approximately equal in the 7 donors. This implies questioning the use of CD63 as a standard exosomal marker since the expression level of this

Detergent-mediated incorporation of transmembrane proteins in giant unilamellar vesicles with controlled physiological contents

PubMed Central

Dezi, Manuela; Di Cicco, Aurelie; Bassereau, Patricia; Lévy, Daniel

2013-01-01

Giant unilamellar vesicles (GUVs) are convenient biomimetic systems of the same size as cells that are increasingly used to quantitatively address biophysical and biochemical processes related to cell functions. However, current approaches to incorporate transmembrane proteins in the membrane of GUVs are limited by the amphiphilic nature or proteins. Here, we report a method to incorporate transmembrane proteins in GUVs, based on concepts developed for detergent-mediated reconstitution in large unilamellar vesicles. Reconstitution is performed either by direct incorporation from proteins purified in detergent micelles or by fusion of purified native vesicles or proteoliposomes in preformed GUVs. Lipid compositions of the membrane and the ionic, protein, or DNA compositions in the internal and external volumes of GUVs can be controlled. Using confocal microscopy and functional assays, we show that proteins are unidirectionally incorporated in the GUVs and keep their functionality. We have successfully tested our method with three types of transmembrane proteins. GUVs containing bacteriorhodopsin, a photoactivable proton pump, can generate large transmembrane pH and potential gradients that are light-switchable and stable for hours. GUVs with FhuA, a bacterial porin, were used to follow the DNA injection by T5 phage upon binding to its transmembrane receptor. GUVs incorporating BmrC/BmrD, a bacterial heterodimeric ATP-binding cassette efflux transporter, were used to demonstrate the protein-dependent translocation of drugs and their interactions with encapsulated DNA. Our method should thus apply to a wide variety of membrane or peripheral proteins for producing more complex biomimetic GUVs. PMID:23589883

Synaptic vesicle distribution by conveyor belt.

PubMed

Moughamian, Armen J; Holzbaur, Erika L F

2012-03-02

The equal distribution of synaptic vesicles among synapses along the axon is critical for robust neurotransmission. Wong et al. show that the continuous circulation of synaptic vesicles throughout the axon driven by molecular motors ultimately yields this even distribution. Copyright © 2012 Elsevier Inc. All rights reserved.

Intraluminal proteome and peptidome of human urinary extracellular vesicles.

PubMed

Liu, Xinyu; Chinello, Clizia; Musante, Luca; Cazzaniga, Marta; Tataruch, Dorota; Calzaferri, Giulio; James Smith, Andrew; De Sio, Gabriele; Magni, Fulvio; Zou, Hequn; Holthofer, Harry

2015-06-01

Urinary extracellular vesicles (UEVs) are a novel source for disease biomarker discovery. However, Tamm-Horsfall protein (THP) is still a challenge for proteomic analysis since it can inhibit detection of low-abundance proteins. Here, we introduce a new approach that does not involve an ultracentrifugation step to enrich vesicles and that reduces the amount of THP to manageable levels. UEVs were dialyzed and ultrafiltered after reduction and alkylation. The retained fraction was digested with trypsin to reduce the remaining THP and incubated with deoxycholate (DOC). The internal peptidome and internal proteome were analyzed by LC-ESI-MS. A total of 942 different proteins and 3115 unique endogenous peptide fragments deriving from 973 different protein isoforms were identified. Around 82% of the key endosomal sorting complex required for transport components of UEVs generation could be detected from the intraluminal content. Our UEVs preparation protocol provides a simplified way to investigate the intraluminal proteome and peptidome, in particular the subpopulation of UEVs of the trypsin-resistant class of exosomes (positive for tumor susceptibility gene101) and eliminates the majority of interfering proteins such as THP. This method allows the possibility to study endoproteome and endopeptidome of UEVs, thus greatly facilitating biomarker discovery. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Role of Ion Selectivity of the Fusion Pore on Transmission and the Exocytosis of Neurotransmitters and Hormones

NASA Astrophysics Data System (ADS)

Delacruz, Joannalyn Bongar

Healthy nervous system function depends on proper transmission. Synaptic transmission occurs by the release of transmitters from vesicles that fuse to the plasma membrane of a pre-synaptic cell. Regulated release of neurotransmitters, neuropeptides, and hormones occurs by exocytosis, initiated by the formation of the fusion pore. The initial fusion pore has molecular dimensions with a diameter of 1-2 nm and a rapid lifetime on the millisecond time scale. It connects the vesicular lumen and extracellular space, serving as an important step for regulating the release of charged transmitters. Comprehending the molecular structure and biophysical properties of the fusion pore is essential for a mechanistic understanding of vesicle-plasma membrane fusion and transmitter release. Release of charged transmitter molecules such as glutamate, acetylcholine, dopamine, or noradrenaline through a narrow fusion pore requires compensation of change in charge. Transmitter release through the fusion pore is therefore an electrodiffusion process. If the fusion pore is selective for specific ions, then its selectivity will affect the rate of transmitter release via the voltage gradient that develops across the fusion pore. The elucidation of these mechanisms can lead to a better understanding of nervous system cell biology, neural and endocrine signaling, learning, memory, motor control, sensory function and integration, and in particular synaptic transmission. This investigation can advance our understanding of neurological disorders in which noradrenergic and dopaminergic exocytosis is disturbed, leading to neurological consequences of developmental disorders, epilepsy, Parkinson's disease, and other neurodegenerative diseases. Ultimately, understanding the role of selectivity in the fusion pore and its effects on exocytosis can contribute to the development of more effective therapies. This study investigates the selectivity of the fusion pore by observing the effects of ion

Binding of SEC11 Indicates Its Role in SNARE Recycling after Vesicle Fusion and Identifies Two Pathways for Vesicular Traffic to the Plasma Membrane[OPEN

PubMed Central

Karnik, Rucha; Zhang, Ben; Waghmare, Sakharam; Aderhold, Christin; Grefen, Christopher; Blatt, Michael R.

2015-01-01

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins drive vesicle fusion in all eukaryotes and contribute to homeostasis, pathogen defense, cell expansion, and growth in plants. Two homologous SNAREs, SYP121 (=SYR1/PEN1) and SYP122, dominate secretory traffic to the Arabidopsis thaliana plasma membrane. Although these proteins overlap functionally, differences between SYP121 and SYP122 have surfaced, suggesting that they mark two discrete pathways for vesicular traffic. The SNAREs share primary cognate partners, which has made separating their respective control mechanisms difficult. Here, we show that the regulatory protein SEC11 (=KEULE) binds selectively with SYP121 to affect secretory traffic mediated by this SNARE. SEC11 rescued traffic block by dominant-negative (inhibitory) fragments of both SNAREs, but only in plants expressing the native SYP121. Traffic and its rescue were sensitive to mutations affecting SEC11 interaction with the N terminus of SYP121. Furthermore, the domain of SEC11 that bound the SYP121 N terminus was itself able to block secretory traffic in the wild type and syp122 but not in syp121 mutant Arabidopsis. Thus, SEC11 binds and selectively regulates secretory traffic mediated by SYP121 and is important for recycling of the SNARE and its cognate partners. PMID:25747882

The presynaptic ribbon maintains vesicle populations at the hair cell afferent fiber synapse

PubMed Central

Becker, Lars; Schnee, Michael E; Niwa, Mamiko; Sun, Willy; Maxeiner, Stephan; Talaei, Sara; Kachar, Bechara; Rutherford, Mark A

2018-01-01

The ribbon is the structural hallmark of cochlear inner hair cell (IHC) afferent synapses, yet its role in information transfer to spiral ganglion neurons (SGNs) remains unclear. We investigated the ribbon’s contribution to IHC synapse formation and function using KO mice lacking RIBEYE. Despite loss of the entire ribbon structure, synapses retained their spatiotemporal development and KO mice had a mild hearing deficit. IHCs of KO had fewer synaptic vesicles and reduced exocytosis in response to brief depolarization; a high stimulus level rescued exocytosis in KO. SGNs exhibited a lack of sustained excitatory postsynaptic currents (EPSCs). We observed larger postsynaptic glutamate receptor plaques, potentially compensating for the reduced EPSC rate in KO. Surprisingly, large-amplitude EPSCs were maintained in KO, while a small population of low-amplitude slower EPSCs was increased in number. The ribbon facilitates signal transduction at physiological stimulus levels by retaining a larger residency pool of synaptic vesicles. PMID:29328021

Topological defects and shapes of triatic liquid crystal vesicles

NASA Astrophysics Data System (ADS)

Serafin, Francesco; Manyuhina, Oksana; Bowick, Mark

Is shape the manifestation of function, or does shape determine function? Since the time of Aristotle, the study of shape has proven to be a fruitful way to understand the behavior of physical systems, from atomic to biological systems scales. Two dimensional soft membranes are a perfect setting to understand the emergence of shape. An interesting possibility is to control and design new self-assemblable supramolecular shapes by coating the surface of soft closed vesicles with liquid crystals (LC) of various symmetries. The microscopic geometry of the liquid crystal molecules, in particular the structure of topological defects, when combined with the topology of the vesicle's surface, ultimately determines the vesicle's shape. Recent work has shown that the minimal energy shapes of smectic and nematic vesicles are faceted polyhedra. A very soft smectic vesicle develops sharp creases and forms a faceted tetrahedron. When the coating LC has the symmetries of the square, the vesicle forms a cube. In this work we extend these results to a 3-fold symmetric LC, proving that the vesicle's ground state is an octahedron. This gives a systematic way of predicting vesicle's shapes as we change the liquid crystal's symmetry. Soft Matter Program of Syracuse University.

Hendra virus fusion protein transmembrane domain contributes to pre-fusion protein stability

PubMed Central

Webb, Stacy; Nagy, Tamas; Moseley, Hunter; Fried, Michael; Dutch, Rebecca

2017-01-01

Enveloped viruses utilize fusion (F) proteins studding the surface of the virus to facilitate membrane fusion with a target cell membrane. Fusion of the viral envelope with a cellular membrane is required for release of viral genomic material, so the virus can ultimately reproduce and spread. To drive fusion, the F protein undergoes an irreversible conformational change, transitioning from a metastable pre-fusion conformation to a more thermodynamically stable post-fusion structure. Understanding the elements that control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Mutations in F protein transmembrane (TM) domains implicated the TM domain in the fusion process, but the structural and molecular details in fusion remain unclear. Previously, analytical ultracentrifugation was utilized to demonstrate that isolated TM domains of Hendra virus F protein associate in a monomer-trimer equilibrium (Smith, E. C., Smith, S. E., Carter, J. R., Webb, S. R., Gibson, K. M., Hellman, L. M., Fried, M. G., and Dutch, R. E. (2013) J. Biol. Chem. 288, 35726–35735). To determine factors driving this association, 140 paramyxovirus F protein TM domain sequences were analyzed. A heptad repeat of β-branched residues was found, and analysis of the Hendra virus F TM domain revealed a heptad repeat leucine-isoleucine zipper motif (LIZ). Replacement of the LIZ with alanine resulted in dramatically reduced TM-TM association. Mutation of the LIZ in the whole protein resulted in decreased protein stability, including pre-fusion conformation stability. Together, our data suggest that the heptad repeat LIZ contributed to TM-TM association and is important for F protein function and pre-fusion stability. PMID:28213515

Metal Sorbing Vesicles: Light Scattering Characterization and Metal Sorbtion Behavior.

NASA Astrophysics Data System (ADS)

van Zanten, John Hollis

1992-01-01

The research described herein consisted of two parts: light scattering characterization of vesicles and kinetic investigations of metal sorbing vesicles. Static light scattering techniques can be used to determine the geometric size, shape and apparent molecular weight of phosphatidylcholine vesicles in aqueous suspension. A Rayleigh-Gans-Debye (RGD) approximation analysis of multiangle scattered light intensity data yields the size and degree of polydispersity of the vesicles in solution, while the Zimm plot technique provides the radius of gyration and apparent weight-average molecular weight. Together the RGD approximation and Zimm plots can be used to confirm the geometric shape of vesicles and can give a good estimate of the vesicle wall thickness in some cases. Vesicles varying from 40 to 115 nm in diameter have been characterized effectively. The static light scattering measurements indicate that, as expected, phosphatidylcholine vesicles in this size range scatter light as isotropic hollow spheres. Additionally, static and dynamic light scattering measurements have been made and compared with one another. The values for geometric radii determined by static light scattering typically agree with those estimated by dynamic light scattering to within a few percent. Interestingly however, dynamic measurements suggest that there is a significant degree of polydispersity present in the vesicle dispersions, while static measurements indicate near size monodisperse dispersions. Metal sorbing vesicles which harbor ionophores, such as antibiotic A23187 and synthetic carriers, in their bilayer membranes have been produced. These vesicles also encapsulate the chelating compound, nitrilotriacetate, to provide the driving force for metal ion uptake. Very dilute dispersions (on the order of 0.03% w/v) of these metal sorbing vesicles were capable of removing Cd ^{2+} and Pb^{2+ } from dilute aqueous solution (5 ppm and less) and concentrating these metal ions several

Formation and size distribution of self-assembled vesicles

PubMed Central

Huang, Changjin; Quinn, David; Suresh, Subra

2017-01-01

When detergents and phospholipid membranes are dispersed in aqueous solutions, they tend to self-assemble into vesicles of various shapes and sizes by virtue of their hydrophobic and hydrophilic segments. A clearer understanding of such vesiculation processes holds promise for better elucidation of human physiology and disease, and paves the way to improved diagnostics, drug development, and drug delivery. Here we present a detailed analysis of the energetics and thermodynamics of vesiculation by recourse to nonlinear elasticity, taking into account large deformation that may arise during the vesiculation process. The effects of membrane size, spontaneous curvature, and membrane stiffness on vesiculation and vesicle size distribution were investigated, and the critical size for vesicle formation was determined and found to compare favorably with available experimental evidence. Our analysis also showed that the critical membrane size for spontaneous vesiculation was correlated with membrane thickness, and further illustrated how the combined effects of membrane thickness and physical properties influenced the size, shape, and distribution of vesicles. These findings shed light on the formation of physiological extracellular vesicles, such as exosomes. The findings also suggest pathways for manipulating the size, shape, distribution, and physical properties of synthetic vesicles, with potential applications in vesicle physiology, the pathobiology of cancer and other diseases, diagnostics using in vivo liquid biopsy, and drug delivery methods. PMID:28265065

Primary vesicles, vesicle-rich segregation structures and recognition of primary and secondary porosities in lava flows from the Paraná igneous province, southern Brazil

NASA Astrophysics Data System (ADS)

Barreto, Carla Joana S.; de Lima, Evandro F.; Goldberg, Karin

2017-04-01

This study focuses on a volcanic succession of pāhoehoe to rubbly lavas of the Paraná-Etendeka Province exposed in a single road profile in southernmost Brazil. This work provides an integrated approach for examining primary vesicles and vesicle-rich segregation structures at the mesoscopic scale. In addition, this study provides a quantitative analysis of pore types in thin section. We documented distinct distribution patterns of vesicle and vesicle-rich segregation structures according to lava thickness. In compound pāhoehoe lavas, the cooling allows only vesicles (<1 cm size) and pipe vesicles to be frozen into place. In inflated pāhoehoe lavas, vesicles of different sizes are common, including pipe vesicles, and also segregation structures such as proto-cylinders, cylinders, cylinder sheets, vesicle sheets, and pods. In rubbly lavas, only vesicles of varying sizes occur. Gas release from melt caused the formation of primary porosity, while hydrothermal alteration and tectonic fracturing are the main processes that generated secondary porosity. Although several forms of porosity were created in the basaltic lava flows, the precipitation of secondary minerals within the pores has tended to reduce the original porosities. Late-stage fractures could create efficient channel networks for possible hydrocarbon/groundwater migration and entrapment owing to their ability to connect single pores. Quantitative permeability data should be gathered in future studies to confirm the potential of these lavas for store hydrocarbons or groundwater.

Crystal Structure of the Pre-fusion Nipah Virus Fusion Glycoprotein Reveals a Novel Hexamer-of-Trimers Assembly.

PubMed

Xu, Kai; Chan, Yee-Peng; Bradel-Tretheway, Birgit; Akyol-Ataman, Zeynep; Zhu, Yongqun; Dutta, Somnath; Yan, Lianying; Feng, YanRu; Wang, Lin-Fa; Skiniotis, Georgios; Lee, Benhur; Zhou, Z Hong; Broder, Christopher C; Aguilar, Hector C; Nikolov, Dimitar B

2015-12-01

Nipah virus (NiV) is a paramyxovirus that infects host cells through the coordinated efforts of two envelope glycoproteins. The G glycoprotein attaches to cell receptors, triggering the fusion (F) glycoprotein to execute membrane fusion. Here we report the first crystal structure of the pre-fusion form of the NiV-F glycoprotein ectodomain. Interestingly this structure also revealed a hexamer-of-trimers encircling a central axis. Electron tomography of Nipah virus-like particles supported the hexameric pre-fusion model, and biochemical analyses supported the hexamer-of-trimers F assembly in solution. Importantly, structure-assisted site-directed mutagenesis of the interfaces between F trimers highlighted the functional relevance of the hexameric assembly. Shown here, in both cell-cell fusion and virus-cell fusion systems, our results suggested that this hexamer-of-trimers assembly was important during fusion pore formation. We propose that this assembly would stabilize the pre-fusion F conformation prior to cell attachment and facilitate the coordinated transition to a post-fusion conformation of all six F trimers upon triggering of a single trimer. Together, our data reveal a novel and functional pre-fusion architecture of a paramyxoviral fusion glycoprotein.

Molecular mechanism of respiratory syncytial virus fusion inhibitors

DOE PAGES

Battles, Michael B.; Langedijk, Johannes P.; Furmanova-Hollenstein, Polina; ...

2015-12-07

Respiratory syncytial virus (RSV) is a leading cause of pneumonia and bronchiolitis in young children and the elderly. Therapeutic small molecules have been developed that bind the RSV F glycoprotein and inhibit membrane fusion, yet their binding sites and molecular mechanisms of action remain largely unknown. In this paper, we show that these inhibitors bind to a three-fold-symmetric pocket within the central cavity of the metastable prefusion conformation of RSV F. Inhibitor binding stabilizes this conformation by tethering two regions that must undergo a structural rearrangement to facilitate membrane fusion. Inhibitor-escape mutations occur in residues that directly contact the inhibitorsmore » or are involved in the conformational rearrangements required to accommodate inhibitor binding. Resistant viruses do not propagate as well as wild-type RSV in vitro, indicating a fitness cost for inhibitor escape. Finally and collectively, these findings provide new insight into class I viral fusion proteins and should facilitate development of optimal RSV fusion inhibitors.« less

Molecular mechanism of respiratory syncytial virus fusion inhibitors

PubMed Central

Battles, Michael B; Langedijk, Johannes P; Furmanova-Hollenstein, Polina; Chaiwatpongsakorn, Supranee; Costello, Heather M; Kwanten, Leen; Vranckx, Luc; Vink, Paul; Jaensch, Steffen; Jonckers, Tim H M; Koul, Anil; Arnoult, Eric; Peeples, Mark E; Roymans, Dirk; McLellan, Jason S

2016-01-01

Respiratory syncytial virus (RSV) is a leading cause of pneumonia and bronchiolitis in young children and the elderly. Therapeutic small molecules have been developed that bind the RSV F glycoprotein and inhibit membrane fusion, yet their binding sites and molecular mechanisms of action remain largely unknown. Here we show that these inhibitors bind to a three-fold-symmetric pocket within the central cavity of the metastable prefusion conformation of RSV F. Inhibitor binding stabilizes this conformation by tethering two regions that must undergo a structural rearrangement to facilitate membrane fusion. Inhibitor-escape mutations occur in residues that directly contact the inhibitors or are involved in the conformational rearrangements required to accommodate inhibitor binding. Resistant viruses do not propagate as well as wild-type RSV in vitro, indicating a fitness cost for inhibitor escape. Collectively, these findings provide new insight into class I viral fusion proteins and should facilitate development of optimal RSV fusion inhibitors. PMID:26641933

Low-resolution simulations of vesicle suspensions in 2D

NASA Astrophysics Data System (ADS)

Kabacaoğlu, Gökberk; Quaife, Bryan; Biros, George

2018-03-01

Vesicle suspensions appear in many biological and industrial applications. These suspensions are characterized by rich and complex dynamics of vesicles due to their interaction with the bulk fluid, and their large deformations and nonlinear elastic properties. Many existing state-of-the-art numerical schemes can resolve such complex vesicle flows. However, even when using provably optimal algorithms, these simulations can be computationally expensive, especially for suspensions with a large number of vesicles. These high computational costs can limit the use of simulations for parameter exploration, optimization, or uncertainty quantification. One way to reduce the cost is to use low-resolution discretizations in space and time. However, it is well-known that simply reducing the resolution results in vesicle collisions, numerical instabilities, and often in erroneous results. In this paper, we investigate the effect of a number of algorithmic empirical fixes (which are commonly used by many groups) in an attempt to make low-resolution simulations more stable and more predictive. Based on our empirical studies for a number of flow configurations, we propose a scheme that attempts to integrate these fixes in a systematic way. This low-resolution scheme is an extension of our previous work [51,53]. Our low-resolution correction algorithms (LRCA) include anti-aliasing and membrane reparametrization for avoiding spurious oscillations in vesicles' membranes, adaptive time stepping and a repulsion force for handling vesicle collisions and, correction of vesicles' area and arc-length for maintaining physical vesicle shapes. We perform a systematic error analysis by comparing the low-resolution simulations of dilute and dense suspensions with their high-fidelity, fully resolved, counterparts. We observe that the LRCA enables both efficient and statistically accurate low-resolution simulations of vesicle suspensions, while it can be 10× to 100× faster.

Deformation of phospholipid vesicles in an optical stretcher.

PubMed

Delabre, Ulysse; Feld, Kasper; Crespo, Eleonore; Whyte, Graeme; Sykes, Cecile; Seifert, Udo; Guck, Jochen

2015-08-14

Phospholipid vesicles are common model systems for cell membranes. Important aspects of the membrane function relate to its mechanical properties. Here we have investigated the deformation behaviour of phospholipid vesicles in a dual-beam laser trap, also called an optical stretcher. This study explicitly makes use of the inherent heating present in such traps to investigate the dependence of vesicle deformation on temperature. By using lasers with different wavelengths, optically induced mechanical stresses and temperature increase can be tuned fairly independently with a single setup. The phase transition temperature of vesicles can be clearly identified by an increase in deformation. In the case of no heating effects, a minimal model for drop deformation in an optical stretcher and a more specific model for vesicle deformation that takes explicitly into account the angular dependence of the optical stress are presented to account for the experimental results. Elastic constants are extracted from the fitting procedures, which agree with literature data. This study demonstrates the utility of optical stretching, which is easily combined with microfluidic delivery, for the future serial, high-throughput study of the mechanical and thermodynamic properties of phospholipid vesicles.

Membrane Transport in Isolated Vesicles from Sugarbeet Taproot 1

PubMed Central

Briskin, Donald P.; Thornley, W. Robert; Wyse, Roger E.

1985-01-01

Sealed membrane vesicles were isolated from homogenates of sugarbeet (Beta vulgaris L.) taproot by a combination of differential centrifugation, extraction with KI, and dextran gradient centrifugation. Relative to the KI-extracted microsomes, the content of plasma membranes, mitochondrial membranes, and Golgi membranes was much reduced in the final vesicle fraction. A component of ATPase activity that was inhibited by nitrate co-enriched with the capacity of the vesicles to form a steady state pH gradient during the purification procedure. This suggests that the nitrate-sensitive ATPase may be involved in driving H+-transport, and this is consistent with the observation that H+-transport, in the final vesicle fraction was inhibited by nitrate. Proton transport in the sugarbeet vesicles was substrate specific for ATP, insensitive to sodium vanadate and oligomycin but was inhibited by diethylstilbestrol and N,N′-dicyclohexylcarbodiimide. The formation of a pH gradient in the vesicles was enhanced by halide ions in the sequence I− > Br− > Cl− while F− was inhibitory. These stimulatory effects occur from both a direct stimulation of the ATPase by anions and a reduction in the vesicle membrane potential. In the presence of Cl−, alkali cations reduce the pH gradient relative to that observed with bis-tris-propane, possibly by H+/alkali cation exchange. Based upon the properties of the H+-transporting vesicles, it is proposed that they are most likely derived from the tonoplast so that this vesicle preparation would represent a convenient system for studying the mechanism of transport at this membrane boundary. PMID:16664342

Destruction of giant cluster-like vesicles by an ultrasonically activated device

NASA Astrophysics Data System (ADS)

Yahagi, Ryosuke; Yoshida, Kenji; Zhang, Yiting; Ebata, Masahiko; Toyota, Taro; Yamaguchi, Tadashi; Hayashi, Hideki

2016-07-01

In this paper, we propose a technically simple method of destroying a tissue marker composed of giant cluster-like vesicles (GCVs) to facilitate laparoscopic surgeries; the method releases various biological tracers contained in GCVs. An ultrasonically activated device (USAD) emitting 55.5 kHz ultrasound was employed for this purpose. Optical microscopy and fluorospectrophotometry revealed the destruction of GCVs after ultrasound irradiation when the blade tip was set 1.0 mm or closer to, but not directly in contact with, a GCV-containing cell. This means that USAD could be safely used for destroying this GCV tissue marker in clinical settings.

ER-associated SNAREs and Sey1p mediate nuclear fusion at two distinct steps during yeast mating.

PubMed

Rogers, Jason V; Arlow, Tim; Inkellis, Elizabeth R; Koo, Timothy S; Rose, Mark D

2013-12-01

During yeast mating, two haploid nuclei fuse membranes to form a single diploid nucleus. However, the known proteins required for nuclear fusion are unlikely to function as direct fusogens (i.e., they are unlikely to directly catalyze lipid bilayer fusion) based on their predicted structure and localization. Therefore we screened known fusogens from vesicle trafficking (soluble N-ethylmaleimide-sensitive factor attachment protein receptors [SNAREs]) and homotypic endoplasmic reticulum (ER) fusion (Sey1p) for additional roles in nuclear fusion. Here we demonstrate that the ER-localized SNAREs Sec20p, Ufe1p, Use1p, and Bos1p are required for efficient nuclear fusion. In contrast, Sey1p is required indirectly for nuclear fusion; sey1Δ zygotes accumulate ER at the zone of cell fusion, causing a block in nuclear congression. However, double mutants of Sey1p and Sec20p, Ufe1p, or Use1p, but not Bos1p, display extreme ER morphology defects, worse than either single mutant, suggesting that retrograde SNAREs fuse ER in the absence of Sey1p. Together these data demonstrate that SNAREs mediate nuclear fusion, ER fusion after cell fusion is necessary to complete nuclear congression, and there exists a SNARE-mediated, Sey1p-independent ER fusion pathway.

Release of Membrane-Bound Vesicles and Inhibition of Tumor Cell Adhesion by the Peptide Neopetrosiamide A

PubMed Central

Austin, Pamela; Heller, Markus; Williams, David E.; McIntosh, Lawrence P.; Vogl, A. Wayne; Foster, Leonard J.; Andersen, Raymond J.; Roberge, Michel; Roskelley, Calvin D.

2010-01-01

Background Neopetrosiamide A (NeoA) is a 28-amino acid tricyclic peptide originally isolated from a marine sponge as a tumor cell invasion inhibitor whose mechanism of action is unknown. Methodology/Principal Findings We show that NeoA reversibly inhibits tumor cell adhesion, disassembles focal adhesions in pre-attached cells, and decreases the level of β1 integrin subunits on the cell surface. NeoA also induces the formation of dynamic, membrane-bound protrusions on the surface of treated cells and the release of membrane-bound vesicles into the culture medium. Proteomic analysis indicates that the vesicles contain EGF and transferrin receptors as well as a number of proteins involved in adhesion and migration including: β1 integrin and numerous α integrin subunits; actin and actin-binding proteins such as cofilin, moesin and myosin 1C; and membrane modulating eps15 homology domain (EHD) proteins. Surface labeling, trafficking inhibition, and real-time imaging experiments all suggest that β1 integrin-containing vesicles are released directly from NeoA-induced cell surface protrusions rather than from vesicles generated intracellularly. The biological activity of NeoA is dependent on its disulfide bond pattern and NMR spectroscopy indicates that the peptide is globular with a continuous ridge of hydrophobic groups flanked by charged amino acid residues that could facilitate a simultaneous interaction with lipids and proteins in the membrane. Conclusions/Significance NeoA is an anti-adhesive peptide that decreases cell surface integrin levels through a novel, yet to be elucidated, mechanism that involves the release of adhesion molecule-containing vesicles from the cell surface. PMID:20520768

The International Society for Extracellular Vesicles launches the first massive open online course on extracellular vesicles.

PubMed

Lässer, Cecilia; Théry, Clotilde; Buzás, Edit I; Mathivanan, Suresh; Zhao, Weian; Gho, Yong Song; Lötvall, Jan

2016-01-01

The International Society for Extracellular Vesicles (ISEV) has organised its first educational online course for students and beginners in the field of extracellular vesicles (EVs). This course, "Basics of Extracellular Vesicles," uses recorded lectures from experts in the field and will be open for an unlimited number of participants. The course is divided into 5 modules and can be accessed at www.coursera.org/learn/extracellular-vesicles. The first module is an introduction to the field covering the nomenclature and history of EVs. Module 2 focuses on the biogenesis and uptake mechanisms of EVs, as well as their RNA, protein and lipid cargo. Module 3 covers the collection and processing of cell culture media and body fluids such as blood, breast milk, cerebrospinal fluid and urine prior to isolation of EVs. Modules 4 and 5 present different isolation methods and characterisation techniques utilised in the EV field. Here, differential ultracentrifugation, size-exclusion chromatography, density gradient centrifugation, kit-based precipitation, electron microscopy, cryo-electron microscopy, flow cytometry, atomic-force microscopy and nanoparticle-tracking analysis are covered. This first massive open online course (MOOC) on EVs was launched on 15 August 2016 at the platform "Coursera" and is free of charge.

Haloarchaea and the Formation of Gas Vesicles

PubMed Central

Pfeifer, Felicitas

2015-01-01

Halophilic Archaea (Haloarchaea) thrive in salterns containing sodium chloride concentrations up to saturation. Many Haloarchaea possess genes encoding gas vesicles, but only a few species, such as Halobacterium salinarum and Haloferax mediterranei, produce these gas-filled, proteinaceous nanocompartments. Gas vesicles increase the buoyancy of cells and enable them to migrate vertically in the water body to regions with optimal conditions. Their synthesis depends on environmental factors, such as light, oxygen supply, temperature and salt concentration. Fourteen gas vesicle protein (gvp) genes are involved in their formation, and regulation of gvp gene expression occurs at the level of transcription, including the two regulatory proteins, GvpD and GvpE, but also at the level of translation. The gas vesicle wall is solely formed of proteins with the two major components, GvpA and GvpC, and seven additional accessory proteins are also involved. Except for GvpI and GvpH, all of these are required to form the gas permeable wall. The applications of gas vesicles include their use as an antigen presenter for viral or pathogen proteins, but also as a stable ultrasonic reporter for biomedical purposes. PMID:25648404

The motion of a train of vesicles in channel flow

NASA Astrophysics Data System (ADS)

Barakat, Joseph; Shaqfeh, Eric

2017-11-01

The inertialess motion of a train of lipid-bilayer vesicles flowing through a channel is simulated using a 3D boundary integral equation method. Steady-state results are reported for vesicles positioned concentrically inside cylindrical channels of circular, square, and rectangular cross sections. The vesicle translational velocity U and excess channel pressure drop Δp+ depend strongly on the ratio of the vesicle radius to the hydraulic radius λ and the vesicle reduced volume υ. ``Deflated vesicles'' of lower reduced volume υ are more streamlined and translate with greater velocity U relative to the mean flow velocity V. Increasing the vesicle size (λ) increases the wall friction force and extra pressure drop Δp+, which in turn reduces the vesicle velocity U. Hydrodynamic interactions between vesicles in a periodic train are largely screened by the channel walls, in accordance with previous results for spheres and drops. The hydraulic resistance is compared across different cross sections, and a simple correction factor is proposed to unify the results. Nonlinear effects are observed when β - the ratio of membrane bending elasticity to viscous traction - is changed. The simulation results show excellent agreement with available experimental measurements as well as a previously reported ``small-gap theory'' valid for large values of λ. NSF CBET 1066263/1066334.

The BAR Domain Proteins: Molding Membranes in Fission, Fusion, and Phagy

PubMed Central

Ren, Gang; Vajjhala, Parimala; Lee, Janet S.; Winsor, Barbara; Munn, Alan L.

2006-01-01

The Bin1/amphiphysin/Rvs167 (BAR) domain proteins are a ubiquitous protein family. Genes encoding members of this family have not yet been found in the genomes of prokaryotes, but within eukaryotes, BAR domain proteins are found universally from unicellular eukaryotes such as yeast through to plants, insects, and vertebrates. BAR domain proteins share an N-terminal BAR domain with a high propensity to adopt α-helical structure and engage in coiled-coil interactions with other proteins. BAR domain proteins are implicated in processes as fundamental and diverse as fission of synaptic vesicles, cell polarity, endocytosis, regulation of the actin cytoskeleton, transcriptional repression, cell-cell fusion, signal transduction, apoptosis, secretory vesicle fusion, excitation-contraction coupling, learning and memory, tissue differentiation, ion flux across membranes, and tumor suppression. What has been lacking is a molecular understanding of the role of the BAR domain protein in each process. The three-dimensional structure of the BAR domain has now been determined and valuable insight has been gained in understanding the interactions of BAR domains with membranes. The cellular roles of BAR domain proteins, characterized over the past decade in cells as distinct as yeasts, neurons, and myocytes, can now be understood in terms of a fundamental molecular function of all BAR domain proteins: to sense membrane curvature, to bind GTPases, and to mold a diversity of cellular membranes. PMID:16524918

Diffusion behavior of lipid vesicles in entangled polymer solutions.

PubMed Central

Cao, X; Bansil, R; Gantz, D; Moore, E W; Niu, N; Afdhal, N H

1997-01-01

Dynamic light scattering was used to follow the tracer diffusion of phospholipid/cholesterol vesicles in aqueous polyacrylamide solutions and compared with the diffusive behavior of polystyrene (PS) latex spheres of comparable diameters. Over the range of the matrix concentration examined (Cp = 0.1-10 mg/ml), the diffusivities of the PS spheres and the large multilamellar vesicles exhibited the Stokes-Einstein (SE) relation, while the diffusivity of the unilamellar vesicles did not follow the increase of the solution's viscosity caused by the presence of the matrix molecules. The difference between the diffusion behaviors of unilamellar vesicles and hard PS spheres of similar size is possibly due to the flexibility of the lipid bilayer of the vesicles. The unilamellar vesicles are capable of changing their shape to move through the entangled polymer solution so that the hindrance to their diffusion due to the presence of the polymer chains is reduced, while the rigid PS spheres have little flexibility and they encounter greater resistance. The multilamellar vesicles are less flexible, thus their diffusion is similar to the hard PS spheres of similar diameter. Images FIGURE 2 PMID:9336189

Anticancer drug delivery with transferrin targeted polymeric chitosan vesicles.

PubMed

Dufes, Christine; Muller, Jean-Marc; Couet, William; Olivier, Jean-Christophe; Uchegbu, Ijeoma F; Schätzlein, Andreas G

2004-01-01

The study reports the initial biological evaluation of targeted polymeric glycol chitosan vesicles as carrier systems for doxorubicin (Dox). Transferrin (Tf) was covalently bound to the Dox-loaded palmitoylated glycol chitosan (GCP) vesicles using dimethylsuberimidate (DMSI). For comparison, glucose targeted niosomes were prepared using N-palmitoyl glucosamine. Biological properties were studied using confocal microscopy, flow cytometry, and cytotoxicity assays as well as a mouse xenograft model. Tf vesicles were taken up rapidly with a plateau after 1-2 h and Dox reached the nucleus after 60-90 min. Uptake was not increased with the use of glucose ligands, but higher uptake and increased cytotoxicity were observed for Tf targeted as compared to GCP Dox alone. In the drug-resistant A2780AD cells and in A431 cells, the relative increase in activity was significantly higher for the Tf-GCP vesicles than would have been expected from the uptake studies. All vesicle formulations had a superior in vivo safety profile compared to the free drug. The in vitro advantage of targeted Tf vesicles did not translate into a therapeutic advantage in vivo. All vesicles reduced tumor size on day 2 but were overall less active than the free drug.

Fusion Imaging for Procedural Guidance.

PubMed

Wiley, Brandon M; Eleid, Mackram F; Thaden, Jeremy J

2018-05-01

The field of percutaneous structural heart interventions has grown tremendously in recent years. This growth has fueled the development of new imaging protocols and technologies in parallel to help facilitate these minimally-invasive procedures. Fusion imaging is an exciting new technology that combines the strength of 2 imaging modalities and has the potential to improve procedural planning and the safety of many commonly performed transcatheter procedures. In this review we discuss the basic concepts of fusion imaging along with the relative strengths and weaknesses of static vs dynamic fusion imaging modalities. This review will focus primarily on echocardiographic-fluoroscopic fusion imaging and its application in commonly performed transcatheter structural heart procedures. Copyright © 2017 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.

Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles.

PubMed

Lötvall, Jan; Hill, Andrew F; Hochberg, Fred; Buzás, Edit I; Di Vizio, Dolores; Gardiner, Christopher; Gho, Yong Song; Kurochkin, Igor V; Mathivanan, Suresh; Quesenberry, Peter; Sahoo, Susmita; Tahara, Hidetoshi; Wauben, Marca H; Witwer, Kenneth W; Théry, Clotilde

2014-01-01

Secreted membrane-enclosed vesicles, collectively called extracellular vesicles (EVs), which include exosomes, ectosomes, microvesicles, microparticles, apoptotic bodies and other EV subsets, encompass a very rapidly growing scientific field in biology and medicine. Importantly, it is currently technically challenging to obtain a totally pure EV fraction free from non-vesicular components for functional studies, and therefore there is a need to establish guidelines for analyses of these vesicles and reporting of scientific studies on EV biology. Here, the International Society for Extracellular Vesicles (ISEV) provides researchers with a minimal set of biochemical, biophysical and functional standards that should be used to attribute any specific biological cargo or functions to EVs.

Phase behavior, rheological property, and transmutation of vesicles in fluorocarbon and hydrocarbon surfactant mixtures.

PubMed

Yuan, Zaiwu; Qin, Menghua; Chen, Xiushan; Liu, Changcheng; Li, Hongguang; Hao, Jingcheng

2012-06-26

We present a detailed study of a salt-free cationic/anionic (catanionic) surfactant system where a strongly alkaline cationic surfactant (tetradecyltrimethylammonium hydroxide, TTAOH) was mixed with a single-chain fluorocarbon acid (nonadecafluorodecanoic acid, NFDA) and a hyperbranched hydrocarbon acid [di-(2-ethylhexyl)phosphoric acid, DEHPA] in water. Typically the concentration of TTAOH is fixed while the total concentration and mixing molar ratio of NFDA and DEHPA is varied. In the absence of DEHPA and at a TTAOH concentration of 80 mmol·L(-1), an isotropic L(1) phase, an L(1)/L(α) two-phase region, and a single L(α) phase were observed successively with increasing mixing molar ratio of NFDA to TTAOH (n(NFDA)/n(TTAOH)). In the NFDA-rich region (n(NFDA)/n(TTAOH) > 1), a small amount of excess NFDA can be solubilized into the L(α) phase while a large excess of NFDA eventually leads to phase separation. When NFDA is replaced gradually by DEHPA, the mixed system of TTAOH/NFDA/DEHPA/H(2)O follows the same phase sequence as that of the TTAOH/NFDA/H(2)O system and the phase boundaries remain almost unchanged. However, the viscoelasticity of the samples in the single L(α) phase region becomes higher at the same total surfactant concentration as characterized by rheological measurements. Cryo-transmission electron microscopic (cryo-TEM) observations revealed a microstructural evolution from unilamellar vesicles to multilamellar ones and finally to gaint onions. The size of the vesicle and number of lamella can be controlled by adjusting the molar ratio of NFDA to DEHPA. The dynamic properties of the vesicular solutions have also been investigated. It is found that the yield stress and the storage modulus are time-dependent after a static mixing process between the two different types of vesicle solutions, indicating the occurrence of a dynamic fusion between the two types of vesicles. The microenvironmental changes induced by aggregate transitions were probed by

Hendra virus fusion protein transmembrane domain contributes to pre-fusion protein stability.

PubMed

Webb, Stacy; Nagy, Tamas; Moseley, Hunter; Fried, Michael; Dutch, Rebecca

2017-04-07

Enveloped viruses utilize fusion (F) proteins studding the surface of the virus to facilitate membrane fusion with a target cell membrane. Fusion of the viral envelope with a cellular membrane is required for release of viral genomic material, so the virus can ultimately reproduce and spread. To drive fusion, the F protein undergoes an irreversible conformational change, transitioning from a metastable pre-fusion conformation to a more thermodynamically stable post-fusion structure. Understanding the elements that control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Mutations in F protein transmembrane (TM) domains implicated the TM domain in the fusion process, but the structural and molecular details in fusion remain unclear. Previously, analytical ultracentrifugation was utilized to demonstrate that isolated TM domains of Hendra virus F protein associate in a monomer-trimer equilibrium (Smith, E. C., Smith, S. E., Carter, J. R., Webb, S. R., Gibson, K. M., Hellman, L. M., Fried, M. G., and Dutch, R. E. (2013) J. Biol. Chem. 288, 35726-35735). To determine factors driving this association, 140 paramyxovirus F protein TM domain sequences were analyzed. A heptad repeat of β-branched residues was found, and analysis of the Hendra virus F TM domain revealed a heptad repeat leucine-isoleucine zipper motif (LIZ). Replacement of the LIZ with alanine resulted in dramatically reduced TM-TM association. Mutation of the LIZ in the whole protein resulted in decreased protein stability, including pre-fusion conformation stability. Together, our data suggest that the heptad repeat LIZ contributed to TM-TM association and is important for F protein function and pre-fusion stability. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Arrest of trans-SNARE zippering uncovers loosely and tightly docked intermediates in membrane fusion.

PubMed

Yavuz, Halenur; Kattan, Iman; Hernandez, Javier Matias; Hofnagel, Oliver; Witkowska, Agata; Raunser, Stefan; Walla, Peter Jomo; Jahn, Reinhard

2018-04-17

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion in the secretory pathway. They contain conserved regions, termed SNARE motifs, that assemble between opposing membranes directionally from their N-termini to their membrane-proximal C-termini in a highly exergonic reaction. However, how this energy is utilized to overcome the energy barriers along the fusion pathway is still under debate. Here we have used mutants of the SNARE synaptobrevin to arrest trans-SNARE zippering at defined stages. We have uncovered two distinct vesicle docking intermediates, where the membranes are loosely and tightly connected, respectively. The tightly connected state is irreversible and independent of maintaining assembled SNARE complexes. Together, our results shed new light on the intermediate stages along the pathway of membrane fusion. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Associative polymers bridging between layers of multilamellar vesicles.

NASA Astrophysics Data System (ADS)

Choi, Seo; Bhatia, Surita

2006-03-01

Multilamellar vesicles can be found in a variety of pharmaceutical formulations, personal care products, and home care products. Hydrophobically modified associative polymers are often used to stabilize the vesicles or to control the rheological properties of these formulations. The hydrophobic groups are expected to insert themselves into the vesicle bilayers. Recent experimental work shows that hydrophobically modified polymers may from bridges between vesicles or may bridge between layers of a single vesicle. The latter configuration forces an interlayer spacing roughly equal to the radius of gyration of the backbone between associative groups. We have performed simple mean-field calculations on ideal telechelic associative polymers between concentric spherical surfaces. We find that the free energy per chain has an attractive minimum when the layer spacing is approximately N^1/2l, which is consistent with experimental results. The depth of the minimum depends on both chain length and curvature, and as expected when the curvature becomes small, the result for telechelic chains between flat surfaces is recovered.

Vesicle Pool Size at the Salamander Cone Ribbon Synapse

PubMed Central

Bartoletti, Theodore M.; Babai, Norbert

2010-01-01

Cone light responses are transmitted to postsynaptic neurons by changes in the rate of synaptic vesicle release. Vesicle pool size at the cone synapse constrains the amount of release and can thus shape contrast detection. We measured the number of vesicles in the rapidly releasable and reserve pools at cone ribbon synapses by performing simultaneous whole cell recording from cones and horizontal or off bipolar cells in the salamander retinal slice preparation. We found that properties of spontaneously occurring miniature excitatory postsynaptic currents (mEPSCs) are representative of mEPSCs evoked by depolarizing presynaptic stimulation. Strong, brief depolarization of the cone stimulated release of the entire rapidly releasable pool (RRP) of vesicles. Comparing charge transfer of the EPSC with mEPSC charge transfer, we determined that the fast component of the EPSC reflects release of ∼40 vesicles. Comparing EPSCs with simultaneous presynaptic capacitance measurements, we found that horizontal cell EPSCs constitute 14% of the total number of vesicles released from a cone terminal. Using a fluorescent ribeye-binding peptide, we counted ∼13 ribbons per cone. Together, these results suggest each cone contacts a single horizontal cell at ∼2 ribbons. The size of discrete components in the EPSC amplitude histogram also suggested ∼2 ribbon contacts per cell pair. We therefore conclude there are ∼20 vesicles per ribbon in the RRP, similar to the number of vesicles contacting the plasma membrane at the ribbon base. EPSCs evoked by lengthy depolarization suggest a reserve pool of ∼90 vesicles per ribbon, similar to the number of additional docking sites further up the ribbon. PMID:19923246

Release of outer membrane vesicles from Bordetella pertussis.

PubMed

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

1999-05-01

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

Plasma membrane aquaporins mediates vesicle stability in broccoli

PubMed Central

Martínez-Ballesta, Maria del Carmen; García-Gomez, Pablo; Yepes-Molina, Lucía; Guarnizo, Angel L.; Teruel, José A.

2018-01-01

The use of in vitro membrane vesicles is attractive because of possible applications in therapies. Here we aimed to compare the stability and functionality of plasma membrane vesicles extracted from control and salt-treated broccoli. The impact of the amount of aquaporins was related to plasma membrane osmotic water permeability and the stability of protein secondary structure. Here, we describe for first time an increase in plant aquaporins acetylation under high salinity. Higher osmotic water permeability in NaCl vesicles has been related to higher acetylation, upregulation of aquaporins, and a more stable environment to thermal denaturation. Based on our findings, we propose that aquaporins play an important role in vesicle stability. PMID:29420651

Yeast Membrane Vesicles: Isolation and General Characteristics1

PubMed Central

Christensen, Michael S.; Cirillo, Vincent P.

1972-01-01

Yeast membrane vesicles are formed when packed yeast are ground manually in a porcelain mortar and pestle with glass beads (0.2 mm diameter). These vesicles can be separated from the other components of the grinding mixture by a combination of centrifugation steps and elution from a column of the same glass beads (0.2 mm diameter). Isolated vesicles are osmotically sensitive, contain cytoplasmic components, and have energy-independent transport function. They are unable to metabolize glucose, but have respiratory function which is thought to be associated with intravesicular mitochondria. Invertase and oligomycin-insensitive adenosine triphosphatase are present in lysed vesicle preparations, and the appropriateness of these enzyme activities as membrane markers is discussed. Images PMID:4337848

Ultrastructural and functional fate of recycled vesicles in hippocampal synapses

PubMed Central

Rey, Stephanie A.; Smith, Catherine A.; Fowler, Milena W.; Crawford, Freya; Burden, Jemima J.; Staras, Kevin

2015-01-01

Efficient recycling of synaptic vesicles is thought to be critical for sustained information transfer at central terminals. However, the specific contribution that retrieved vesicles make to future transmission events remains unclear. Here we exploit fluorescence and time-stamped electron microscopy to track the functional and positional fate of vesicles endocytosed after readily releasable pool (RRP) stimulation in rat hippocampal synapses. We show that most vesicles are recovered near the active zone but subsequently take up random positions in the cluster, without preferential bias for future use. These vesicles non-selectively queue, advancing towards the release site with further stimulation in an actin-dependent manner. Nonetheless, the small subset of vesicles retrieved recently in the stimulus train persist nearer the active zone and exhibit more privileged use in the next RRP. Our findings reveal heterogeneity in vesicle fate based on nanoscale position and timing rules, providing new insights into the origins of future pool constitution. PMID:26292808

Magnetic vesicles as MRI-trackable biogenic nanovectors

NASA Astrophysics Data System (ADS)

Andriola Silva, Amanda K.; Luciani, Nathalie; Gazeau, Florence; Wilhelm, Claire

2012-03-01

Magnetic labeling renders cells MRI-detectable which provides attractive solutions for tracking the fate of a transplanted cell population. Understanding the interplay of magnetic nanoparticles and cells is then an important point that should not be neglected. Here we show that in the condition of food starvation, macrophage cells emit vesicles containing nanoparticles. First, we inferred the intracellular iron oxide load from the magnetophoretic velocity of cells at a calibrated magnetic field gradient. After magnetic labeling and culture in stress conditions, the intracellular iron oxide load was once more determined and a detectable difference was observed before and after stress. Moreover, we identified in the stress conditioned medium membrane vesicle structures carrying magnetic particles. Besides pointing out the role of cell-derived vesicles in the sequestration of the intracellular magnetic label, experiments also demonstrated that vesicles were able to chaperone the magnetic cargo into naïve cells.

CO2-filled vesicles in mid-ocean basalt

USGS Publications Warehouse

Moore, J.G.; Batchelder, J.N.; Cunningham, C.G.

1977-01-01

Volatile-filled vesicles are present in minor amounts in all samples of mid-ocean basalt yet collected (and presumably erupted) down to depths of 4.8 km. When such vesicles are pierced in liquid under standard conditions, the volume expansion of the gas is 0.2 ?? 0.05 times the eruption pressure in bars or 20 ?? 5 times the eruption depth in km. Such expansion could be used as a measure of eruption depth. A variety of techniques: (1) vacuum crushing and gas chromatographic, freezing separation, and mass spectrographic analyses; (2) measurements of phase changes on a freezing microscope stage; (3) microscopic chemical and solubility observations; and (4) volume change measurements, all indicate that CO2 comprises more than 95% by volume of the vesicle gas in several submarine basalt samples from the Atlantic and Pacific. The CO2 held in vesicles is present in quantities about equal to or greater than that presumed to be dissolved in the glass (melt) and amounts to 400-900 ppm of the rock. The rigid temperature of the glass is 800-1000??C and increases for shallower samples. A sulfur gas was originally present in subordinate amounts in the vesicles, but has largely reacted with iron in the vesicle walls to produce sulfide spherules. ?? 1977.

Studies of vesicle distribution patterns in Hawaiian lavas

NASA Technical Reports Server (NTRS)

Walker, George P. L.

1987-01-01

Basaltic lava flows are generally vesicular, and the broader facts relating to vesicle distribution have long been established; few studies have yet been made with a view to determining how and when vesicles form in the cooling history of the lava, explaining vesicle shape and size distribution, and gaining enough understanding to employ vesicles as a geological tool. Various avenues of approach exist by which one may seek to gain a better understanding of these ubiquitous structures and make a start towards developing a general theory, and three such avenues have recently been explored. One avenue involves the study of pipe vesicles; these are a well known feature of lava flows and are narrow pipes which occur near the base of many pahoehoe flow units. Another avenue of approach is that presented by the distinctive spongy pahoehoe facies of lava that is common in distal locations on Hawaiian volcanoes. A third avenue of approach is that of the study of gas blisters in lava. Gas blisters are voids, which can be as much as tens of meters wide, where the lava split along a vesicle-rich layer and the roof up-arched by gas pressure. These three avenues are briefly discussed.

Active elastohydrodynamics of vesicles in narrow blind constrictions

NASA Astrophysics Data System (ADS)

Fai, T. G.; Kusters, R.; Harting, J.; Rycroft, C. H.; Mahadevan, L.

2017-11-01

Fluid-resistance limited transport of vesicles through narrow constrictions is a recurring theme in many biological and engineering applications. Inspired by the motor-driven movement of soft membrane-bound vesicles into closed neuronal dendritic spines, here we study this problem using a combination of passive three-dimensional simulations and a simplified semianalytical theory for the active transport of vesicles forced through constrictions by molecular motors. We show that the motion of these objects is characterized by two dimensionless quantities related to the geometry and to the strength of forcing relative to the vesicle elasticity. We use numerical simulations to characterize the transit time for a vesicle forced by fluid pressure through a constriction in a channel and find that relative to an open channel, transport into a blind end leads to the formation of a smaller forward-flowing lubrication layer that strongly impedes motion. When the fluid pressure forcing is complemented by forces due to molecular motors that are responsible for vesicle trafficking into dendritic spines, we find that the competition between motor forcing and fluid drag results in multistable dynamics reminiscent of the real system. Our study highlights the role of nonlocal hydrodynamic effects in determining the kinetics of vesicular transport in constricted geometries.

CTP:phosphocholine cytidylyltransferase binds anionic phospholipid vesicles in a cross-bridging mode.

PubMed

Taneva, Svetla G; Patty, Philipus J; Frisken, Barbara J; Cornell, Rosemary B

2005-07-05

CTP:phosphocholine cytidylyltransferase (CCT) catalyzes the rate-limiting step in phosphatidylcholine (PC) synthesis, and its activity is regulated by reversible association with membranes, mediated by an amphipathic helical domain M. Here we describe a new feature of the CCTalpha isoform, vesicle tethering. We show, using dynamic light scattering and transmission electron microscopy, that dimers of CCTalpha can cross-bridge separate vesicles to promote vesicle aggregation. The vesicles contained either class I activators (anionic phospholipids) or the less potent class II activators, which favor nonlamellar phase formation. CCT increased the apparent hydrodynamic radius and polydispersity of anionic phospholipid vesicles even at low CCT concentrations corresponding to only one or two dimers per vesicle. Electron micrographs of negatively stained phosphatidylglycerol (PG) vesicles confirmed CCT-mediated vesicle aggregation. CCT conjugated to colloidal gold accumulated on the vesicle surfaces and in areas of vesicle-vesicle contact. PG vesicle aggregation required both the membrane-binding domain and the intact CCT dimer, suggesting binding of CCT to apposed membranes via the two M domains situated on opposite sides of the dimerization domain. In contrast to the effects on anionic phospholipid vesicles, CCT did not induce aggregation of PC vesicles containing the class II lipids, oleic acid, diacylglycerol, or phosphatidylethanolamine. The different behavior of the two lipid classes reflected differences in measured binding affinity, with only strongly binding phospholipid vesicles being susceptible to CCT-induced aggregation. Our findings suggest a new model for CCTalpha domain organization and membrane interaction, and a potential involvement of the enzyme in cellular events that implicate close apposition of membranes.

Physical determinants of vesicle mobility and supply at a central synapse

PubMed Central

Rothman, Jason Seth; Kocsis, Laszlo; Herzog, Etienne; Nusser, Zoltan; Silver, Robin Angus

2016-01-01

Encoding continuous sensory variables requires sustained synaptic signalling. At several sensory synapses, rapid vesicle supply is achieved via highly mobile vesicles and specialized ribbon structures, but how this is achieved at central synapses without ribbons is unclear. Here we examine vesicle mobility at excitatory cerebellar mossy fibre synapses which sustain transmission over a broad frequency bandwidth. Fluorescent recovery after photobleaching in slices from VGLUT1Venus knock-in mice reveal 75% of VGLUT1-containing vesicles have a high mobility, comparable to that at ribbon synapses. Experimentally constrained models establish hydrodynamic interactions and vesicle collisions are major determinants of vesicle mobility in crowded presynaptic terminals. Moreover, models incorporating 3D reconstructions of vesicle clouds near active zones (AZs) predict the measured releasable pool size and replenishment rate from the reserve pool. They also show that while vesicle reloading at AZs is not diffusion-limited at the onset of release, diffusion limits vesicle reloading during sustained high-frequency signalling. DOI: http://dx.doi.org/10.7554/eLife.15133.001 PMID:27542193

Cancerous epithelial cell lines shed extracellular vesicles with a bimodal size distribution that is sensitive to glutamine inhibition

NASA Astrophysics Data System (ADS)

Santana, Steven Michael; Antonyak, Marc A.; Cerione, Richard A.; Kirby, Brian J.

2014-12-01

Extracellular shed vesicles (ESVs) facilitate a unique mode of cell-cell communication wherein vesicle uptake can induce a change in the recipient cell's state. Despite the intensity of ESV research, currently reported data represent the bulk characterization of concentrated vesicle samples with little attention paid to heterogeneity. ESV populations likely represent diversity in mechanisms of formation, cargo and size. To better understand ESV subpopulations and the signaling cascades implicated in their formation, we characterize ESV size distributions to identify subpopulations in normal and cancerous epithelial cells. We have discovered that cancer cells exhibit bimodal ESV distributions, one small-diameter and another large-diameter population, suggesting that two mechanisms may govern ESV formation, an exosome population and a cancer-specific microvesicle population. Altered glutamine metabolism in cancer is thought to fuel cancer growth but may also support metastatic niche formation through microvesicle production. We describe the role of a glutaminase inhibitor, compound 968, in ESV production. We have discovered that inhibiting glutamine metabolism significantly impairs large-diameter microvesicle production in cancer cells.

Real-time sensor validation and fusion for distributed autonomous sensors

NASA Astrophysics Data System (ADS)

Yuan, Xiaojing; Li, Xiangshang; Buckles, Bill P.

2004-04-01

Multi-sensor data fusion has found widespread applications in industrial and research sectors. The purpose of real time multi-sensor data fusion is to dynamically estimate an improved system model from a set of different data sources, i.e., sensors. This paper presented a systematic and unified real time sensor validation and fusion framework (RTSVFF) based on distributed autonomous sensors. The RTSVFF is an open architecture which consists of four layers - the transaction layer, the process fusion layer, the control layer, and the planning layer. This paradigm facilitates distribution of intelligence to the sensor level and sharing of information among sensors, controllers, and other devices in the system. The openness of the architecture also provides a platform to test different sensor validation and fusion algorithms and thus facilitates the selection of near optimal algorithms for specific sensor fusion application. In the version of the model presented in this paper, confidence weighted averaging is employed to address the dynamic system state issue noted above. The state is computed using an adaptive estimator and dynamic validation curve for numeric data fusion and a robust diagnostic map for decision level qualitative fusion. The framework is then applied to automatic monitoring of a gas-turbine engine, including a performance comparison of the proposed real-time sensor fusion algorithms and a traditional numerical weighted average.

Controlling Two-dimensional Tethered Vesicle Motion Using an Electric Field

PubMed Central

Yoshina-Ishii, Chiaki; Boxer, Steven G.

2008-01-01

We recently introduced methods to tether phospholipid vesicles or proteoliposomes onto a fluid supported lipid bilayer using DNA hybridization. These intact tethered vesicles diffuse in two dimensions parallel to the supporting membrane surface. In this paper, we report the dynamic response of individual tethered vesicles to an electric field applied parallel to the bilayer surface. Vesicles respond to the field by moving in the direction of electro-osmotic flow, and this can be used to reversibly concentrate tethered vesicles against a barrier. By adding increasing amounts of negatively charged phosphatidylserine to the supporting bilayer to increase electro-osmosis, the electrophoretic mobility of the tethered vesicles can be increased. The electro-osmotic contribution can be modeled well by a sphere connected to a cylindrical anchor in a viscous membrane with charged head groups. The electrophoretic force on the negatively charged tethered vesicles opposes the electro-osmotic force. By increasing the amount of negative charge on the tethered vesicle, drift in the direction of electro-osmotic flow can be slowed; at high negative charge on the tethered vesicle, motion can be forced in the direction of electrophoresis. The balance between these forces can be visualized on a patterned supporting bilayer containing negatively charged lipids which themselves reorganize in an externally applied electric field to create a gradient of charge within a corralled region. The charge gradient at the surface creates a gradient of electro-osmotic flow, and vesicles carrying similar amounts of negative charge can be focused to a region perpendicular to the applied field where electrophoresis is balanced by electro-osmosis, away from the corral boundary. Electric fields are effective tools to direct tethered vesicles, concentrate them and to measure the tethered vesicle’s electrostatic properties. PMID:16489833

α-SNAP interferes with the zippering of the SNARE protein membrane fusion machinery.

PubMed

Park, Yongsoo; Vennekate, Wensi; Yavuz, Halenur; Preobraschenski, Julia; Hernandez, Javier M; Riedel, Dietmar; Walla, Peter Jomo; Jahn, Reinhard

2014-06-06

Neuronal exocytosis is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Before fusion, SNARE proteins form complexes bridging the membrane followed by assembly toward the C-terminal membrane anchors, thus initiating membrane fusion. After fusion, the SNARE complex is disassembled by the AAA-ATPase N-ethylmaleimide-sensitive factor that requires the cofactor α-SNAP to first bind to the assembled SNARE complex. Using chromaffin granules and liposomes we now show that α-SNAP on its own interferes with the zippering of membrane-anchored SNARE complexes midway through the zippering reaction, arresting SNAREs in a partially assembled trans-complex and preventing fusion. Intriguingly, the interference does not result in an inhibitory effect on synaptic vesicles, suggesting that membrane properties also influence the final outcome of α-SNAP interference with SNARE zippering. We suggest that binding of α-SNAP to the SNARE complex affects the ability of the SNARE complex to harness energy or transmit force to the membrane. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Feruloyl Dioleoyglycerol Antioxidant Capacity in Phospholipid Vesicles

USDA-ARS?s Scientific Manuscript database

Ferulic acid and its esters are known to be effective antioxidants. Feruloyl dioleoylglycerol was assessed for its ability to serve as an antioxidant in model membrane phospholipid vesicles. The molecule was incorporated into single-lamellar vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine at ...

Molecular genetic and physical analysis of gas vesicles in buoyant enterobacteria

PubMed Central

Tashiro, Yosuke; Monson, Rita E.; Ramsay, Joshua P.

2016-01-01

Summary Different modes of bacterial taxis play important roles in environmental adaptation, survival, colonization and dissemination of disease. One mode of taxis is flotation due to the production of gas vesicles. Gas vesicles are proteinaceous intracellular organelles, permeable only to gas, that enable flotation in aquatic niches. Gene clusters for gas vesicle biosynthesis are partially conserved in various archaea, cyanobacteria, and some proteobacteria, such as the enterobacterium, S erratia sp. ATCC 39006 (S39006). Here we present the first systematic analysis of the genes required to produce gas vesicles in S39006, identifying how this differs from the archaeon H alobacterium salinarum. We define 11 proteins essential for gas vesicle production. Mutation of gvpN or gvpV produced small bicone gas vesicles, suggesting that the cognate proteins are involved in the morphogenetic assembly pathway from bicones to mature cylindrical forms. Using volumetric compression, gas vesicles were shown to comprise 17% of S39006 cells, whereas in E scherichia coli heterologously expressing the gas vesicle cluster in a deregulated environment, gas vesicles can occupy around half of cellular volume. Gas vesicle production in S39006 and E . coli was exploited to calculate the instantaneous turgor pressure within cultured bacterial cells; the first time this has been performed in either strain. PMID:26743231

A modular platform for one-step assembly of multi-component membrane systems by fusion of charged proteoliposomes

NASA Astrophysics Data System (ADS)

Ishmukhametov, Robert R.; Russell, Aidan N.; Berry, Richard M.

2016-10-01

An important goal in synthetic biology is the assembly of biomimetic cell-like structures, which combine multiple biological components in synthetic lipid vesicles. A key limiting assembly step is the incorporation of membrane proteins into the lipid bilayer of the vesicles. Here we present a simple method for delivery of membrane proteins into a lipid bilayer within 5 min. Fusogenic proteoliposomes, containing charged lipids and membrane proteins, fuse with oppositely charged bilayers, with no requirement for detergent or fusion-promoting proteins, and deliver large, fragile membrane protein complexes into the target bilayers. We demonstrate the feasibility of our method by assembling a minimal electron transport chain capable of adenosine triphosphate (ATP) synthesis, combining Escherichia coli F1Fo ATP-synthase and the primary proton pump bo3-oxidase, into synthetic lipid vesicles with sizes ranging from 100 nm to ~10 μm. This provides a platform for the combination of multiple sets of membrane protein complexes into cell-like artificial structures.

The International Society for Extracellular Vesicles launches the first massive open online course on extracellular vesicles

PubMed Central

Lässer, Cecilia; Théry, Clotilde; Buzás, Edit I.; Mathivanan, Suresh; Zhao, Weian; Gho, Yong Song; Lötvall, Jan

2016-01-01

The International Society for Extracellular Vesicles (ISEV) has organised its first educational online course for students and beginners in the field of extracellular vesicles (EVs). This course, “Basics of Extracellular Vesicles,” uses recorded lectures from experts in the field and will be open for an unlimited number of participants. The course is divided into 5 modules and can be accessed at www.coursera.org/learn/extracellular-vesicles. The first module is an introduction to the field covering the nomenclature and history of EVs. Module 2 focuses on the biogenesis and uptake mechanisms of EVs, as well as their RNA, protein and lipid cargo. Module 3 covers the collection and processing of cell culture media and body fluids such as blood, breast milk, cerebrospinal fluid and urine prior to isolation of EVs. Modules 4 and 5 present different isolation methods and characterisation techniques utilised in the EV field. Here, differential ultracentrifugation, size-exclusion chromatography, density gradient centrifugation, kit-based precipitation, electron microscopy, cryo-electron microscopy, flow cytometry, atomic-force microscopy and nanoparticle-tracking analysis are covered. This first massive open online course (MOOC) on EVs was launched on 15 August 2016 at the platform “Coursera” and is free of charge. PMID:27989272

ABC triblock copolymer vesicles with mesh-like morphology.

PubMed

Zhao, Wei; Chen, Dian; Hu, Yunxia; Grason, Gregory M; Russell, Thomas P

2011-01-25

Polymer vesicles made from poly(isoprene-b-styrene-b-2-vinyl pyridine) (PI-b-PS-b-P2VP) triblock copolymer confined within the nanopores of an anodic aluminum oxide (AAO) membrane are studied. It was found that these vesicles have well-defined, nanoscopic size, and complex microphase-separated hydrophobic membranes, comprised of the PS and PI blocks, while the coronas are formed by the P2VP block. Vesicle formation was tracked using both transmission and scanning electron microscopy. A mesh-like morphology formed in the membrane at a well-defined composition of the three blocks that can be tuned by changing the copolymer composition. The nanoscale confinement, copolymer composition, and subtle molecular interactions contribute to the generation of these vesicles with such unusual morphologies.

Dynamic Properties of the Alkaline Vesicle Population at Hippocampal Synapses

PubMed Central

Röther, Mareike; Brauner, Jan M.; Ebert, Katrin; Welzel, Oliver; Jung, Jasmin; Bauereiss, Anna; Kornhuber, Johannes; Groemer, Teja W.

2014-01-01

In compensatory endocytosis, scission of vesicles from the plasma membrane to the cytoplasm is a prerequisite for intravesicular reacidification and accumulation of neurotransmitter molecules. Here, we provide time-resolved measurements of the dynamics of the alkaline vesicle population which appears upon endocytic retrieval. Using fast perfusion pH-cycling in live-cell microscopy, synapto-pHluorin expressing rat hippocampal neurons were electrically stimulated. We found that the relative size of the alkaline vesicle population depended significantly on the electrical stimulus size: With increasing number of action potentials the relative size of the alkaline vesicle population expanded. In contrast to that, increasing the stimulus frequency reduced the relative size of the population of alkaline vesicles. Measurement of the time constant for reacification and calculation of the time constant for endocytosis revealed that both time constants were variable with regard to the stimulus condition. Furthermore, we show that the dynamics of the alkaline vesicle population can be predicted by a simple mathematical model. In conclusion, here a novel methodical approach to analyze dynamic properties of alkaline vesicles is presented and validated as a convenient method for the detection of intracellular events. Using this method we show that the population of alkaline vesicles is highly dynamic and depends both on stimulus strength and frequency. Our results implicate that determination of the alkaline vesicle population size may provide new insights into the kinetics of endocytic retrieval. PMID:25079223

Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis

PubMed Central

Dragovic, Rebecca A.; Gardiner, Christopher; Brooks, Alexandra S.; Tannetta, Dionne S.; Ferguson, David J.P.; Hole, Patrick; Carr, Bob; Redman, Christopher W.G.; Harris, Adrian L.; Dobson, Peter J.; Harrison, Paul; Sargent, Ian L.

2011-01-01

Cellular microvesicles and nanovesicles (exosomes) are involved in many disease processes and have major potential as biomarkers. However, developments in this area are constrained by limitations in the technology available for their measurement. Here we report on the use of fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles. In this system vesicles are visualized by light scattering using a light microscope. A video is taken, and the NTA software tracks the brownian motion of individual vesicles and calculates their size and total concentration. Using human placental vesicles and plasma, we have demonstrated that NTA can measure cellular vesicles as small as ∼50 nm and is far more sensitive than conventional flow cytometry (lower limit ∼300 nm). By combining NTA with fluorescence measurement we have demonstrated that vesicles can be labeled with specific antibody-conjugated quantum dots, allowing their phenotype to be determined. From the Clinical Editor The authors of this study utilized fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles, demonstrating that NTA is far more sensitive than conventional flow cytometry. PMID:21601655

Complexin Mutants Reveal Partial Segregation between Recycling Pathways That Drive Evoked and Spontaneous Neurotransmission

PubMed Central

Sabeva, Nadezhda; Cho, Richard W.; Vasin, Alexander; Gonzalez, Agustin; Littleton, J. Troy

2017-01-01

Synaptic vesicles fuse at morphological specializations in the presynaptic terminal termed active zones (AZs). Vesicle fusion can occur spontaneously or in response to an action potential. Following fusion, vesicles are retrieved and recycled within nerve terminals. It is still unclear whether vesicles that fuse spontaneously or following evoked release share similar recycling mechanisms. Genetic deletion of the SNARE-binding protein complexin dramatically increases spontaneous fusion, with the protein serving as the synaptic vesicle fusion clamp at Drosophila synapses. We examined synaptic vesicle recycling pathways at complexin null neuromuscular junctions, where spontaneous release is dramatically enhanced. We combined loading of the lipophilic dye FM1–43 with photoconversion, electron microscopy, and electrophysiology to monitor evoked and spontaneous recycling vesicle pools. We found that the total number of recycling vesicles was equal to those retrieved through spontaneous and evoked pools, suggesting that retrieval following fusion is partially segregated for spontaneous and evoked release. In addition, the kinetics of FM1–43 destaining and synaptic depression measured in the presence of the vesicle-refilling blocker bafilomycin indicated that spontaneous and evoked recycling pools partially intermix during the release process. Finally, FM1–43 photoconversion combined with electron microscopy analysis indicated that spontaneous recycling preferentially involves synaptic vesicles in the vicinity of AZs, whereas vesicles recycled following evoked release involve a larger intraterminal pool. Together, these results suggest that spontaneous and evoked vesicles use separable recycling pathways and then partially intermix during subsequent rounds of fusion. SIGNIFICANCE STATEMENT Neurotransmitter release involves fusion of synaptic vesicles with the plasma membrane in response to an action potential, or spontaneously in the absence of stimulation. Upon

Membrane fusion between baculovirus budded virus-enveloped particles and giant liposomes generated using a droplet-transfer method for the incorporation of recombinant membrane proteins.

PubMed

Nishigami, Misako; Mori, Takaaki; Tomita, Masahiro; Takiguchi, Kingo; Tsumoto, Kanta

2017-07-01

Giant proteoliposomes are generally useful as artificial cell membranes in biochemical and biophysical studies, and various procedures for their preparation have been reported. We present here a novel preparation technique that involves the combination of i) cell-sized lipid vesicles (giant unilamellar vesicles, GUVs) that are generated using the droplet-transfer method, where lipid monolayer-coated water-in-oil microemulsion droplets interact with oil/water interfaces to form enclosed bilayer vesicles, and ii) budded viruses (BVs) of baculovirus (Autographa californica nucleopolyhedrovirus) that express recombinant transmembrane proteins on their envelopes. GP64, a fusogenic glycoprotein on viral envelopes, is activated by weak acids and is thought to cause membrane fusion with liposomes. Using confocal laser scanning microscopy (CLSM), we observed that the single giant liposomes fused with octadecyl rhodamine B chloride (R18)-labeled wild-type BV envelopes with moderate leakage of entrapped soluble compounds (calcein), and the fusion profile depended on the pH of the exterior solution: membrane fusion occurred at pH ∼4-5. We further demonstrated that recombinant transmembrane proteins, a red fluorescent protein (RFP)-tagged GPCR (corticotropin-releasing hormone receptor 1, CRHR1) and envelope protein GP64 could be partly incorporated into membranes of the individual giant liposomes with a reduction of the pH value, though there were also some immobile fluorescent spots observed on their circumferences. This combination may be useful for preparing giant proteoliposomes containing the desired membranes and inner phases. Copyright © 2017 Elsevier B.V. All rights reserved.

Phosphorylation of Synaptojanin Differentially Regulates Endocytosis of Functionally Distinct Synaptic Vesicle Pools

PubMed Central

Geng, Junhua; Wang, Liping; Lee, Joo Yeun; Chen, Chun-Kan

2016-01-01

The rapid replenishment of synaptic vesicles through endocytosis is crucial for sustaining synaptic transmission during intense neuronal activity. Synaptojanin (Synj), a phosphoinositide phosphatase, is known to play an important role in vesicle recycling by promoting the uncoating of clathrin following synaptic vesicle uptake. Synj has been shown to be a substrate of the minibrain (Mnb) kinase, a fly homolog of the dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A); however, the functional impacts of Synj phosphorylation by Mnb are not well understood. Here we identify that Mnb phosphorylates Synj at S1029 in Drosophila. We find that phosphorylation of Synj at S1029 enhances Synj phosphatase activity, alters interaction between Synj and endophilin, and promotes efficient endocytosis of the active cycling vesicle pool (also referred to as exo-endo cycling pool) at the expense of reserve pool vesicle endocytosis. Dephosphorylated Synj, on the other hand, is deficient in the endocytosis of the active recycling pool vesicles but maintains reserve pool vesicle endocytosis to restore total vesicle pool size and sustain synaptic transmission. Together, our findings reveal a novel role for Synj in modulating reserve pool vesicle endocytosis and further indicate that dynamic phosphorylation and dephosphorylation of Synj differentially maintain endocytosis of distinct functional synaptic vesicle pools. SIGNIFICANCE STATEMENT Synaptic vesicle endocytosis sustains communication between neurons during a wide range of neuronal activities by recycling used vesicle membrane and protein components. Here we identify that Synaptojanin, a protein with a known role in synaptic vesicle endocytosis, is phosphorylated at S1029 in vivo by the Minibrain kinase. We further demonstrate that the phosphorylation status of Synaptojanin at S1029 differentially regulates its participation in the recycling of distinct synaptic vesicle pools. Our results reveal a new role for

ER-associated SNAREs and Sey1p mediate nuclear fusion at two distinct steps during yeast mating

PubMed Central

Rogers, Jason V.; Arlow, Tim; Inkellis, Elizabeth R.; Koo, Timothy S.; Rose, Mark D.

2013-01-01

During yeast mating, two haploid nuclei fuse membranes to form a single diploid nucleus. However, the known proteins required for nuclear fusion are unlikely to function as direct fusogens (i.e., they are unlikely to directly catalyze lipid bilayer fusion) based on their predicted structure and localization. Therefore we screened known fusogens from vesicle trafficking (soluble N-ethylmaleimide–sensitive factor attachment protein receptors [SNAREs]) and homotypic endoplasmic reticulum (ER) fusion (Sey1p) for additional roles in nuclear fusion. Here we demonstrate that the ER-localized SNAREs Sec20p, Ufe1p, Use1p, and Bos1p are required for efficient nuclear fusion. In contrast, Sey1p is required indirectly for nuclear fusion; sey1Δ zygotes accumulate ER at the zone of cell fusion, causing a block in nuclear congression. However, double mutants of Sey1p and Sec20p, Ufe1p, or Use1p, but not Bos1p, display extreme ER morphology defects, worse than either single mutant, suggesting that retrograde SNAREs fuse ER in the absence of Sey1p. Together these data demonstrate that SNAREs mediate nuclear fusion, ER fusion after cell fusion is necessary to complete nuclear congression, and there exists a SNARE-mediated, Sey1p-independent ER fusion pathway. PMID:24152736

Cytosol-dependent membrane fusion in ER, nuclear envelope and nuclear pore assembly: biological implications.

PubMed

Rafikova, Elvira R; Melikov, Kamran; Chernomordik, Leonid V

2010-01-01

Endoplasmic reticulum and nuclear envelope rearrangements after mitosis are often studied in the reconstitution system based on Xenopus egg extract. In our recent work we partially replaced the membrane vesicles in the reconstitution mix with protein-free liposomes to explore the relative contributions of cytosolic and transmembrane proteins. Here we discuss our finding that cytosolic proteins mediate fusion between membranes lacking functional transmembrane proteins and the role of membrane fusion in endoplasmic reticulum and nuclear envelope reorganization. Cytosol-dependent liposome fusion has allowed us to restore, without adding transmembrane nucleoporins, functionality of nuclear pores, their spatial distribution and chromatin decondensation in nuclei formed at insufficient amounts of membrane material and characterized by only partial decondensation of chromatin and lack of nuclear transport. Both the mechanisms and the biological implications of the discovered coupling between spatial distribution of nuclear pores, chromatin decondensation and nuclear transport are discussed.

[Possibilities of sonographic image fusion: Current developments].

PubMed

Jung, E M; Clevert, D-A

2015-11-01

For diagnostic and interventional procedures ultrasound (US) image fusion can be used as a complementary imaging technique. Image fusion has the advantage of real time imaging and can be combined with other cross-sectional imaging techniques. With the introduction of US contrast agents sonography and image fusion have gained more importance in the detection and characterization of liver lesions. Fusion of US images with computed tomography (CT) or magnetic resonance imaging (MRI) facilitates the diagnostics and postinterventional therapy control. In addition to the primary application of image fusion in the diagnosis and treatment of liver lesions, there are more useful indications for contrast-enhanced US (CEUS) in routine clinical diagnostic procedures, such as intraoperative US (IOUS), vascular imaging and diagnostics of other organs, such as the kidneys and prostate gland.

Wheat TaNPSN SNARE homologues are involved in vesicle-mediated resistance to stripe rust (Puccinia striiformis f. sp. tritici)

PubMed Central

Wang, Xiaodong; Wang, Xiaojie; Deng, Lin; Chang, Haitao; Dubcovsky, Jorge; Feng, Hao; Han, Qingmei; Huang, Lili; Kang, Zhensheng

2014-01-01

Subcellular localisation of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and their ability to form SNARE complexes are critical for determining the specificity of vesicle fusion. NPSN11, a Novel Plant SNARE (NPSN) gene, has been reported to be involved in the delivery of cell wall precursors to the newly formed cell plate during cytokinesis. However, functions of NPSN genes in plant–pathogen interactions are largely unknown. In this study, we cloned and characterized three NPSN genes (TaNPSN11, TaNPSN12, and TaNPSN13) and three plant defence-related SNARE homologues (TaSYP132, TaSNAP34, and TaMEMB12). TaSYP132 showed a highly specific interaction with TaNPSN11 in both yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. We hypothesize that this interaction may indicate a partnership in vesicle trafficking. Expressions of the three TaNPSNs and TaSYP132 were differentially induced in wheat leaves when challenged by Puccinia striiformis f. sp. tritici (Pst). In virus-induced gene silencing (VIGS) assays, resistance of wheat (Triticum aestivum) cultivar Xingzi9104 to the Pst avirulent race CYR23 was reduced by knocking down TaNPSN11, TaNPSN13 and TaSYP132, but not TaNPSN12, implying diversified functions of these wheat SNARE homologues in prevention of Pst infection and hyphal elongation. Immuno-localization results showed that TaNPSN11 or its structural homologues were mainly distributed in vesicle structures near cell membrane toward Pst hypha. Taken together, our data suggests a role of TaNPSN11 in vesicle-mediated resistance to stripe rust. PMID:24963004

Frequency-dependent electrodeformation of giant phospholipid vesicles in AC electric field

PubMed Central

2010-01-01

A model of vesicle electrodeformation is described which obtains a parametrized vesicle shape by minimizing the sum of the membrane bending energy and the energy due to the electric field. Both the vesicle membrane and the aqueous media inside and outside the vesicle are treated as leaky dielectrics, and the vesicle itself is modeled as a nearly spherical shape enclosed within a thin membrane. It is demonstrated (a) that the model achieves a good quantitative agreement with the experimentally determined prolate-to-oblate transition frequencies in the kilohertz range and (b) that the model can explain a phase diagram of shapes of giant phospholipid vesicles with respect to two parameters: the frequency of the applied alternating current electric field and the ratio of the electrical conductivities of the aqueous media inside and outside the vesicle, explored in a recent paper (S. Aranda et al., Biophys J 95:L19–L21, 2008). A possible use of the frequency-dependent shape transitions of phospholipid vesicles in conductometry of microliter samples is discussed. PMID:21886342

Neuronal Depolarization Drives Increased Dopamine Synaptic Vesicle Loading via VGLUT

PubMed Central

Aguilar, Jenny I.; Dunn, Matthew; Mingote, Susana; Karam, Caline S.; Farino, Zachary J.; Sonders, Mark S.; Choi, Se Joon; Grygoruk, Anna; Zhang, Yuchao; Cela, Carolina; Choi, Ben Jiwon; Flores, Jorge; Freyberg, Robin J.; McCabe, Brian D.; Mosharov, Eugene V.; Krantz, David E.; Javitch, Jonathan A.; Sulzer, David; Sames, Dalibor; Rayport, Stephen; Freyberg, Zachary

2017-01-01

SUMMARY The ability of presynaptic dopamine terminals to tune neurotransmitter release to meet the demands of neuronal activity is critical to neurotransmission. Although vesicle content has been assumed to be static, in vitro data increasingly suggest that cell activity modulates vesicle content. Here, we use a coordinated genetic, pharmacological, and imaging approach in Drosophila to study the presynaptic machinery responsible for these vesicular processes in vivo. We show that cell depolarization increases synaptic vesicle dopamine content prior to release via vesicular hyperacidification. This depolarization-induced hyperacidification is mediated by the vesicular glutamate transporter (VGLUT). Remarkably, both depolarization-induced dopamine vesicle hyperacidification and its dependence on VGLUT2 are seen in ventral midbrain dopamine neurons in the mouse. Together, these data suggest that in response to depolarization, dopamine vesicles utilize a cascade of vesicular transporters to dynamically increase the vesicular pH gradient, thereby increasing dopamine vesicle content. PMID:28823729

Activation of calcineurin by phosphotidylserine containing vesicles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Politino, M.; King, M.M.

1986-05-01

Calcineurin (CaN) is a Ca/sup 2 +/- and calmodulin-regulated phosphatase. Recent findings suggested an association of CaN with biological membranes and prompted the present investigation into the interactions of the phosphatase with phospholipids in vitro. In the absence of calmodulin, sonicated preparations of phosphatidylserine (PS) provided a five-fold activation of the Ni- and Mn-supported activities of CaN towards (/sup 32/P) histone Hl; activation in the presence of calmodulin was much less pronounced. Half-maximal activation in the absence of calmodulin required approximately 0.1 mg/ml of PS. Activation of CaN was also observed with mixed vesicles of phosphatidylcholine (PC) containing 20% PSmore » but not with PC alone, or with phosphatidylethanolamine (PE). Molecular sieve chromatography on Ultrogel AcA 34 provided further evidence that CaN associates with phospholipid vesicles composed of PS, or PC containing 20% PS, but not with vesicles of PC or PE. Complete association with medium sized vesicles of PS and PC/PS required Ca/sup 2 +/ ions; in the absence of the metal ion at least 60% of the enzyme failed to interact with the lipids while the remainder preferentially migrated with larger vesicles. These results suggest a role for Ca/sup 2 +/ in regulating CaN's interaction with phospholipids.« less

Experimental study of nuclear fusion reactions in muonic molecular systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bogdanova, L. N., E-mail: ludmila@itep.ru

2013-03-15

Since the pioneering discovery of the muon catalysis by Alvarez [L. W. Alvarez, K. Brander, F. S. Crawford, et al., Phys. Rev. 105, 1127 (1957)], considerable efforts were aimed at observation of various fusion processes. Results of these studies facilitated understanding the properties of lightest nuclei and dynamics of low-energy fusion reactions. There still remain unsolved theoretical and experimental problems, especially in case of pt fusion.

Prominin-1-containing membrane vesicles: origins, formation, and utility.

PubMed

Marzesco, Anne-Marie

2013-01-01

The stem cell antigen prominin-1 (CD133) is associated with two major types (small and large) of extracellular membrane vesicles in addition to its selective concentration in various kinds of plasma membrane protrusion. During development of the mammalian central nervous system, differentiating neuroepithelial stem cells release these vesicles into the embryonic cerebrospinal fluid. In glioblastoma patients, an increase of such vesicles, particularly the smaller ones, have been also observed in cerebrospinal fluid. Similarly, hematopoietic stem and progenitor cells release small ones concomitantly with their differentiation. Although the functional significance of these prominin-1-containing membrane vesicles is poorly understood, a link between differentiation of stem (and cancer stem) cells and their release is emerging. In this chapter, I will summarize our knowledge about prominin-1-containing membrane vesicles including a potential role in cell-cell communication and highlight their prospective value as a new biomarker for tumorigenesis diagnostics.

Compartmentalization and Transport in Synthetic Vesicles

PubMed Central

Schmitt, Christine; Lippert, Anna H.; Bonakdar, Navid; Sandoghdar, Vahid; Voll, Lars M.

2016-01-01

Nanoscale vesicles have become a popular tool in life sciences. Besides liposomes that are generated from phospholipids of natural origin, polymersomes fabricated of synthetic block copolymers enjoy increasing popularity, as they represent more versatile membrane building blocks that can be selected based on their specific physicochemical properties, such as permeability, stability, or chemical reactivity. In this review, we focus on the application of simple and nested artificial vesicles in synthetic biology. First, we provide an introduction into the utilization of multicompartmented vesosomes as compartmentalized nanoscale bioreactors. In the bottom-up development of protocells from vesicular nanoreactors, the specific exchange of pathway intermediates across compartment boundaries represents a bottleneck for future studies. To date, most compartmented bioreactors rely on unspecific exchange of substrates and products. This is either based on changes in permeability of the coblock polymer shell by physicochemical triggers or by the incorporation of unspecific porin proteins into the vesicle membrane. Since the incorporation of membrane transport proteins into simple and nested artificial vesicles offers the potential for specific exchange of substances between subcompartments, it opens new vistas in the design of protocells. Therefore, we devote the main part of the review to summarize the technical advances in the use of phospholipids and block copolymers for the reconstitution of membrane proteins. PMID:26973834

The Bretherton Problem for a Vesicle

NASA Astrophysics Data System (ADS)

Barakat, Joseph; Spann, Andrew; Shaqfeh, Eric

2016-11-01

The motion of a lipid bilayer vesicle through a circular tube is investigated by singular perturbation theory in the limit of vanishing clearance. The vesicle is treated as a sac of fluid enclosed by a thin, elastic sheet that admits a bending stiffness. It is assumed that the vesicle is axisymmetric and swollen to a near-critical volume such that the clearance "e" between the membrane and the tube wall is very small. In this limit, bending resistance is of negligible importance compared to the isotropic tension, allowing the vesicle to be treated as a "no-slip bubble." The effective membrane tension is found to scale inversely with "e" raised to the 3/2 power with a comparatively weak Marangoni gradient. The extra pressure drop is found to have a leading contribution due to the cylindrical midsection, which scales inversely with "e," as well as a correction due to the end caps, which scales inversely with the square root of "e." The apparent viscosity is predicted as a unique function of the geometry. The theory exhibits excellent agreement with a simplified, "quasi-parallel" theory and with direct numerical simulations using the boundary element method. The results of this work are compared to those for bubbles, rigid particles, and red blood cells in confined flows.

From Vesicles to Protocells: The Roles of Amphiphilic Molecules

PubMed Central

Sakuma, Yuka; Imai, Masayuki

2015-01-01

It is very challenging to construct protocells from molecular assemblies. An important step in this challenge is the achievement of vesicle dynamics that are relevant to cellular functions, such as membrane trafficking and self-reproduction, using amphiphilic molecules. Soft matter physics will play an important role in the development of vesicles that have these functions. Here, we show that simple binary phospholipid vesicles have the potential to reproduce the relevant functions of adhesion, pore formation and self-reproduction of vesicles, by coupling the lipid geometries (spontaneous curvatures) and the phase separation. This achievement will elucidate the pathway from molecular assembly to cellular life. PMID:25738256

Sulfur vesicles from Thermococcales: A possible role in sulfur detoxifying mechanisms

PubMed Central

Gorlas, A.; Marguet, E.; Gill, S.; Geslin, C.; Guigner, J.-M.; Guyot, F.; Forterre, P.

2015-01-01

The euryarchaeon Thermococcus prieurii inhabits deep-sea hydrothermal vents, one of the most extreme environments on Earth, which is reduced and enriched with heavy metals. Transmission electron microscopy and cryo-electron microscopy imaging of T. prieurii revealed the production of a plethora of diverse membrane vesicles (MVs) (from 50 nm to 400 nm), as is the case for other Thermococcales. T. prieurii also produces particularly long nanopods/nanotubes, some of them containing more than 35 vesicles encased in a S-layer coat. Notably, cryo-electron microscopy of T. prieurii cells revealed the presence of numerous intracellular dark vesicles that bud from the host cells via interaction with the cytoplasmic membrane. These dark vesicles are exclusively found in conjunction with T. prieurii cells and never observed in the purified membrane vesicles preparations. Energy-Dispersive-X-Ray analyses revealed that these dark vesicles are filled with sulfur. Furthermore, the presence of these sulfur vesicles (SVs) is exclusively observed when elemental sulfur was added into the growth medium. In this report, we suggest that these atypical vesicles sequester the excess sulfur not used for growth, thus preventing the accumulation of toxic levels of sulfur in the host's cytoplasm. These SVs transport elemental sulfur out of the cell where they are rapidly degraded. Intriguingly, closely related archaeal species, Thermococcus nautili and Thermococcus kodakaraensis, show some differences about the production of sulfur vesicles. Whereas T. kodakaraensis produces less sulfur vesicles than T. prieurii, T. nautili does not produce such sulfur vesicles, suggesting that Thermococcales species exhibit significant differences in their sulfur metabolic pathways. PMID:26234734

Inkjet formation of unilamellar lipid vesicles for cell-like encapsulation†

PubMed Central

Stachowiak, Jeanne C.; Richmond, David L.; Li, Thomas H.; Brochard-Wyart, Françoise

2010-01-01

Encapsulation of macromolecules within lipid vesicles has the potential to drive biological discovery and enable development of novel, cell-like therapeutics and sensors. However, rapid and reliable production of large numbers of unilamellar vesicles loaded with unrestricted and precisely-controlled contents requires new technologies that overcome size, uniformity, and throughput limitations of existing approaches. Here we present a high-throughput microfluidic method for vesicle formation and encapsulation using an inkjet printer at rates up to 200 Hz. We show how multiple high-frequency pulses of the inkjet’s piezoelectric actuator create a microfluidic jet that deforms a bilayer lipid membrane, controlling formation of individual vesicles. Variations in pulse number, pulse voltage, and solution viscosity are used to control the vesicle size. As a first step toward cell-like reconstitution using this method, we encapsulate the cytoskeletal protein actin and use co-encapsulated microspheres to track its polymerization into a densely entangled cytoskeletal network upon vesicle formation. PMID:19568667

Exosome-like vesicles with dipeptidyl peptidase IV in human saliva.

PubMed

Ogawa, Yuko; Kanai-Azuma, Masami; Akimoto, Yoshihiro; Kawakami, Hayato; Yanoshita, Ryohei

2008-06-01

Saliva contains a large number of proteins that participate in the protection of oral tissue. We found, for the first time, small vesicles (30-130 nm in diameter) in human whole saliva. Vesicles from saliva were identified by electron microscopy after isolation by gel-filtration on Sepharose CL-4B. They resemble exosomes, which are vesicles with an endosome-derived limiting membrane that are secreted by a diverse range of cell types. We performed a biochemical characterization of these vesicles by amino acid sequence analysis and Western blot analysis. We found that they contain dipeptidyl peptidase IV (DPP IV), galectin-3 and immunoglobulin A, which have potential to influence immune response. The DPP IV in the vesicles was metabolically active in cleaving substance P and glucose-dependent insulinotropic polypeptide to release N-terminal dipeptides. Our results demonstrate that human whole saliva contains exosome-like vesicles; they might participate in the catabolism of bioactive peptides and play a regulatory role in local immune defense in the oral cavity.

Neuronal Depolarization Drives Increased Dopamine Synaptic Vesicle Loading via VGLUT.

PubMed

Aguilar, Jenny I; Dunn, Matthew; Mingote, Susana; Karam, Caline S; Farino, Zachary J; Sonders, Mark S; Choi, Se Joon; Grygoruk, Anna; Zhang, Yuchao; Cela, Carolina; Choi, Ben Jiwon; Flores, Jorge; Freyberg, Robin J; McCabe, Brian D; Mosharov, Eugene V; Krantz, David E; Javitch, Jonathan A; Sulzer, David; Sames, Dalibor; Rayport, Stephen; Freyberg, Zachary

2017-08-30

The ability of presynaptic dopamine terminals to tune neurotransmitter release to meet the demands of neuronal activity is critical to neurotransmission. Although vesicle content has been assumed to be static, in vitro data increasingly suggest that cell activity modulates vesicle content. Here, we use a coordinated genetic, pharmacological, and imaging approach in Drosophila to study the presynaptic machinery responsible for these vesicular processes in vivo. We show that cell depolarization increases synaptic vesicle dopamine content prior to release via vesicular hyperacidification. This depolarization-induced hyperacidification is mediated by the vesicular glutamate transporter (VGLUT). Remarkably, both depolarization-induced dopamine vesicle hyperacidification and its dependence on VGLUT2 are seen in ventral midbrain dopamine neurons in the mouse. Together, these data suggest that in response to depolarization, dopamine vesicles utilize a cascade of vesicular transporters to dynamically increase the vesicular pH gradient, thereby increasing dopamine vesicle content. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of 17alpha-methyltestosterone on seminal vesicle development and semen release response in the African catfish, Clarias gariepinus.

PubMed

Viveiros, A T; Eding, E H; Komen, J

2001-11-01

The effects of 17alpha-methyltestosterone on seminal vesicle development in the African catfish, Clarias gariepinus, were investigated in an attempt to improve semen collection from this species. Treatment of larvae with dietary 17alpha-methyltestosterone at 50 mg kg(-1) for days 12-33 or days 12-40 after hatching, or at 20 mg kg(-1) for days 12-26, 12-33, 12-40 or 12-47 after hatching inhibited the development of the seminal vesicle finger-like extensions in male catfish, but did not affect the sex ratio. The minimum effective dose and period of treatment to inhibit seminal vesicle development in all male catfish treated with 17alpha-methyltestosterone was 20 mg kg(-1) for days 12-40 after hatching. Male catfish from this treatment group developed normal testes that, in some cases, contained a few oocytes, which tended to disappear before sexual maturation. After sexual maturation, the semen release response was evaluated in males with incomplete seminal vesicles. Fluid with viable spermatozoa was obtained after two consecutive injections of carp pituitary suspension, from 10 of 19 males that had been fed 20 mg 17alpha-methyltestosterone kg(-1) for days 12-40 or days 12-47 after hatching, but from only 4 of 15 males that did not receive any dietary steroid. Intratesticular semen quality was not affected by 17alpha-methyltestosterone treatment. The results of this study demonstrate that the absence of seminal vesicle extensions induced by treatment with 17alpha-methyltestosterone facilitated the collection of semen by stripping from this species of fish.

Asymmetric osmotic water permeation through a vesicle membrane

NASA Astrophysics Data System (ADS)

Su, Jiaye; Zhao, Yunzhen; Fang, Chang; Shi, Yue

2017-05-01

Understanding the water permeation through a cell membrane is of primary importance for biological activities and a key step to capture its shape transformation in salt solution. In this work, we reveal the dynamical behaviors of osmotically driven transport of water molecules across a vesicle membrane by molecular dynamics simulations. Of particular interest is that the water transport in and out of vesicles is highly distinguishable given the osmotic force are the same, suggesting an asymmetric osmotic transportation. This asymmetric phenomenon exists in a broad range of parameter space such as the salt concentration, temperature, and vesicle size and can be ascribed to the similar asymmetric potential energy of lipid-ion, lipid-water, lipid-solution, lipid-lipid, and the lipid-lipid energy fluctuation. Specifically, the water flux has a linear increase with the salt concentration, similar to the prediction by Nernst-Planck equation or Fick's first law. Furthermore, due to the Arrhenius relation between the membrane permeability and temperature, the water flux also exhibits excellent Arrhenius dependence on the temperature. Meanwhile, the water flux shows a linear increase with the vesicle surface area since the flux amount across a unit membrane area should be a constant. Finally, we also present the anonymous diffusion behaviors for the vesicle itself, where transitions from normal diffusion at short times to subdiffusion at long times are identified. Our results provide significant new physical insights for the osmotic water permeation through a vesicle membrane and are helpful for future experimental studies.

Overall energy conversion efficiency of a photosynthetic vesicle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sener, Melih; Strumpfer, Johan; Singharoy, Abhishek

The chromatophore of purple bacteria is an intracellular spherical vesicle that exists in numerous copies in the cell and that efficiently converts sunlight into ATP synthesis, operating typically under low light conditions. Building on an atomic-level structural model of a low-light-adapted chromatophore vesicle from Rhodobacter sphaeroides, we investigate the cooperation between more than a hundred protein complexes in the vesicle. The steady-state ATP production rate as a function of incident light intensity is determined after identifying quinol turnover at the cytochrome bc1 complex (cytbc1) as rate limiting and assuming that the quinone/quinol pool of about 900 molecules acts in amore » quasi-stationary state. For an illumination condition equivalent to 1% of full sunlight, the vesicle exhibits an ATP production rate of 82 ATP molecules/s. The energy conversion efficiency of ATP synthesis at illuminations corresponding to 1%–5% of full sunlight is calculated to be 0.12-0.04, respectively. The vesicle stoichiometry, evolutionarily adapted to the low light intensities in the habitat of purple bacteria, is suboptimal for steady-state ATP turnover for the benefit of protection against over-illumination.« less

Overall energy conversion efficiency of a photosynthetic vesicle

DOE PAGES

Sener, Melih; Strumpfer, Johan; Singharoy, Abhishek; ...

2016-08-26

The chromatophore of purple bacteria is an intracellular spherical vesicle that exists in numerous copies in the cell and that efficiently converts sunlight into ATP synthesis, operating typically under low light conditions. Building on an atomic-level structural model of a low-light-adapted chromatophore vesicle from Rhodobacter sphaeroides, we investigate the cooperation between more than a hundred protein complexes in the vesicle. The steady-state ATP production rate as a function of incident light intensity is determined after identifying quinol turnover at the cytochrome bc1 complex (cytbc1) as rate limiting and assuming that the quinone/quinol pool of about 900 molecules acts in amore » quasi-stationary state. For an illumination condition equivalent to 1% of full sunlight, the vesicle exhibits an ATP production rate of 82 ATP molecules/s. The energy conversion efficiency of ATP synthesis at illuminations corresponding to 1%–5% of full sunlight is calculated to be 0.12-0.04, respectively. The vesicle stoichiometry, evolutionarily adapted to the low light intensities in the habitat of purple bacteria, is suboptimal for steady-state ATP turnover for the benefit of protection against over-illumination.« less

Immunomodulatory role for membrane vesicles released by THP-1 macrophages and respiratory pathogens during macrophage infection.

PubMed

Volgers, Charlotte; Benedikter, Birke J; Grauls, Gert E; Savelkoul, Paul H M; Stassen, Frank R M

2017-11-13

During infection, inflammation is partially driven by the release of mediators which facilitate intercellular communication. Amongst these mediators are small membrane vesicles (MVs) that can be released by both host cells and Gram-negative and -positive bacteria. Bacterial membrane vesicles are known to exert immuno-modulatory and -stimulatory actions. Moreover, it has been proposed that host cell-derived vesicles, released during infection, also have immunostimulatory properties. In this study, we assessed the release and activity of host cell-derived and bacterial MVs during the first hours following infection of THP-1 macrophages with the common respiratory pathogens non-typeable Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, and Pseudomonas aeruginosa. Using a combination of flow cytometry, tunable resistive pulse sensing (TRPS)-based analysis and electron microscopy, we demonstrated that the release of MVs occurs by both host cells and bacteria during infection. MVs released during infection and bacterial culture were found to induce a strong pro-inflammatory response by naive THP-1 macrophages. Yet, these MVs were also found to induce tolerance of host cells to secondary immunogenic stimuli and to enhance bacterial adherence and the number of intracellular bacteria. Bacterial MVs may play a dual role during infection, as they can both trigger and dampen immune responses thereby contributing to immune defence and bacterial survival.

Phosphorylation of Synaptojanin Differentially Regulates Endocytosis of Functionally Distinct Synaptic Vesicle Pools.

PubMed

Geng, Junhua; Wang, Liping; Lee, Joo Yeun; Chen, Chun-Kan; Chang, Karen T

2016-08-24

The rapid replenishment of synaptic vesicles through endocytosis is crucial for sustaining synaptic transmission during intense neuronal activity. Synaptojanin (Synj), a phosphoinositide phosphatase, is known to play an important role in vesicle recycling by promoting the uncoating of clathrin following synaptic vesicle uptake. Synj has been shown to be a substrate of the minibrain (Mnb) kinase, a fly homolog of the dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A); however, the functional impacts of Synj phosphorylation by Mnb are not well understood. Here we identify that Mnb phosphorylates Synj at S1029 in Drosophila We find that phosphorylation of Synj at S1029 enhances Synj phosphatase activity, alters interaction between Synj and endophilin, and promotes efficient endocytosis of the active cycling vesicle pool (also referred to as exo-endo cycling pool) at the expense of reserve pool vesicle endocytosis. Dephosphorylated Synj, on the other hand, is deficient in the endocytosis of the active recycling pool vesicles but maintains reserve pool vesicle endocytosis to restore total vesicle pool size and sustain synaptic transmission. Together, our findings reveal a novel role for Synj in modulating reserve pool vesicle endocytosis and further indicate that dynamic phosphorylation and dephosphorylation of Synj differentially maintain endocytosis of distinct functional synaptic vesicle pools. Synaptic vesicle endocytosis sustains communication between neurons during a wide range of neuronal activities by recycling used vesicle membrane and protein components. Here we identify that Synaptojanin, a protein with a known role in synaptic vesicle endocytosis, is phosphorylated at S1029 in vivo by the Minibrain kinase. We further demonstrate that the phosphorylation status of Synaptojanin at S1029 differentially regulates its participation in the recycling of distinct synaptic vesicle pools. Our results reveal a new role for Synaptojanin in

Isolation of Tonoplast Vesicles from Tomato Fruit Pericarp

PubMed Central

Snowden, Christopher J.; Thomas, Benjamin; Baxter, Charles J.; Smith, J. Andrew C.; Sweetlove, Lee J.

2017-01-01

This protocol describes the isolation of tonoplast vesicles from tomato fruit. The vesicles isolated using this procedure are of sufficiently high purity for downstream proteomic analysis whilst remaining transport competent for functional assays. The methodology was used to study the transport of amino acids during tomato fruit ripening (Snowden et al., 2015) and based on the procedure used by Betty and Smith (Bettey and Smith, 1993). Such vesicles may be useful in further studies into the dynamic transfer of metabolites across the tonoplast for storage and metabolism during tomato fruit development. PMID:29085859

Coarse-Grain Simulations Reveal Movement of the Synaptobrevin C-Terminus in Response to Piconewton Forces

PubMed Central

Lindau, Manfred; Hall, Benjamin A.; Chetwynd, Alan; Beckstein, Oliver; Sansom, Mark S.P.

2012-01-01

Fusion of neurosecretory vesicles with the plasma membrane is mediated by SNARE proteins, which transfer a force to the membranes. However, the mechanism by which this force transfer induces fusion pore formation is still unknown. The neuronal vesicular SNARE protein synaptobrevin 2 (syb2) is anchored in the vesicle membrane by a single C-terminal transmembrane (TM) helix. In coarse-grain molecular-dynamics simulations, self-assembly of the membrane occurred with the syb2 TM domain inserted, as expected from experimental data. The free-energy profile for the position of the syb2 membrane anchor in the membrane was determined using umbrella sampling. To predict the free-energy landscapes for a reaction pathway pulling syb2 toward the extravesicular side of the membrane, which is the direction of the force transfer from the SNARE complex, harmonic potentials were applied to the peptide in its unbiased position, pulling it toward new biased equilibrium positions. Application of piconewton forces to the extravesicular end of the TM helix in the simulation detached the synaptobrevin C-terminus from the vesicle's inner-leaflet lipid headgroups and pulled it deeper into the membrane. This C-terminal movement was facilitated and hindered by specific mutations in parallel with experimentally observed facilitation and inhibition of fusion. Direct application of such forces to the intravesicular end of the TM domain resulted in tilting motion of the TM domain through the membrane with an activation energy of ∼70 kJ/mol. The results suggest a mechanism whereby fusion pore formation is induced by movement of the charged syb2 C-terminus within the membrane in response to pulling and tilting forces generated by C-terminal zippering of the SNARE complex. PMID:23009845

Coarse-grain simulations reveal movement of the synaptobrevin C-terminus in response to piconewton forces.

PubMed

Lindau, Manfred; Hall, Benjamin A; Chetwynd, Alan; Beckstein, Oliver; Sansom, Mark S P

2012-09-05

Fusion of neurosecretory vesicles with the plasma membrane is mediated by SNARE proteins, which transfer a force to the membranes. However, the mechanism by which this force transfer induces fusion pore formation is still unknown. The neuronal vesicular SNARE protein synaptobrevin 2 (syb2) is anchored in the vesicle membrane by a single C-terminal transmembrane (TM) helix. In coarse-grain molecular-dynamics simulations, self-assembly of the membrane occurred with the syb2 TM domain inserted, as expected from experimental data. The free-energy profile for the position of the syb2 membrane anchor in the membrane was determined using umbrella sampling. To predict the free-energy landscapes for a reaction pathway pulling syb2 toward the extravesicular side of the membrane, which is the direction of the force transfer from the SNARE complex, harmonic potentials were applied to the peptide in its unbiased position, pulling it toward new biased equilibrium positions. Application of piconewton forces to the extravesicular end of the TM helix in the simulation detached the synaptobrevin C-terminus from the vesicle's inner-leaflet lipid headgroups and pulled it deeper into the membrane. This C-terminal movement was facilitated and hindered by specific mutations in parallel with experimentally observed facilitation and inhibition of fusion. Direct application of such forces to the intravesicular end of the TM domain resulted in tilting motion of the TM domain through the membrane with an activation energy of ∼70 kJ/mol. The results suggest a mechanism whereby fusion pore formation is induced by movement of the charged syb2 C-terminus within the membrane in response to pulling and tilting forces generated by C-terminal zippering of the SNARE complex. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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

2015-08-01

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.

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

Mover Is a Homomeric Phospho-Protein Present on Synaptic Vesicles

PubMed Central

Kremer, Thomas; Hoeber, Jan; Kiran Akula, Asha; Urlaub, Henning; Islinger, Markus; Kirsch, Joachim; Dean, Camin; Dresbach, Thomas

2013-01-01

With remarkably few exceptions, the molecules mediating synaptic vesicle exocytosis at active zones are structurally and functionally conserved between vertebrates and invertebrates. Mover was found in a yeast-2-hybrid assay using the vertebrate-specific active zone scaffolding protein bassoon as a bait. Peptides of Mover have been reported in proteomics screens for self-interacting proteins, phosphorylated proteins, and synaptic vesicle proteins, respectively. Here, we tested the predictions arising from these screens. Using flotation assays, carbonate stripping of peripheral membrane proteins, mass spectrometry, immunogold labelling of purified synaptic vesicles, and immuno-organelle isolation, we found that Mover is indeed a peripheral synaptic vesicle membrane protein. In addition, by generating an antibody against phosphorylated Mover and Western blot analysis of fractionated rat brain, we found that Mover is a bona fide phospho-protein. The localization of Mover to synaptic vesicles is phosphorylation dependent; treatment with a phosphatase caused Mover to dissociate from synaptic vesicles. A yeast-2-hybrid screen, co-immunoprecipitation and cell-based optical assays of homomerization revealed that Mover undergoes homophilic interaction, and regions within both the N- and C- terminus of the protein are required for this interaction. Deleting a region required for homomeric interaction abolished presynaptic targeting of recombinant Mover in cultured neurons. Together, these data prove that Mover is associated with synaptic vesicles, and implicate phosphorylation and multimerization in targeting of Mover to synaptic vesicles and presynaptic sites. PMID:23723986

Sec34p, a Protein Required for Vesicle Tethering to the Yeast Golgi Apparatus, Is in a Complex with Sec35p

PubMed Central

VanRheenen, Susan M.; Cao, Xiaochun; Sapperstein, Stephanie K.; Chiang, Elbert C.; Lupashin, Vladimir V.; Barlowe, Charles; Waters, M. Gerard

1999-01-01

A screen for mutants of Saccharomyces cerevisiae secretory pathway components previously yielded sec34, a mutant that accumulates numerous vesicles and fails to transport proteins from the ER to the Golgi complex at the restrictive temperature (Wuestehube, L.J., R. Duden, A. Eun, S. Hamamoto, P. Korn, R. Ram, and R. Schekman. 1996. Genetics. 142:393–406). We find that SEC34 encodes a novel protein of 93-kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec34-2 is suppressed by the rab GTPase Ypt1p that functions early in the secretory pathway, or by the dominant form of the ER to Golgi complex target-SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor)–associated protein Sly1p, Sly1-20p. Weaker suppression is evident upon overexpression of genes encoding the vesicle tethering factor Uso1p or the vesicle-SNAREs Sec22p, Bet1p, or Ykt6p. This genetic suppression profile is similar to that of sec35-1, a mutant allele of a gene encoding an ER to Golgi vesicle tethering factor and, like Sec35p, Sec34p is required in vitro for vesicle tethering. sec34-2 and sec35-1 display a synthetic lethal interaction, a genetic result explained by the finding that Sec34p and Sec35p can interact by two-hybrid analysis. Fractionation of yeast cytosol indicates that Sec34p and Sec35p exist in an ∼750-kD protein complex. Finally, we describe RUD3, a novel gene identified through a genetic screen for multicopy suppressors of a mutation in USO1, which suppresses the sec34-2 mutation as well. PMID:10562277

Laser-induced fusion of human embryonic stem cells with optical tweezers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen Shuxun; Wang Xiaolin; Sun Dong

2013-07-15

We report a study on the laser-induced fusion of human embryonic stem cells (hESCs) at the single-cell level. Cells were manipulated by optical tweezers and fused under irradiation with pulsed UV laser at 355 nm. Successful fusion was indicated by green fluorescence protein transfer. The influence of laser pulse energy on the fusion efficiency was investigated. The fused products were viable as gauged by live cell staining. Successful fusion of hESCs with somatic cells was also demonstrated. The reported fusion outcome may facilitate studies of cell differentiation, maturation, and reprogramming.

Nuclear Fusion prize laudation Nuclear Fusion prize laudation

NASA Astrophysics Data System (ADS)

Burkart, W.

2011-01-01

Clean energy in abundance will be of critical importance to the pursuit of world peace and development. As part of the IAEA's activities to facilitate the dissemination of fusion related science and technology, the journal Nuclear Fusion is intended to contribute to the realization of such energy from fusion. In 2010, we celebrated the 50th anniversary of the IAEA journal. The excellence of research published in the journal is attested to by its high citation index. The IAEA recognizes excellence by means of an annual prize awarded to the authors of papers judged to have made the greatest impact. On the occasion of the 2010 IAEA Fusion Energy Conference in Daejeon, Republic of Korea at the welcome dinner hosted by the city of Daejeon, we celebrated the achievements of the 2009 and 2010 Nuclear Fusion prize winners. Steve Sabbagh, from the Department of Applied Physics and Applied Mathematics, Columbia University, New York is the winner of the 2009 award for his paper: 'Resistive wall stabilized operation in rotating high beta NSTX plasmas' [1]. This is a landmark paper which reports record parameters of beta in a large spherical torus plasma and presents a thorough investigation of the physics of resistive wall mode (RWM) instability. The paper makes a significant contribution to the critical topic of RWM stabilization. John Rice, from the Plasma Science and Fusion Center, MIT, Cambridge is the winner of the 2010 award for his paper: 'Inter-machine comparison of intrinsic toroidal rotation in tokamaks' [2]. The 2010 award is for a seminal paper that analyzes results across a range of machines in order to develop a universal scaling that can be used to predict intrinsic rotation. This paper has already triggered a wealth of experimental and theoretical work. I congratulate both authors and their colleagues on these exceptional papers. W. Burkart Deputy Director General Department of Nuclear Sciences and Applications International Atomic Energy Agency, Vienna

Functional transferred DNA within extracellular vesicles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cai, Jin; Department of Neurology, Jinling Hospital, Nanjing University School of Medicine, Jiangsu Province; Wu, Gengze

Extracellular vesicles (EVs) are small membrane vesicles including exosomes and shedding vesicles that mediated a cell-to-cell communication. EVs are released from almost all cell types under both physiological and pathological conditions and incorporate nuclear and cytoplasmic molecules for intercellular delivery. Besides protein, mRNA, and microRNA of these molecules, as recent studies show, specific DNA are prominently packaged into EVs. It appears likely that some of exosomes or shedding vesicles, bearing nuclear molecules are released upon bubble-like blebs. Specific interaction of EVs with susceptible recipients performs the uptake of EVs into the target cells, discharging their cargo including nuclear and cytoplasmicmore » macromolecules into the cytosol. These findings expand the nucleic acid content of EVs to include increased levels of specific DNA. Thus, EVs contain a repertoire of genetic information available for horizontal gene transfer and potential use as blood biomarkers for cancer and atherosclerosis. In this review, the focus is on the characteristics, biological functions, and roles in diseases of DNA within EVs. - Highlights: • This review is focused on the DNA within EVs including its characteristics, biological functions, and roles in diseases. • It is clear that DNA within EVs might have important physiological and pathological roles in various diseases. • Knowledge in this area may provides us alternative methods for disease diagnosis or therapy in the future.« less

Effects of magnetization on fusion product trapping and secondary neutron spectraa)

NASA Astrophysics Data System (ADS)

Knapp, P. F.; Schmit, P. F.; Hansen, S. B.; Gomez, M. R.; Hahn, K. D.; Sinars, D. B.; Peterson, K. J.; Slutz, S. A.; Sefkow, A. B.; Awe, T. J.; Harding, E.; Jennings, C. A.; Desjarlais, M. P.; Chandler, G. A.; Cooper, G. W.; Cuneo, M. E.; Geissel, M.; Harvey-Thompson, A. J.; Porter, J. L.; Rochau, G. A.; Rovang, D. C.; Ruiz, C. L.; Savage, M. E.; Smith, I. C.; Stygar, W. A.; Herrmann, M. C.

2015-05-01

By magnetizing the fusion fuel in inertial confinement fusion (ICF) systems, the required stagnation pressure and density can be relaxed dramatically. This happens because the magnetic field insulates the hot fuel from the cold pusher and traps the charged fusion burn products. This trapping allows the burn products to deposit their energy in the fuel, facilitating plasma self-heating. Here, we report on a comprehensive theory of this trapping in a cylindrical DD plasma magnetized with a purely axial magnetic field. Using this theory, we are able to show that the secondary fusion reactions can be used to infer the magnetic field-radius product, BR, during fusion burn. This parameter, not ρR, is the primary confinement parameter in magnetized ICF. Using this method, we analyze data from recent Magnetized Liner Inertial Fusion experiments conducted on the Z machine at Sandia National Laboratories. We show that in these experiments BR ≈ 0.34(+0.14/-0.06) MG . cm, a ˜ 14× increase in BR from the initial value, and confirming that the DD-fusion tritons are magnetized at stagnation. This is the first experimental verification of charged burn product magnetization facilitated by compression of an initial seed magnetic flux.

Effects of magnetization on fusion product trapping and secondary neutron spectra

DOE PAGES

Knapp, Patrick F.; Schmit, Paul F.; Hansen, Stephanie B.; ...

2015-05-14

In magnetizing the fusion fuel in inertial confinement fusion (ICF) systems, we found that the required stagnation pressure and density can be relaxed dramatically. This happens because the magnetic field insulates the hot fuel from the cold pusher and traps the charged fusion burn products. This trapping allows the burn products to deposit their energy in the fuel, facilitating plasma self-heating. Here, we report on a comprehensive theory of this trapping in a cylindrical DD plasma magnetized with a purely axial magnetic field. Using this theory, we are able to show that the secondary fusion reactions can be used tomore » infer the magnetic field-radius product, BR, during fusion burn. This parameter, not ρR, is the primary confinement parameter in magnetized ICF. Using this method, we analyze data from recent Magnetized Liner InertialFusion experiments conducted on the Z machine at Sandia National Laboratories. Furthermore, we show that in these experiments BR ≈ 0.34(+0.14/-0.06) MG · cm, a ~ 14× increase in BR from the initial value, and confirming that the DD-fusion tritons are magnetized at stagnation. Lastly, this is the first experimental verification of charged burn product magnetization facilitated by compression of an initial seed magnetic flux.« less

The parasite Toxoplasma sequesters diverse Rab host vesicles within an intravacuolar network

PubMed Central

2017-01-01

Many intracellular pathogens subvert host membrane trafficking pathways to promote their replication. Toxoplasma multiplies in a membrane-bound parasitophorous vacuole (PV) that interacts with mammalian host organelles and intercepts Golgi Rab vesicles to acquire sphingolipids. The mechanisms of host vesicle internalization and processing within the PV remain undefined. We demonstrate that Toxoplasma sequesters a broad range of Rab vesicles into the PV. Correlative light and electron microscopy analysis of infected cells illustrates that intravacuolar Rab1A vesicles are surrounded by the PV membrane, suggesting a phagocytic-like process for vesicle engulfment. Rab11A vesicles concentrate to an intravacuolar network (IVN), but this is reduced in Δgra2 and Δgra2Δgra6 parasites, suggesting that tubules stabilized by the TgGRA2 and TgGRA6 proteins secreted by the parasite within the PV contribute to host vesicle sequestration. Overexpression of a phospholipase TgLCAT, which is localized to the IVN, results in a decrease in the number of intravacuolar GFP-Rab11A vesicles, suggesting that TgLCAT controls lipolytic degradation of Rab vesicles for cargo release. PMID:29070609

Chromatic response of polydiacetylene vesicle induced by the permeation of methotrexate.

PubMed

Shin, Min Jae; Kim, Ye Jin; Kim, Jong-Duk

2015-07-07

The noble vesicular system of polydiacetylene showed a red shift using two types of detecting systems. One of the systems involves the absorption of target materials from the outer side of the vesicle, and the other system involves the permeation through the vesicular layers from within the vesicle. The chromatic mixed vesicles of N-(2-aminoethyl)pentacosa-10,12-diynamide (AEPCDA) and dimethyldioctadecylammonium chloride (DODAC) were fabricated by sonication, followed by polymerization by UV irradiation. The stability of monomeric vesicles was observed to increase with the polymerization of the vesicles. Methotrexate was used as a target material. The polymerized mixed vesicles having a blue color were exposed to a concentration gradient of methotrexate, and a red shift was observed indicating the adsorption of methotrexate on the polydiacetylene bilayer. In order to check the chromatic change by the permeation of methotrexate, we separated the vesicle portion, which contained methotrexate inside the vesicle, and checked chromatic change during the permeation of methotrexate through the vesicle. The red shift apparently indicates the disturbance in the bilayer induced by the permeation of methotrexate. The maximum contrast of color appeared at the equal molar ratio of AEPCDA and DODAC, indicating that the formation of flexible and deformable vesicular layers is important for red shift. Therefore, it is hypothesized that the system can be applicable for the chromatic detection of the permeation of methotrexate through the polydiacetylene layer.

Janus dendrimersomes coassembled from fluorinated, hydrogenated, and hybrid Janus dendrimers as models for cell fusion and fission.

PubMed

Xiao, Qi; Sherman, Samuel E; Wilner, Samantha E; Zhou, Xuhao; Dazen, Cody; Baumgart, Tobias; Reed, Ellen H; Hammer, Daniel A; Shinoda, Wataru; Klein, Michael L; Percec, Virgil

2017-08-22

A three-component system of Janus dendrimers (JDs) including hydrogenated, fluorinated, and hybrid hydrogenated-fluorinated JDs are reported to coassemble by film hydration at specific ratios into an unprecedented class of supramolecular Janus particles (JPs) denoted Janus dendrimersomes (JDSs). They consist of a dumbbell-shaped structure composed of an onion-like hydrogenated vesicle and an onion-like fluorinated vesicle tethered together. The synthesis of dye-tagged analogs of each JD component enabled characterization of JDS architectures with confocal fluorescence microscopy. Additionally, a simple injection method was used to prepare submicron JDSs, which were imaged with cryogenic transmission electron microscopy (cryo-TEM). As reported previously, different ratios of the same three-component system yielded a variety of structures including homogenous onion-like vesicles, core-shell structures, and completely self-sorted hydrogenated and fluorinated vesicles. Taken together with the JDSs reported herein, a self-sorting pathway is revealed as a function of the relative concentration of the hybrid JD, which may serve to stabilize the interface between hydrogenated and fluorinated bilayers. The fission-like pathway suggests the possibility of fusion and fission processes in biological systems that do not require the assistance of proteins but instead may result from alterations in the ratios of membrane composition.

Comparison of Extruded and Sonicated Vesicles for Planar Bilayer Self-Assembly

PubMed Central

Cho, Nam-Joon; Hwang, Lisa Y.; Solandt, Johan J.R.; Frank, Curtis W.

2013-01-01

Lipid vesicles are an important class of biomaterials that have a wide range of applications, including drug delivery, cosmetic formulations and model membrane platforms on solid supports. Depending on the application, properties of a vesicle population such as size distribution, charge and permeability need to be optimized. Preparation methods such as mechanical extrusion and sonication play a key role in controlling these properties, and yet the effects of vesicle preparation method on vesicular properties and integrity (e.g., shape, size, distribution and tension) remain incompletely understood. In this study, we prepared vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid by either extrusion or sonication, and investigated the effects on vesicle size distribution over time as well as the concomitant effects on the self-assembly of solid-supported planar lipid bilayers. Dynamic light scattering (DLS), quartz crystal microbalance with dissipation (QCM-D) monitoring, fluorescence recovery after photobleaching (FRAP) and atomic force microscopy (AFM) experiments were performed to characterize vesicles in solution as well as their interactions with silicon oxide substrates. Collectively, the data support that sonicated vesicles offer more robust control over the self-assembly of homogenous planar lipid bilayers, whereas extruded vesicles are vulnerable to aging and must be used soon after preparation. PMID:28811437

Stability, folding dynamics, and long-range conformational transition of the synaptic t-SNARE complex

PubMed Central

Zhang, Xinming; Rebane, Aleksander A.; Ma, Lu; Li, Feng; Jiao, Junyi; Qu, Hong; Pincet, Frederic; Rothman, James E.

2016-01-01

Synaptic soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) couple their stepwise folding to fusion of synaptic vesicles with plasma membranes. In this process, three SNAREs assemble into a stable four-helix bundle. Arguably, the first and rate-limiting step of SNARE assembly is the formation of an activated binary target (t)-SNARE complex on the target plasma membrane, which then zippers with the vesicle (v)-SNARE on the vesicle to drive membrane fusion. However, the t-SNARE complex readily misfolds, and its structure, stability, and dynamics are elusive. Using single-molecule force spectroscopy, we modeled the synaptic t-SNARE complex as a parallel three-helix bundle with a small frayed C terminus. The helical bundle sequentially folded in an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a central ionic layer, with total unfolding energy of ∼17 kBT, where kB is the Boltzmann constant and T is 300 K. Peptide binding to the CTD activated the t-SNARE complex to initiate NTD zippering with the v-SNARE, a mechanism likely shared by the mammalian uncoordinated-18-1 protein (Munc18-1). The NTD zippering then dramatically stabilized the CTD, facilitating further SNARE zippering. The subtle bidirectional t-SNARE conformational switch was mediated by the ionic layer. Thus, the t-SNARE complex acted as a switch to enable fast and controlled SNARE zippering required for synaptic vesicle fusion and neurotransmission. PMID:27911771

Gold nanoparticles covalently assembled onto vesicle structures as possible biosensing platform

PubMed Central

Barroso, M Fátima; Luna, M Alejandra; Tabares, Juan S Flores; Delerue-Matos, Cristina; Correa, N Mariano

2016-01-01

Summary In this contribution a strategy is shown to covalently immobilize gold nanoparticles (AuNPs) onto vesicle bilayers with the aim of using this nanomaterial as platform for the future design of immunosensors. A novel methodology for the self-assembly of AuNPs onto large unilamellar vesicle structures is described. The vesicles were formed with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-undecanethiol (SH). After, the AuNPs photochemically synthesized in pure glycerol were mixed and anchored onto SH–DOPC vesicles. The data provided by voltammetry, spectrometry and microscopy techniques indicated that the AuNPs were successfully covalently anchored onto the vesicle bilayer and decorated vesicles exhibit a spherical shape with a size of 190 ± 10 nm. The developed procedure is easy, rapid and reproducible to start designing a possible immunosensor by using environmentally friendly procedures. PMID:27335755

Overall energy conversion efficiency of a photosynthetic vesicle

PubMed Central

Sener, Melih; Strumpfer, Johan; Singharoy, Abhishek; Hunter, C Neil; Schulten, Klaus

2016-01-01

The chromatophore of purple bacteria is an intracellular spherical vesicle that exists in numerous copies in the cell and that efficiently converts sunlight into ATP synthesis, operating typically under low light conditions. Building on an atomic-level structural model of a low-light-adapted chromatophore vesicle from Rhodobacter sphaeroides, we investigate the cooperation between more than a hundred protein complexes in the vesicle. The steady-state ATP production rate as a function of incident light intensity is determined after identifying quinol turnover at the cytochrome bc1 complex (cytb⁢c1) as rate limiting and assuming that the quinone/quinol pool of about 900 molecules acts in a quasi-stationary state. For an illumination condition equivalent to 1% of full sunlight, the vesicle exhibits an ATP production rate of 82 ATP molecules/s. The energy conversion efficiency of ATP synthesis at illuminations corresponding to 1%–5% of full sunlight is calculated to be 0.12–0.04, respectively. The vesicle stoichiometry, evolutionarily adapted to the low light intensities in the habitat of purple bacteria, is suboptimal for steady-state ATP turnover for the benefit of protection against over-illumination. DOI: http://dx.doi.org/10.7554/eLife.09541.001 PMID:27564854

Synaptophysin regulates the kinetics of synaptic vesicle endocytosis in central neurons

PubMed Central

Kwon, Sung E.; Chapman, Edwin R.

2011-01-01

Summary Despite being the most abundant synaptic vesicle membrane protein, the function of synaptophysin remains enigmatic. For example, synaptic transmission was reported to be completely normal in synaptophysin knockout mice; however, direct experiments to monitor the synaptic vesicle cycle have not been carried out. Here, using optical imaging and electrophysiological experiments, we demonstrate that synaptophysin is required for kinetically efficient endocytosis of synaptic vesicles in cultured hippocampal neurons. Truncation analysis revealed that distinct structural elements of synaptophysin differentially regulate vesicle retrieval during and after stimulation. Thus, synaptophysin regulates at least two phases of endocytosis to ensure vesicle availability during and after sustained neuronal activity. PMID:21658579

Interaction and rheology of vesicle suspensions in confined shear flow

NASA Astrophysics Data System (ADS)

Shen, Zaiyi; Farutin, Alexander; Thiébaud, Marine; Misbah, Chaouqi

2017-10-01

Dynamics and rheology of a confined suspension of vesicles (a model for red blood cells) are studied numerically in two dimensions by using an immersed boundary lattice Boltzmann method. We pay particular attention to the link between the spatiotemporal organization and the rheology of the suspension. Besides confinement, we analyze the effect of concentration of the suspension, ϕ (defined as the area fraction occupied by the vesicles in the simulation domain), as well as the viscosity contrast λ (defined as the ratio between the viscosity of the fluid inside the vesicles, ηint, and that of the suspending fluid, ηext). The hydrodynamic interaction between two vesicles is shown to play a key role in determining the spatial organization. For λ =1 , the pair of vesicles settles into an equilibrium state with constant interdistance, which is regulated by the confinement. The equilibrium interdistance increases with the gap between walls, following a linear relationship. However, no stable equilibrium interdistance between two tumbling vesicles is observed for λ =10 . A quite ordered suspension is observed concomitant with the existence of an equilibrium interdistance between a vesicle pair. However, a disordered suspension prevails when no pair equilibrium interdistance exists, as occurs for tumbling vesicles. We then analyze the rheology, focusing on the effective viscosity, denoted as η , as well as on normalized viscosity, defined as [η ] =(η -ηext) /(ηextϕ ) . Ordering of the suspension is accompanied by a nonmonotonic behavior of [η ] with ϕ , while η exhibits plateaus. The nonmonotonic behavior of [η ] is suppressed when a disordered pattern prevails.

The zebrafish mutant vps18 as a model for vesicle-traffic related hypopigmentation diseases.

PubMed

Maldonado, Ernesto; Hernandez, Fabiola; Lozano, Carlos; Castro, Marta E; Navarro, Rosa E

2006-08-01

Hypopigmentation is a characteristic of several diseases associated with vesicle traffic defects, like the Hermansky-Pudlak, Chediak-Higashi, and Griscelli syndromes. Hypopigmentation is also a characteristic of the zebrafish mutant vps18(hi2499A), which is affected in the gene vps18, a component of the homotypic fusion and protein sorting complex that is involved in tethering during vesicular traffic. Vps18, as part of this complex, participates in the formation of early endosomes, late endosomes, and lysosomes. Here, we show that Vps18 is also involved in the formation of melanosomes. In the zebrafish mutant vps18(hi2499A) the retroviral insertion located at exon 4 of vps18, leads to the formation of two abnormal splicing variants lacking the coding sequence for the clathrin repeat and the RING finger conserved domains. A deficiency of Vps18 in zebrafish larvae results in hepatomegaly and skin hypopigmentation. We also observed a drastic reduction in the number of melanosomes in the eye's retinal pigmented epithelium along with the accumulation of immature melanosomes. A significant reduction in the vps18(hi2499A) larvae visual system capacity was found using the optokinetic response assay. We propose that the insertional mutant vps18(hi2499A) can be used as a model for studying hypopigmentation diseases in which vesicle traffic problems exist.

Activity-Dependent Calpain Activation Plays a Critical Role in Synaptic Facilitation and Post-Tetanic Potentiation

ERIC Educational Resources Information Center

Khoutorsky, Arkady; Spira, Micha E.

2009-01-01

Synaptic facilitation and post-tetanic potentiation (PTP) are believed to necessitate active regeneration of the release machinery and supply of synaptic vesicles to a ready-releasable site. The prevailing hypothesis assumes that synapsins play pivotal roles in these processes. Using a cholinergic synapse formed between cultured "Aplysia" neurons…

The mechanism of a nuclear pore assembly: a molecular biophysics view.

PubMed

Kuvichkin, Vasily V

2011-06-01

The basic problem of nuclear pore assembly is the big perinuclear space that must be overcome for nuclear membrane fusion and pore creation. Our investigations of ternary complexes: DNA-PC liposomes-Mg²⁺, and modern conceptions of nuclear pore structure allowed us to introduce a new mechanism of nuclear pore assembly. DNA-induced fusion of liposomes (membrane vesicles) with a single-lipid bilayer or two closely located nuclear membranes is considered. After such fusion on the lipid bilayer surface, traces of a complex of ssDNA with lipids were revealed. At fusion of two identical small liposomes (membrane vesicles) < 100 nm in diameter, a "big" liposome (vesicle) with ssDNA on the vesicle equator is formed. ssDNA occurrence on liposome surface gives a biphasic character to the fusion kinetics. The "big" membrane vesicle surrounded by ssDNA is the base of nuclear pore assembly. Its contact with the nuclear envelope leads to fast fusion of half of the vesicles with one nuclear membrane; then ensues a fusion delay when ssDNA reaches the membrane. The next step is to turn inside out the second vesicle half and its fusion to other nuclear membrane. A hole is formed between the two membranes, and nucleoporins begin pore complex assembly around the ssDNA. The surface tension of vesicles and nuclear membranes along with the kinetic energy of a liquid inside a vesicle play the main roles in this process. Special cases of nuclear pore formation are considered: pore formation on both nuclear envelope sides, the difference of pores formed in various cell-cycle phases and linear nuclear pore clusters.

Development and characterization of nanopore system for nano-vesicle analysis

NASA Astrophysics Data System (ADS)

Goyal, Gaurav

Nano-vesicles have recently attracted a lot of attention in research and medical communities and are very promising next-generation drug delivery vehicles. This is due to their biocompatibility, biodegradability and their ability to protect drug cargo and deliver it to site-specific locations, while maintaining the desired pharmacokinetic profile. The interaction of these drug loaded vesicles with the recipient cells via adsorption, endocytosis or receptor mediated internalization involve significant bending and deformation and is governed by mechanical properties of the nano-vesicles. Currently, the mechanical characteristics of nano-vesicles are left unexplored because of the difficulties associated with vesicle analysis at sub-100 nm length scale. The need for a complete understanding of nano-vesicle interaction with each other and the recipient cells warrants development of an analytical tool capable of mechanical investigation of individual vesicles at sub-100 nm scale. This dissertation presents investigation of nano-vesicle deformability using resistive pulse sensing and solid-state nanopore devices. The dissertation is divided into four chapters. Chapter 1 discusses the motivation, specific aims and presents an overview of nanoparticle characterization techniques, resistive pulse sensing background and principles, techniques for fabricating solid-state nanopores, as well the deformation behavior of giant vesicles when placed in electric field. Chapter 2 is dedicated to understanding of the scientific principles governing transport of sub-100 nm particles in dilute solutions. We investigated the translocation of rigid nanoparticles through nanopores at salt concentrations < 50 mM. When using low electrolyte strength, surface effects become predominant and resulted in unconventional current signatures in our experiments. It prompted us to explore the effects of different experimental parameters using Multiphysics simulations, in order to optimize our system

Adhesion signals of phospholipid vesicles at an electrified interface.

PubMed

DeNardis, Nadica Ivošević; Žutić, Vera; Svetličić, Vesna; Frkanec, Ruža

2012-09-01

General adhesion behavior of phospholipid vesicles was examined in a wide range of potentials at the mercury electrode by recording time-resolved adhesion signals. It was demonstrated that adhesion-based detection is sensitive to polar headgroups in phospholipid vesicles. We identified a narrow potential window around the point of zero charge of the electrode where the interaction of polar headgroups of phosphatidylcholine vesicles with the substrate is manifested in the form of bidirectional signals. The bidirectional signal is composed of the charge flow due to the nonspecific interaction of vesicle adhesion and spreading and of the charge flow due to a specific interaction of the negatively charged electrode and the most exposed positively charged choline headgroups. These signals are expected to appear only when the electrode surface charge density is less than the surface charge density of the choline groups at the contact interface. In comparison, for the negatively charged phosphatidylserine vesicles, we identified the potential window at the mercury electrode where charge compensation takes place, and bidirectional signals were not detected.

The LRRK2 G2385R variant is a partial loss-of-function mutation that affects synaptic vesicle trafficking through altered protein interactions.

PubMed

Carrion, Maria Dolores Perez; Marsicano, Silvia; Daniele, Federica; Marte, Antonella; Pischedda, Francesca; Di Cairano, Eliana; Piovesana, Ester; von Zweydorf, Felix; Kremmer, Elisabeth; Gloeckner, Christian Johannes; Onofri, Franco; Perego, Carla; Piccoli, Giovanni

2017-07-14

Mutations in the Leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial Parkinson's disease (PD). LRRK2 protein contains several functional domains, including protein-protein interaction domains at its N- and C-termini. In this study, we analyzed the functional features attributed to LRRK2 by its N- and C-terminal domains. We combined TIRF microscopy and synaptopHluorin assay to visualize synaptic vesicle trafficking. We found that N- and C-terminal domains have opposite impact on synaptic vesicle dynamics. Biochemical analysis demonstrated that different proteins are bound at the two extremities, namely β3-Cav2.1 at N-terminus part and β-Actin and Synapsin I at C-terminus domain. A sequence variant (G2385R) harboured within the C-terminal WD40 domain increases the risk for PD. Complementary biochemical and imaging approaches revealed that the G2385R variant alters strength and quality of LRRK2 interactions and increases fusion of synaptic vesicles. Our data suggest that the G2385R variant behaves like a loss-of-function mutation that mimics activity-driven events. Impaired scaffolding capabilities of mutant LRRK2 resulting in perturbed vesicular trafficking may arise as a common pathophysiological denominator through which different LRRK2 pathological mutations cause disease.

Quantification of mixing in vesicle suspensions using numerical simulations in two dimensions.

PubMed

Kabacaoğlu, G; Quaife, B; Biros, G

2017-02-01

We study mixing in Stokesian vesicle suspensions in two dimensions on a cylindrical Couette apparatus using numerical simulations. The vesicle flow simulation is done using a boundary integral method, and the advection-diffusion equation for the mixing of the solute is solved using a pseudo-spectral scheme. We study the effect of the area fraction, the viscosity contrast between the inside (the vesicles) and the outside (the bulk) fluid, the initial condition of the solute, and the mixing metric. We compare mixing in the suspension with mixing in the Couette apparatus without vesicles. On the one hand, the presence of vesicles in most cases slightly suppresses mixing. This is because the solute can be only diffused across the vesicle interface and not advected. On the other hand, there exist spatial distributions of the solute for which the unperturbed Couette flow completely fails to mix whereas the presence of vesicles enables mixing. We derive a simple condition that relates the velocity and solute and can be used to characterize the cases in which the presence of vesicles promotes mixing.

Quantification of mixing in vesicle suspensions using numerical simulations in two dimensions

PubMed Central

Quaife, B.; Biros, G.

2017-01-01

We study mixing in Stokesian vesicle suspensions in two dimensions on a cylindrical Couette apparatus using numerical simulations. The vesicle flow simulation is done using a boundary integral method, and the advection-diffusion equation for the mixing of the solute is solved using a pseudo-spectral scheme. We study the effect of the area fraction, the viscosity contrast between the inside (the vesicles) and the outside (the bulk) fluid, the initial condition of the solute, and the mixing metric. We compare mixing in the suspension with mixing in the Couette apparatus without vesicles. On the one hand, the presence of vesicles in most cases slightly suppresses mixing. This is because the solute can be only diffused across the vesicle interface and not advected. On the other hand, there exist spatial distributions of the solute for which the unperturbed Couette flow completely fails to mix whereas the presence of vesicles enables mixing. We derive a simple condition that relates the velocity and solute and can be used to characterize the cases in which the presence of vesicles promotes mixing. PMID:28344432

Vesicle biomechanics in a time-varying magnetic field.

PubMed

Ye, Hui; Curcuru, Austen

2015-01-01

Cells exhibit distortion when exposed to a strong electric field, suggesting that the field imposes control over cellular biomechanics. Closed pure lipid bilayer membranes (vesicles) have been widely used for the experimental and theoretical studies of cellular biomechanics under this electrodeformation. An alternative method used to generate an electric field is by electromagnetic induction with a time-varying magnetic field. References reporting the magnetic control of cellular mechanics have recently emerged. However, theoretical analysis of the cellular mechanics under a time-varying magnetic field is inadequate. We developed an analytical theory to investigate the biomechanics of a modeled vesicle under a time-varying magnetic field. Following previous publications and to simplify the calculation, this model treated the inner and suspending media as lossy dielectrics, the membrane thickness set at zero, and the electric resistance of the membrane assumed to be negligible. This work provided the first analytical solutions for the surface charges, electric field, radial pressure, overall translational forces, and rotational torques introduced on a vesicle by the time-varying magnetic field. Frequency responses of these measures were analyzed, particularly the frequency used clinically by transcranial magnetic stimulation (TMS). The induced surface charges interacted with the electric field to produce a biomechanical impact upon the vesicle. The distribution of the induced surface charges depended on the orientation of the coil and field frequency. The densities of these charges were trivial at low frequency ranges, but significant at high frequency ranges. The direction of the radial force on the vesicle was dependent on the conductivity ratio between the vesicle and the medium. At relatively low frequencies (<200 KHz), including the frequency used in TMS, the computed radial pressure and translational forces on the vesicle were both negligible. This work

Standardization of sample collection, isolation and analysis methods in extracellular vesicle research

PubMed Central

Witwer, Kenneth W.; Buzás, Edit I.; Bemis, Lynne T.; Bora, Adriana; Lässer, Cecilia; Lötvall, Jan; Nolte-‘t Hoen, Esther N.; Piper, Melissa G.; Sivaraman, Sarada; Skog, Johan; Théry, Clotilde; Wauben, Marca H.; Hochberg, Fred

2013-01-01

The emergence of publications on extracellular RNA (exRNA) and extracellular vesicles (EV) has highlighted the potential of these molecules and vehicles as biomarkers of disease and therapeutic targets. These findings have created a paradigm shift, most prominently in the field of oncology, prompting expanded interest in the field and dedication of funds for EV research. At the same time, understanding of EV subtypes, biogenesis, cargo and mechanisms of shuttling remains incomplete. The techniques that can be harnessed to address the many gaps in our current knowledge were the subject of a special workshop of the International Society for Extracellular Vesicles (ISEV) in New York City in October 2012. As part of the “ISEV Research Seminar: Analysis and Function of RNA in Extracellular Vesicles (evRNA)”, 6 round-table discussions were held to provide an evidence-based framework for isolation and analysis of EV, purification and analysis of associated RNA molecules, and molecular engineering of EV for therapeutic intervention. This article arises from the discussion of EV isolation and analysis at that meeting. The conclusions of the round table are supplemented with a review of published materials and our experience. Controversies and outstanding questions are identified that may inform future research and funding priorities. While we emphasize the need for standardization of specimen handling, appropriate normative controls, and isolation and analysis techniques to facilitate comparison of results, we also recognize that continual development and evaluation of techniques will be necessary as new knowledge is amassed. On many points, consensus has not yet been achieved and must be built through the reporting of well-controlled experiments. PMID:24009894

Dynamics of coarsening in multicomponent lipid vesicles with non-uniform mechanical properties

NASA Astrophysics Data System (ADS)

Funkhouser, Chloe M.; Solis, Francisco J.; Thornton, K.

2014-04-01

Multicomponent lipid vesicles are commonly used as a model system for the complex plasma membrane. One phenomenon that is studied using such model systems is phase separation. Vesicles composed of simple lipid mixtures can phase-separate into liquid-ordered and liquid-disordered phases, and since these phases can have different mechanical properties, this separation can lead to changes in the shape of the vesicle. In this work, we investigate the dynamics of phase separation in multicomponent lipid vesicles, using a model that couples composition to mechanical properties such as bending rigidity and spontaneous curvature. The model allows the vesicle surface to deform while conserving surface area and composition. For vesicles initialized as spheres, we study the effects of phase fraction and spontaneous curvature. We additionally initialize two systems with elongated, spheroidal shapes. Dynamic behavior is contrasted in systems where only one phase has a spontaneous curvature similar to the overall vesicle surface curvature and systems where the spontaneous curvatures of both phases are similar to the overall curvature. The bending energy contribution is typically found to slow the dynamics by stabilizing configurations with multiple domains. Such multiple-domain configurations are found more often in vesicles with spheroidal shapes than in nearly spherical vesicles.

Study of the plant COPII vesicle coat subunits by functional complementation of yeast Saccharomyces cerevisiae mutants.

PubMed

De Craene, Johan-Owen; Courte, Fanny; Rinaldi, Bruno; Fitterer, Chantal; Herranz, Mari Carmen; Schmitt-Keichinger, Corinne; Ritzenthaler, Christophe; Friant, Sylvie

2014-01-01

The formation and budding of endoplasmic reticulum ER-derived vesicles depends on the COPII coat protein complex that was first identified in yeast Saccharomyces cerevisiae. The ER-associated Sec12 and the Sar1 GTPase initiate the COPII coat formation by recruiting the Sec23-Sec24 heterodimer following the subsequent recruitment of the Sec13-Sec31 heterotetramer. In yeast, there is usually one gene encoding each COPII protein and these proteins are essential for yeast viability, whereas the plant genome encodes multiple isoforms of all COPII subunits. Here, we used a systematic yeast complementation assay to assess the functionality of Arabidopsis thaliana COPII proteins. In this study, the different plant COPII subunits were expressed in their corresponding temperature-sensitive yeast mutant strain to complement their thermosensitivity and secretion phenotypes. Secretion was assessed using two different yeast cargos: the soluble α-factor pheromone and the membranous v-SNARE (vesicle-soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor) Snc1 involved in the fusion of the secretory vesicles with the plasma membrane. This complementation study allowed the identification of functional A. thaliana COPII proteins for the Sec12, Sar1, Sec24 and Sec13 subunits that could represent an active COPII complex in plant cells. Moreover, we found that AtSec12 and AtSec23 were co-immunoprecipitated with AtSar1 in total cell extract of 15 day-old seedlings of A. thaliana. This demonstrates that AtSar1, AtSec12 and AtSec23 can form a protein complex that might represent an active COPII complex in plant cells.

Thermodynamically stable vesicle formation from glycolipid biosurfactant sponge phase.

PubMed

Imura, Tomohiro; Yanagishita, Hiroshi; Ohira, Junko; Sakai, Hideki; Abe, Masahiko; Kitamoto, Dai

2005-06-25

Thermodynamically stable vesicle (L(alpha1)) formation from glycolipid biosurfactant sponge phase (L(3)) and its mechanism were investigated using a "natural" biocompatible mannosyl-erythritol lipid-A (MEL-A)/L-alpha-dilauroylphosphatidylcholine (DLPC) mixture by varying the composition. The trapping efficiency for calcein and turbidity measurements clearly indicated the existence of three regions: while the trapping efficiencies of the mixed MEL-A/DLPC assemblies at the compositions with X(DLPC)< or =0.1 or X(DLPC)> or =0.8 were almost zero, the mixed assemblies at the compositions with 0.1 or =0.8 were multilamellar vesicles (L(alpha)) with diameter from 2 to 10 microm. Meanwhile, dynamic light scattering (DLS) measurement revealed that the average size of the vesicles at the composition of X(DLPC)=0.3 was 633.2 nm, which is remarkably small compared to other compositions. Moreover, the mixed vesicle solution at the composition of X(DLPC)=0.3 was slightly bluish and turbid and kept its dispersion stability at 25 degrees C for more than 3 months, indicating the formation of a thermodynamically stable vesicle (L(alpha1)). These results exhibited the formation of a thermodynamically stable vesicle (L(alpha1)) with a high dispersibility from the MEL-A/DLPC mixture. The asymmetric distribution of MEL-A and DLPC in the two vesicle monolayers caused by the difference in geometrical structures is very likely to have changed their self-assembled structure from a sponge phase (L(3)) to a thermodynamically stable vesicle (L(alpha1)).

Deformation of giant vesicles in AC electric fields —Dependence of the prolate-to-oblate transition frequency on vesicle radius

NASA Astrophysics Data System (ADS)

Antonova, K.; Vitkova, V.; Mitov, M. D.

2010-02-01

The electrodeformation of giant vesicles is studied as a function of their radii and the frequency of the applied AC field. At low frequency the shape is prolate, at sufficiently high frequency it is oblate and at some frequency, fc, the shape changes from prolate to oblate. A linear dependence of the prolate-to-oblate transition inverse frequency, 1/fc, on the vesicle radius is found. The nature of this phenomenon does not change with the variation of both the solution conductivity, σ, and the type of the fluid enclosed by the lipid membrane (water, sucrose or glucose aqueous solution). When σ increases, the value of fc increases while the slope of the line 1/fc(r) decreases. For vesicles in symmetrical conditions (the same conductivity of the inner and the outer solution) a linear dependence between σ and the critical frequency, fc, is obtained for conductivities up to σ=114 μS/cm. For vesicles with sizes below a certain minimum radius, depending on the solution conductivity, no shape transition could be observed.

Origins of microstructural transformations in charged vesicle suspensions: the crowding hypothesis.

PubMed

Seth, Mansi; Ramachandran, Arun; Murch, Bruce P; Leal, L Gary

2014-09-02

It is observed that charged unilamellar vesicles in a suspension can spontaneously deflate and subsequently transition to form bilamellar vesicles, even in the absence of externally applied triggers such as salt or temperature gradients. We provide strong evidence that the driving force for this deflation-induced transition is the repulsive electrostatic pressure between charged vesicles in concentrated suspensions, above a critical effective volume fraction. We use volume fraction measurements and cryogenic transmission electron microscopy imaging to quantitatively follow both the macroscopic and microstructural time-evolution of cationic diC18:1 DEEDMAC vesicle suspensions at different surfactant and salt concentrations. A simple model is developed to estimate the extent of deflation of unilamellar vesicles caused by electrostatic interactions with neighboring vesicles. It is determined that when the effective volume fraction of the suspension exceeds a critical value, charged vesicles in a suspension can experience "crowding" due to overlap of their electrical double layers, which can result in deflation and subsequent microstructural transformations to reduce the effective volume fraction of the suspension. Ordinarily in polydisperse colloidal suspensions, particles interacting via a repulsive potential transform into a glassy state above a critical volume fraction. The behavior of charged vesicle suspensions reported in this paper thus represents a new mechanism for the relaxation of repulsive interactions in crowded situations.

Integral equation methods for vesicle electrohydrodynamics in three dimensions

NASA Astrophysics Data System (ADS)

Veerapaneni, Shravan

2016-12-01

In this paper, we develop a new boundary integral equation formulation that describes the coupled electro- and hydro-dynamics of a vesicle suspended in a viscous fluid and subjected to external flow and electric fields. The dynamics of the vesicle are characterized by a competition between the elastic, electric and viscous forces on its membrane. The classical Taylor-Melcher leaky-dielectric model is employed for the electric response of the vesicle and the Helfrich energy model combined with local inextensibility is employed for its elastic response. The coupled governing equations for the vesicle position and its transmembrane electric potential are solved using a numerical method that is spectrally accurate in space and first-order in time. The method uses a semi-implicit time-stepping scheme to overcome the numerical stiffness associated with the governing equations.

The Flocculating Cationic Polypetide from Moringa oleifera Seeds Damages Bacterial Cell Membranes by Causing Membrane Fusion.

PubMed

Shebek, Kevin; Schantz, Allen B; Sines, Ian; Lauser, Kathleen; Velegol, Stephanie; Kumar, Manish

2015-04-21

A cationic protein isolated from the seeds of the Moringa oleifera tree has been extensively studied for use in water treatment in developing countries and has been proposed for use in antimicrobial and therapeutic applications. However, the molecular basis for the antimicrobial action of this peptide, Moringa oleifera cationic protein (MOCP), has not been previously elucidated. We demonstrate here that a dominant mechanism of MOCP antimicrobial activity is membrane fusion. We used a combination of cryogenic electron microscopy (cryo-EM) and fluorescence assays to observe and study the kinetics of fusion of membranes in liposomes representing model microbial cells. We also conducted cryo-EM experiments on E. coli cells where MOCP was seen to fuse the inner and outer membranes. Coarse-grained molecular dynamics simulations of membrane vesicles with MOCP molecules were used to elucidate steps in peptide adsorption, stalk formation, and fusion between membranes.

Single-step isolation of extracellular vesicles by size-exclusion chromatography

PubMed Central

Böing, Anita N.; van der Pol, Edwin; Grootemaat, Anita E.; Coumans, Frank A. W.; Sturk, Auguste; Nieuwland, Rienk

2014-01-01

Background Isolation of extracellular vesicles from plasma is a challenge due to the presence of proteins and lipoproteins. Isolation of vesicles using differential centrifugation or density-gradient ultracentrifugation results in co-isolation of contaminants such as protein aggregates and incomplete separation of vesicles from lipoproteins, respectively. Aim To develop a single-step protocol to isolate vesicles from human body fluids. Methods Platelet-free supernatant, derived from platelet concentrates, was loaded on a sepharose CL-2B column to perform size-exclusion chromatography (SEC; n=3). Fractions were collected and analysed by nanoparticle tracking analysis, resistive pulse sensing, flow cytometry and transmission electron microscopy. The concentrations of high-density lipoprotein cholesterol (HDL) and protein were measured in each fraction. Results Fractions 9–12 contained the highest concentrations of particles larger than 70 nm and platelet-derived vesicles (46%±6 and 61%±2 of totals present in all collected fractions, respectively), but less than 5% of HDL and less than 1% of protein (4.8%±1 and 0.65%±0.3, respectively). HDL was present mainly in fractions 18–20 (32%±2 of total), and protein in fractions 19–21 (36%±2 of total). Compared to the starting material, recovery of platelet-derived vesicles was 43%±23 in fractions 9–12, with an 8-fold and 70-fold enrichment compared to HDL and protein. Conclusions SEC efficiently isolates extracellular vesicles with a diameter larger than 70 nm from platelet-free supernatant of platelet concentrates. Application SEC will improve studies on the dimensional, structural and functional properties of extracellular vesicles. PMID:25279113

Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant.

PubMed

Alves, Nathan J; Turner, Kendrick B; DiVito, Kyle A; Daniele, Michael A; Walper, Scott A

To facilitate the rapid purification of bacterial outer membrane vesicles (OMVs), we developed two plasmid constructs that utilize a truncated, transmembrane protein to present an exterior histidine repeat sequence. We chose OmpA, a highly abundant porin protein, as the protein scaffold and utilized the lac promoter to allow for inducible control of the epitope-presenting construct. OMVs containing mutant OmpA-His6 were purified directly from Escherichia coli culture media on an immobilized metal affinity chromatography (IMAC) Ni-NTA resin. This enabling technology can be combined with other molecular tools directed at OMV packaging to facilitate the separation of modified/cargo-loaded OMV from their wt counterparts. In addition to numerous applications in the pharmaceutical and environmental remediation industries, this technology can be utilized to enhance basic research capabilities in the area of elucidating endogenous OMV function. Published by Elsevier Masson SAS.

Deformation analysis of vesicles in an alternating-current electric field.

PubMed

Tang, Yu-Gang; Liu, Ying; Feng, Xi-Qiao

2014-08-01

In this paper the shape equation for axisymmetric vesicles subjected to an ac electric field is derived on the basis of the liquid-crystal model. The equilibrium morphology of a lipid vesicle is determined by the minimization of its free energy in coupled mechanical and ac electric fields. Besides elastic bending, the effects of the osmotic pressure difference, surface tension, Maxwell pressure, and flexoelectric and dielectric properties of phospholipid membrane as well are taken into account. The influences of elastic bending, osmotic pressure difference, and surface tension on the frequency-dependent behavior of a vesicle membrane in an ac electric field are examined. The singularity of the ac electric field is also investigated. Our theoretical results of vesicle deformation agree well with previous experimental and numerical results. The present study provides insights into the physical mechanisms underpinning the frequency-dependent morphological evolution of vesicles in the electric and mechanical fields.

Vesicle Stability and Dynamics: An Undergraduate Biochemistry Laboratory

ERIC Educational Resources Information Center

Del Bianco, Cristina; Torino, Domenica; Mansy, Sheref S.

2014-01-01

A laboratory exercise is described that helps students learn about lipid self-assembly by making vesicles under different solution conditions. Concepts covering the chemical properties of different lipids, the dynamics of lipids, and vesicle stability are explored. Further, the described protocol is easy and cheap to implement. One to two…

Biological reference materials for extracellular vesicle studies.

PubMed

Valkonen, S; van der Pol, E; Böing, A; Yuana, Y; Yliperttula, M; Nieuwland, R; Laitinen, S; Siljander, P R M

2017-02-15

Extracellular vesicles (EVs) mediate normal physiological homeostasis and pathological processes by facilitating intercellular communication. Research of EVs in basic science and clinical settings requires both methodological standardization and development of reference materials (RM). Here, we show insights and results of biological RM development for EV studies. We used a three-step approach to find and develop a biological RM. First, a literature search was done to find candidates for biological RMs. Second, a questionnaire was sent to EV researchers querying the preferences for RM and their use. Third, a biological RM was selected, developed, characterized, and evaluated. The responses to the survey demonstrated a clear and recognized need for RM optimized for the calibration of EV measurements. Based on the literature, naturally occurring and produced biological RM, such as virus particles and liposomes, were proposed as RM. However, none of these candidate RMs have properties completely matching those of EVs, such as size and refractive index distribution. Therefore, we evaluated the use of nanoerythrosomes (NanoE), vesicles produced from erythrocytes, as a potential biological RM. The strength of NanoE is their resemblance to EVs. Compared to the erythrocyte-derived EVs (eryEVs), NanoE have similar morphology, a similar refractive index (1.37), larger diameter (70% of the NanoE are over 200nm), and increased positive staining for CD235a and lipids (Di-8-ANEPPS) (58% and 67% in NanoE vs. 21% and 45% in eryEVs, respectively). Altogether, our results highlight the general need to develop and validate new RM with similar physical and biochemical properties as EVs to standardize EV measurements between instruments and laboratories. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Theory of Disk-to-Vesicle Transformation

NASA Astrophysics Data System (ADS)

Li, Jianfeng; Shi, An-Chang

2009-03-01

Self-assembled membranes from amphiphilic molecules, such as lipids and block copolymers, can assume a variety of morphologies dictated by energy minimization of system. The membrane energy is characterized by a bending modulus (κ), a Gaussian modulus (κG), and the line tension (γ) of the edge. Two basic morphologies of membranes are flat disks that minimize the bending energy at the cost of the edge energy, and enclosed vesicles that minimize the edge energy at the cost of bending energy. In our work, the transition from disk to vesicle is studied theoretically using the string method, which is designed to find the minimum energy path (MEP) or the most probable transition path between two local minima of an energy landscape. Previous studies of disk-to-vesicle transition usually approximate the transitional states by a series of spherical cups, and found that the spherical cups do not correspond to stable or meta-stable states of the system. Our calculation demonstrates that the intermediate shapes along the MEP are very different from spherical cups. Furthermore, some of these transitional states can be meta-stable. The disk-to-vesicle transition pathways are governed by two scaled parameters, κG/κ and γR0/4κ, where R0 is the radius of the disk. In particular, a meta-stable intermediate state is predicted, which may correspond to the open morphologies observed in experiments and simulations.

Case report: endoscopic management of seminal vesicle stones with cutaneous fistula.

PubMed

Modi, Pranjal R

2006-06-01

Stones in the seminal vesicle are rare. Open surgery to remove either the seminal vesicle or the stone usually is required. We report a case of seminal-vesicle stones compounded by cutaneous fistula that was treated by ureteroscopy, intracorporeal lithotripsy, and fulguration of the fistulous tract.

Water secretion associated with exocytosis in endocrine cells revealed by micro forcemetry and evanescent wave microscopy.

PubMed Central

Tsuboi, Takashi; Kikuta, Toshiteru; Sakurai, Takashi; Terakawa, Susumu

2002-01-01

It has been a long belief that release of substances from the cell to the extracellular milieu by exocytosis is completed by diffusion of the substances from secretory vesicles through the fusion pore. Involvement of any mechanical force that may be superposed on the diffusion to enhance the releasing process has not been elucidated to date. We tackled this problem in cultured bovine chromaffin cells using direct and sensitive methods: the laser-trap forcemetry and the evanescent-wave fluorescence microscopy. With a laser beam, we trapped a micro bead in the vicinity of a cell (with 1 microm of separation) and observed movements of the bead optically. Electrical stimulation of the cell induced many of rapid and transient movements of the bead in a direction away from the cell surface. Upon the same stimulation, secretory vesicles stained with a fluorescent probe, acridine orange, and excited under the evanescent field illumination, showed a flash-like response: a transient increase in fluorescence intensity associated with a diffuse cloud of brightness, followed by a complete disappearance. These mechanical and fluorescence transients indicate a directional flow of substances. Blockers of the Cl(-) channel suppressed the rates of both responses in a characteristic way but not exocytotic fusion itself. Immunocytochemical studies revealed the presence of Cl(-) and K(+) channels on the vesicle membranes. These results suggest that the externalization of hormones or transmitters upon exocytosis of vesicles is augmented by secretion of water from the vesicle membrane through the widened fusion pore, possibly modulating the rate and reach of the hormone or transmitter release and facilitating transport of the signal molecules in intercellular spaces. PMID:12080110

Vesicle dynamics in a confined Poiseuille flow: from steady state to chaos.

PubMed

Aouane, Othmane; Thiébaud, Marine; Benyoussef, Abdelilah; Wagner, Christian; Misbah, Chaouqi

2014-09-01

Red blood cells (RBCs) are the major component of blood, and the flow of blood is dictated by that of RBCs. We employ vesicles, which consist of closed bilayer membranes enclosing a fluid, as a model system to study the behavior of RBCs under a confined Poiseuille flow. We extensively explore two main parameters: (i) the degree of confinement of vesicles within the channel and (ii) the flow strength. Rich and complex dynamics for vesicles are revealed, ranging from steady-state shapes (in the form of parachute and slipper shapes) to chaotic dynamics of shape. Chaos occurs through a cascade of multiple periodic oscillations of the vesicle shape. We summarize our results in a phase diagram in the parameter plane (degree of confinement and flow strength). This finding highlights the level of complexity of a flowing vesicle in the small Reynolds number where the flow is laminar in the absence of vesicles and can be rendered turbulent due to elasticity of vesicles.

The membrane fusion enigma: SNAREs, Sec1/Munc18 proteins, and their accomplices--guilty as charged?

PubMed

Rizo, Josep; Südhof, Thomas C

2012-01-01

Neurotransmitter release is governed by proteins that have homo-logs in most types of intracellular membrane fusion, including the Sec1/Munc18 protein Munc18-1 and the SNARE proteins syntaxin-1, synaptobrevin/VAMP, and SNAP-25. The SNAREs initiate fusion by forming tight SNARE complexes that bring the vesicle and plasma membranes together. SNARE maintenance in a functional state depends on two chaperone systems (Hsc70/αCSP/SGT and synuclein); defects in these systems lead to neurodegeneration. Munc18-1 binds to an autoinhibitory closed conformation of syntaxin-1, gating formation of SNARE complexes, and also binds to SNARE complexes, which likely underlies the crucial function of Munc18-1 in membrane fusion by an as-yet unclear mechanism. Syntaxin-1 opening is mediated by Munc13s through their MUN domain, which is homologous to diverse tethering factors and may also have a general role in fusion. MUN domain activity is likely modulated in diverse presynaptic plasticity processes that depend on Ca(2+) and RIM proteins, among others.

Effects of exosome-like vesicles on cumulus expansion in pigs in vitro.

PubMed

Matsuno, Yuta; Onuma, Asuka; Fujioka, Yoshie A; Yasuhara, Kazuma; Fujii, Wataru; Naito, Kunihiko; Sugiura, Koji

2017-02-16

Cell-secreted vesicles, such as exosomes, have recently been recognized as mediators of cell communication. A recent study in cattle showed the involvement of exosome-like vesicles in the control of cumulus expansion, a prerequisite process for normal ovulation; however, whether this is the case in other mammalian species is not known. Therefore, this study aimed to examine the presence of exosome-like vesicles in ovarian follicles and their effects on cumulus expansion in vitro in pigs. The presence of exosome-like vesicles in porcine follicular fluid (pFF) was confirmed by transmission electron microscopic observation, the detection of marker proteins, and RNA profiles specific to exosomes. Fluorescently labeled exosome-like vesicles isolated from pFF were incorporated into both cumulus and mural granulosa cells in vitro. Exosome-like vesicles were not capable of inducing cumulus expansion to a degree comparable to that induced by follicle-stimulating hormone (FSH). Moreover, exosome-like vesicles had no significant effects on the expression levels of transcripts required for the normal expansion process (HAS2, TNFAIP6, and PTGS2). Interestingly, FSH-induced expression of HAS2 and TNFAIP6 mRNA, but not of PTGS2 mRNA, was significantly increased by the presence of exosome-like vesicles; however, the degree of FSH-induced expansion was not affected. In addition, porcine exosome-like vesicles had no significant effects on the expansion of mouse cumulus-oocyte complexes. Collectively, the present results suggest that exosome-like vesicles are present in pFF, but they are not efficient in inducing cumulus expansion in pigs.

Effects of exosome-like vesicles on cumulus expansion in pigs in vitro

PubMed Central