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Sample records for ap-3 transport vesicles

  1. Mutation in AP-3 delta in the mocha mouse links endosomal transport to storage deficiency in platelets, melanosomes, and synaptic vesicles.

    PubMed

    Kantheti, P; Qiao, X; Diaz, M E; Peden, A A; Meyer, G E; Carskadon, S L; Kapfhamer, D; Sufalko, D; Robinson, M S; Noebels, J L; Burmeister, M

    1998-07-01

    The mouse mutant mocha, a model for the Hermansky-Pudlak storage pool deficiency syndrome, is characterized by defective platelets, coat and eye color dilution, lysosomal abnormalities, inner ear degeneration, and neurological deficits. Here, we show that mocha is a null allele of the delta subunit of the adaptor-like protein complex AP-3, which is associated with coated vesicles budding from the trans-Golgi network, and that AP-3 is missing in mocha tissues. In mocha brain, the ZnT-3 transporter is reduced, resulting in a lack of zinc-associated Timm historeactivity in hippocampal mossy fibers. Our results demonstrate that the AP-3 complex is responsible for cargo selection to lysosome-related organelles such as melanosomes and platelet dense granules as well as to neurotransmitter vesicles.

  2. Vesicles derived via AP-3 dependent recycling contribute to asynchronous release and influence information transfer

    PubMed Central

    Evstratova, Alesya; Chamberland, Simon; Faundez, Victor; Tóth, Katalin

    2014-01-01

    Summary Action potentials trigger synchronous and asynchronous neurotransmitter release. Temporal properties of both types of release could be altered in an activity-dependent manner. While the effects of activity-dependent changes in synchronous release on postsynaptic signal integration have been studied, the contribution of asynchronous release to information transfer during natural stimulus patterns is unknown. Here we find that during trains of stimulations, asynchronous release contributes to the precision of action potential firing. Our data show that this form of release is selectively diminished in AP-3b2 KO animals, which lack functional neuronal AP-3, an adaptor protein regulating vesicle formation from endosomes generated during bulk endocytosis. We find that in the absence of neuronal AP-3, asynchronous release is attenuated and the activity-dependent increase in the precision of action potential timing is compromised. Lack of asynchronous release decreases the capacity of synaptic information transfer and renders synaptic communication less reliable in response to natural stimulus patterns. PMID:25410111

  3. The AP-3 Complex Required for Endosomal Synaptic Vesicle Biogenesis is Associated with a Casein Kinase Ια-Like Isoform

    PubMed Central

    Faundez, Victor V.; Kelly, Regis B.

    2000-01-01

    The formation of small vesicles is mediated by cytoplasmic coats the assembly of which is regulated by the activity of GTPases, kinases, and phosphatases. A heterotetrameric AP-3 adaptor complex has been implicated in the formation of synaptic vesicles from PC12 endosomes (Faundez et al., 1998). When the small GTPase ARF1 is prevented from hydrolyzing GTP, we can reconstitute AP-3 recruitment to synaptic vesicle membranes in an assembly reaction that requires temperatures above 15°C and the presence of ATP suggesting that an enzymatic step is involved in the coat assembly. We have now found an enzymatic reaction, the phosphorylation of the AP-3 adaptor complex, that is linked with synaptic vesicle coating. Phosphorylation occurs in the β3 subunit of the complex by a kinase similar to casein kinase 1α. The kinase copurifies with neuronal-specific AP-3. In vitro, purified casein kinase I selectively phosphorylates the β3A and β3B subunit at its hinge domain. Inhibiting the kinase hinders the recruitment of AP-3 to synaptic vesicles. The same inhibitors that prevent coat assembly in vitro also inhibit the formation of synaptic vesicles in PC12 cells. The data suggest, therefore, that the mechanism of AP-3-mediated vesiculation from neuroendocrine endosomes requires the phosphorylation of the adaptor complex at a step during or after AP-3 recruitment to membranes. PMID:10930456

  4. Intracellular transport of MHC class II and associated invariant chain in antigen presenting cells from AP-3-deficient mocha mice.

    PubMed

    Sevilla, L M; Richter, S S; Miller, J

    2001-06-15

    MHC class II-restricted antigen presentation requires trafficking of newly synthesized class II-invariant chain complexes from the trans-Golgi network to endosomal, peptide-loading compartments. This transport is mediated by dileucine-like motifs within the cytosolic tail of the invariant chain. Although these signals have been well characterized, the cytosolic proteins that interact with these dileucine signals and mediate Golgi sorting and endosomal transport have not been identified. Recently, an adaptor complex, AP-3, has been identified that interacts with dileucine motifs and mediates endosomal/lysosomal transport in yeast, Drosophila, and mammals. In this report, we have assessed class II-invariant chain trafficking in a strain of mice (mocha) which lacks expression of AP-3. Our studies demonstrate that the lack of AP-3 does not affect the kinetics of invariant chain degradation, the route of class II-invariant chain transport, or the rate and extent of class II-peptide binding as assessed by the generation of SDS-stable dimers. The possible role of other known or unknown adaptor complexes in class II-invariant chain transport is discussed.

  5. A dileucine-like sorting signal directs transport into an AP-3-dependent, clathrin-independent pathway to the yeast vacuole.

    PubMed Central

    Vowels, J J; Payne, G S

    1998-01-01

    Transport of yeast alkaline phosphatase (ALP) to the vacuole depends on the clathrin adaptor-like complex AP-3, but does not depend on proteins necessary for transport through pre-vacuolar endosomes. We have identified ALP sequences that direct sorting into the AP-3-dependent pathway using chimeric proteins containing residues from the ALP cytoplasmic domain fused to sequences from a Golgi-localized membrane protein, guanosine diphosphatase (GDPase). The full-length ALP cytoplasmic domain, or ALP amino acids 1-16 separated from the transmembrane domain by a spacer, directed GDPase chimeric proteins from the Golgi complex to the vacuole via the AP-3 pathway. Mutation of residues Leu13 and Val14 within the ALP cytoplasmic domain prevented AP-3-dependent vacuolar transport of both chimeric proteins and full-length ALP. This Leucine-Valine (LV)-based sorting signal targeted chimeric proteins and native ALP to the vacuole in cells lacking clathrin function. These results identify an LV-based sorting signal in the ALP cytoplasmic domain that directs transport into a clathrin-independent, AP-3-dependent pathway to the vacuole. The similarity of the ALP sorting signal to mammalian dileucine sorting motifs, and the evolutionary conservation of AP-3 subunits, suggests that dileucine-like signals constitute a core element for AP-3-dependent transport to lysosomal compartments in all eukaryotic cells. PMID:9564031

  6. The AP-3 adaptor complex mediates sorting of yeast and mammalian PQ-loop-family basic amino acid transporters to the vacuolar/lysosomal membrane

    PubMed Central

    Llinares, Elisa; Barry, Abdoulaye Oury; André, Bruno

    2015-01-01

    The limiting membrane of lysosomes in animal cells and that of the vacuole in yeast include a wide variety of transporters, but little is known about how these proteins reach their destination membrane. The mammalian PQLC2 protein catalyzes efflux of basic amino acids from the lysosome, and the similar Ypq1, −2, and −3 proteins of yeast perform an equivalent function at the vacuole. We here show that the Ypq proteins are delivered to the vacuolar membrane via the alkaline phosphatase (ALP) trafficking pathway, which requires the AP-3 adaptor complex. When traffic via this pathway is deficient, the Ypq proteins pass through endosomes from where Ypq1 and Ypq2 properly reach the vacuolar membrane whereas Ypq3 is missorted to the vacuolar lumen via the multivesicular body pathway. When produced in yeast, PQLC2 also reaches the vacuolar membrane via the ALP pathway, but tends to sort to the vacuolar lumen if AP-3 is defective. Finally, in HeLa cells, inhibiting the synthesis of an AP-3 subunit also impairs sorting of PQLC2 to lysosomes. Our results suggest the existence of a conserved AP-3-dependent trafficking pathway for proper delivery of basic amino acid exporters to the yeast vacuole and to lysosomes of human cells. PMID:26577948

  7. Calcyon, a mammalian specific NEEP21 family member, interacts with adaptor protein complex 3 (AP-3) and regulates targeting of AP-3 cargoes.

    PubMed

    Muthusamy, Nagendran; Faundez, Victor; Bergson, Clare

    2012-10-01

    Calcyon is a neural enriched, single transmembrane protein that interacts with clathrin light chain and stimulates clathrin assembly and clathrin-mediated endocytosis. A similar property is shared by the heterotetrameric adaptor protein (AP) complexes AP-1, AP-2, and AP-3 which recruit cargoes for insertion into clathrin coated transport vesicles. Here we report that AP medium (μ) subunits interact with a YXXØ-type tyrosine motif located at residues 133-136 in the cytoplasmic domain of calcyon. Site specific mutagenesis of the critical tyrosine and bulky hydrophobic residues tyrosine 133 and methionine 136 preferentially abrogated binding of the ubiquitous and neuronal isoforms of μ3, and also impacted μ1 and μ2 binding to a lesser degree. The relevance of these interactions was explored in vivo using mice harboring null alleles of calcyon. As seen in the mutagenesis studies, calcyon deletion in mice preferentially altered the subcellular distribution of AP-3 suggesting that calcyon could regulate membrane-bound pools of AP-3 and AP-3 function. To test this hypothesis, we focused on the hilar region of hippocampus, where levels of calcyon, AP-3, and AP-3 cargoes are abundant. We analyzed brain cryosections from control and calcyon null mice for zinc transporter 3 (ZnT3), and phosphatidylinositol-4-kinase type II alpha (PI4KIIα), two well-defined AP-3 cargoes. Confocal microscopy indicated that ZnT3 and PI4KIIα are significantly reduced in the hippocampal mossy fibers of calcyon knock-out brain, a phenotype previously described in AP-3 deficiencies. Altogether, our data suggest that calcyon directly interacts with μ3A and μ3B, and regulates the subcellular distribution of AP-3 and the targeting of AP-3 cargoes.

  8. CALCYON, A MAMMALIAN SPECIFIC NEEP21 FAMILY MEMBER, INTERACTS WITH ADAPTOR PROTEIN COMPLEX 3 (AP-3) AND REGULATES TARGETING OF AP-3 CARGOES

    PubMed Central

    Muthusamy, Nagendran; Faundez, Victor; Bergson, Clare

    2013-01-01

    Calcyon is a neural enriched, single transmembrane protein that interacts with clathrin light chain (CLC) and stimulates clathrin assembly and clathrin mediated endocytosis (CME). A similar property is shared by the heterotetrameric adaptor protein (AP) complexes AP-1, AP-2, and AP-3 which recruit cargoes for insertion into clathrin coated transport vesicles. Here we report that AP medium (µ) subunits interact with a YXXØ-type tyrosine motif located at residues 133–136 in the cytoplasmic domain of calcyon. Site specific mutagenesis of the critical tyrosine and bulky hydrophobic residues tyrosine 133 and methionine 136 preferentially abrogated binding of the ubiquitous and neuronal isoforms of µ3, and also impacted µ1 and µ2 binding but to a lesser degree. The relevance of these interactions was explored in vivo using mice harboring null-alleles of calcyon. As seen in the mutagenesis studies, calcyon deletion in mice preferentially altered the subcellular distribution of AP-3 suggesting that calcyon could regulate membrane-bound pools of AP-3 and AP-3 function. To test this hypothesis, we focused on the hilar region of hippocampus, where levels of calcyon, AP-3, and AP-3 cargoes are abundant. We analyzed brain cryosections from control and calcyon null mice for zinc transporter 3 (ZnT3), and phosphatidylinositol-4-kinase type II alpha (PI4KIIα), two well-defined AP-3 cargoes. Confocal microscopy indicated that ZnT3 and PI4KIIα are significantly reduced in the hippocampal mossy fibers of calcyon knock-out brain, a phenotype previously described in AP-3 deficiencies. Altogether, our data suggest that calcyon directly interacts with µ3A and µ3B, and regulates the subcellular distribution of AP-3 and the targeting of AP-3 cargoes. PMID:22650988

  9. 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

  10. An AP-3-dependent mechanism drives synaptic-like microvesicle biogenesis in pancreatic islet beta-cells.

    PubMed

    Suckow, Arthur T; Craige, Branch; Faundez, Victor; Cain, William J; Chessler, Steven D

    2010-07-01

    Pancreatic islet beta-cells contain synaptic-like microvesicles (SLMVs). The origin, trafficking, and role of these SLMVs are poorly understood. In neurons, synaptic vesicle (SV) biogenesis is mediated by two different cytosolic adaptor protein complexes, a ubiquitous AP-2 complex and the neuron-specific AP-3B complex. Mice lacking AP-3B subunits exhibit impaired GABAergic (inhibitory) neurotransmission and reduced neuronal vesicular GABA transporter (VGAT) content. Since beta-cell maturation and exocytotic function seem to parallel that of the inhibitory synapse, we predicted that AP-3B-associated vesicles would be present in beta-cells. Here, we test the hypothesis that AP-3B is expressed in islets and mediates beta-cell SLMV biogenesis. A secondary aim was to test whether the sedimentation properties of INS-1 beta-cell microvesicles are identical to those of bona fide SLMVs isolated from PC12 cells. Our results show that the two neuron-specific AP-3 subunits beta3B and mu3B are expressed in beta-cells, the first time these proteins have been found to be expressed outside the nervous system. We found that beta-cell SLMVs share the same sedimentation properties as PC12 SLMVs and contain SV proteins that sort specifically to AP-3B-associated vesicles in the brain. Brefeldin A, a drug that interferes with AP-3-mediated SV biogenesis, inhibits the delivery of AP-3 cargoes to beta-cell SLMVs. Consistent with a role for AP-3 in the biogenesis of GABAergic SLMV in beta-cells, INS-1 cell VGAT content decreases upon inhibition of AP-3 delta-subunit expression. Our findings suggest that beta-cells and neurons share molecules and mechanisms important for mediating the neuron-specific membrane trafficking pathways that underlie synaptic vesicle formation.

  11. Altered retinal cell differentiation in the AP-3 delta mutant (Mocha) mouse.

    PubMed

    Baguma-Nibasheka, Mark; Kablar, Boris

    2009-11-01

    Adaptor-related protein complex 3 delta 1 (Ap3d1) encodes the delta 1 subunit of an adaptor protein regulating intracellular vesicle-mediated transport, and the Ap3d-deletion mutant (Mocha) mouse undergoes rapid photoreceptor degeneration leading to blindness soon after birth. Previous microarray analysis revealed Ap3d down-regulation in the retina of mouse embryos specifically lacking cholinergic amacrine cells as a result of the absence of skeletal musculature. To investigate the role of Ap3d in the determination of retinal cell fate, the present study examined retinal morphology in newborn Ap3d-/- mice. The Ap3d-/- retina showed a complete absence of cholinergic amacrine cells and a decrease in parvalbumin-expressing amacrine cells and syntaxin- and VC1.1-expressing amacrine precursor cells, but had a normal number of cell layers and number of cells in each layer with no detectable difference in cell proliferation or apoptosis. These findings indicate that, despite having no apparent effect on the basic spatial organization of the retina at this stage of development, Ap3d could be involved in the regulation of progenitor cell competence and the eventual ratio of resulting differentiated cells. Finding the mouse mutant which phenocopies the eye defect seen in fetuses with no striated muscle was accomplished by the Systematic Subtractive Microarray Analysis Approach (SSMAA), explained in the discussion section.

  12. Glucocorticoid affects dendritic transport of BDNF-containing vesicles.

    PubMed

    Adachi, Naoki; Numakawa, Tadahiro; Nakajima, Shingo; Fukuoka, Masashi; Odaka, Haruki; Katanuma, Yusuke; Ooshima, Yoshiko; Hohjoh, Hirohiko; Kunugi, Hiroshi

    2015-08-04

    Brain-derived neurotrophic factor (BDNF) is essential for neuronal survival, differentiation, and functions in the central nervous system (CNS). Because BDNF protein is sorted into secretory vesicles at the trans-Golgi network in the cell body after translation, transport of BDNF-containing vesicles to the secretion sites is an important process for its function. Here we examined the effect of dexamethasone (DEX), a synthetic glucocorticoid, on BDNF-containing vesicle transport and found that DEX decreased the proportion of stationary vesicles and increased velocity of the microtubule-based vesicle transport in dendrites of cortical neurons. Furthermore, DEX increased huntingtin (Htt) protein levels via glucocorticoid receptor (GR) activation, and reduction in the amount of Htt by a specific shRNA reversed the action of DEX on BDNF vesicle transport. Given that Htt protein is a positive regulator for the microtubule-dependent vesicular transport in neurons, our data suggest that glucocorticoid stimulates BDNF vesicle transport through upregulation of Htt protein levels.

  13. Characteristics of endoplasmic reticulum-derived transport vesicles

    PubMed Central

    1994-01-01

    We have isolated vesicles that mediate protein transport from the ER to Golgi membranes in perforated yeast. These vesicles, which form de novo during in vitro incubations, carry lumenal and membrane proteins that include core-glycosylated pro-alpha-factor, Bet1, Sec22, and Bos1, but not ER-resident Kar2 or Sec61 proteins. Thus, lumenal and membrane proteins in the ER are sorted prior to transport vesicle scission. Inhibition of Ypt1p-function, which prevents newly formed vesicles from docking to cis-Golgi membranes, was used to block transport. Vesicles that accumulate are competent for fusion with cis-Golgi membranes, but not with ER membranes, and thus are functionally committed to vectorial transport. A 900-fold enrichment was developed using differential centrifugation and a series of velocity and equilibrium density gradients. Electron microscopic analysis shows a uniform population of 60 nm vesicles that lack peripheral protein coats. Quantitative Western blot analysis indicates that protein markers of cytosol and cellular membranes are depleted throughout the purification, whereas the synaptobrevin-like Bet1, Sec22, and Bos1 proteins are highly enriched. Uncoated ER-derived transport vesicles (ERV) contain twelve major proteins that associate tightly with the membrane. The ERV proteins may represent abundant cargo and additional targeting molecules. PMID:8063853

  14. Recognition and tethering of transport vesicles at the Golgi apparatus.

    PubMed

    Witkos, Tomasz M; Lowe, Martin

    2017-02-23

    The Golgi apparatus occupies a central position within the secretory pathway where it is a hub for vesicle trafficking. Distinct classes of transport vesicles traffic diverse cargoes into and out of this organelle, as well as between the different Golgi subcompartments. A key feature of Golgi trafficking is the specific recognition of transport vesicles at the different regions of the Golgi apparatus, required for the correct cargo delivery. Specificity is ensured by coiled-coil golgins and multi-subunit tethering complexes (MTCs), which act together to capture vesicles and promote their subsequent fusion with the Golgi membrane. In this review we discuss our current understanding of how golgins and MTCs function together to mediate the specific recognition of vesicles at the Golgi apparatus.

  15. 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

  16. Cholinergic synaptic vesicle heterogeneity: evidence for regulation of acetylcholine transport

    SciTech Connect

    Gracz, L.M.; Wang, W.; Parsons, S.M.

    1988-07-12

    Crude cholinergic synaptic vesicles from a homogenate of the electric organ of Torpedo californica were centrifuged to equilibrium in an isosmotic sucrose density gradient. The classical VP/sub 1/ synaptic vesicles banding at 1.055 g/mL actively transported (/sup 3/H)acetylcholine (AcCh). An organelle banding at about 1.071 g/mL transported even more (/sup 3/H)AcCh. Transport by both organelles was inhibited by the known AcCh storage blockers trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183) and nigericin. Relative to VP/sub 1/ vesicles the denser organelle was slightly smaller as shown by size-exclusion chromatography. It is concluded that the denser organelle corresponds to the recycling VP/sub 2/ synaptic vesicle originally described in intact Torpedo marmorata electric organ. The properties of the receptor for vesamicol were studied by measuring binding of (/sup 3/H)vesamicol, and the amount of SV2 antigen characteristic of secretory vesicles was assayed with a monoclonal antibody directed against it. Relative to VP/sub 1/ vesicles the VP/sub 2/ vesicles had a ratio of (/sup 3/H)AcCh transport activity to vesamicol receptor concentration that typically was 4-7-fold higher, whereas the ratio of SV2 antigen concentration to vesamicol receptor concentration was about 2-fold higher. The Hill coefficients ..cap alpha../sub H/ and equilibrium dissociation constants K for vesamicol binding to VP/sub 1/ and VP/sub 2/ vesicles were essentially the same. The positive Hill coefficient suggests that the vesamicol receptor exists as a homotropic oligomeric complex. The results demonstrate that VP/sub 1/ and VP/sub 2/ synaptic vesicles exhibit functional differences in the AcCh transport system, presumably as a result of regulatory phenomena.

  17. Directed vesicle transport by diffusio-osmosis

    NASA Astrophysics Data System (ADS)

    Michler, D.; Shahidzadeh, N.; Sprik, R.; Bonn, D.

    2015-04-01

    We present a study on surfactant vesicles that spontaneously move towards an oil droplet that is deposited on a glass substrate. Tracer particles in the surfactant solution show that the motion is not self-propelled: the vesicles are entrained by a macroscopic hydrodynamic flow. Measurements of the flow velocity suggest that the flow is of diffusio-osmotic nature. The surfactant is observed to move into the oil phase which creates a gradient in ion concentration in the vicinity of the droplet. As the diffusion coefficients of the surfactant's co- and counter-ions differ, a charge separation takes place and an electric field arises. This electric field then generates a hydrodynamic flow along the charged glass substrate in which the vesicles are entrained.

  18. Fewer active motors per vesicle may explain slowed vesicle transport in chick motoneurons after three days in vitro.

    PubMed

    Macosko, Jed C; Newbern, Jason M; Rockford, Jean; Chisena, Ernest N; Brown, Charlotte M; Holzwarth, George M; Milligan, Carol E

    2008-05-23

    Vesicle transport in cultured chick motoneurons was studied over a period of 3 days using motion-enhanced differential interference contrast (MEDIC) microscopy, an improved version of video-enhanced DIC. After 3 days in vitro (DIV), the average vesicle velocity was about 30% less than after 1 DIV. In observations at 1, 2 and 3 DIV, larger vesicles moved more slowly than small vesicles, and retrograde vesicles were larger than anterograde vesicles. The number of retrograde vesicles increased relative to anterograde vesicles after 3 DIV, but this fact alone could not explain the decrease in velocity, since the slowing of vesicle transport in maturing motoneurons was observed independently for both anterograde and retrograde vesicles. In order to better understand the slowing trend, the distance vs. time trajectories of individual vesicles were examined at a frame rate of 8.3/s. Qualitatively, these trajectories consisted of short (1-2 s) segments of constant velocity, and the changes in velocity between segments were abrupt (<0.2 s). The trajectories were therefore fit to a series of connected straight lines. Surprisingly, the slopes of theses lines, i.e. the vesicle velocities, were often found to be multiples of ~0.6 mum/s. The velocity histogram showed multiple peaks, which, when fit with Gaussians using a least squares minimization, yielded an average spacing of 0.57 mum/s (taken as the slope of a fit to peak position vs. peak number, R(2)=0.994). We propose that the abrupt velocity changes occur when 1 or 2 motors suddenly begin or cease actively participating in vesicle transport. Under this hypothesis, the decrease in average vesicle velocity observed for maturing motoneurons is due to a decrease in the average number of active motors per vesicle.

  19. Mapping of the spontaneous deletion in the Ap3d1 gene of mocha mice: fast and reliable genotyping

    PubMed Central

    Drasbek, Kim Ryun; Holm, Mai Marie; Delenclos, Marion; Jensen, Kimmo

    2008-01-01

    Background The mocha mouse carries a spontaneous deletion in the Ap3d1 gene, encoding the delta 1 subunit of the adaptor related protein complex 3, (Ap3d1), and subsequently lack the expression of functional AP-3. This leads to a deficiency in vesicle transport and storage, which affects neurotransmitter vesicle turnover and release in the central nervous system. Since the genomic sequence of the Ap3d1 gene of mocha mouse is not known, precise mapping of the deletion as well as reliable genotyping protocols are lacking. Findings We sequenced the Ap3d1 gene (HGNC GeneID: 8943) around the deletion site in the mocha mouse and revealed a 10639 bp deletion covering exon 2 to 6. Subsequently, new PCR primers were designed yielding a reliable genotyping protocol of both newborn and adult tissue. To examine the genotypes further, hippocampal neurons were cultured from mocha and control mice. Patch-clamp recordings showed that mocha neurons had a higher input resistance, and that autaptic EPSC in mocha cultures depressed faster and stronger as compared with control cultures. Conclusion Our study reports the sequence of the deleted part of the Ap3d1 gene in mocha mice, as well as a reliable PCR-based genotyping protocol. We cultured hippocampal neurons from control and mocha mice, and found a difference in input resistance of the neurons, and in the synaptic short-term plasticity of glutamatergic autapses showing a larger synaptic depression than controls. The described procedures may be useful for the future utilization of the mocha mouse as a model of defective vesicle biogenesis. Importantly, as genotyping by eye color is complicated in newborn mice, the designed protocol is so fast and reliable that newborn mice could rapidly be genotyped and hippocampal neurons dissociated and cultured, which is normally best done at P0-P2. PMID:19032734

  20. Vesicular Glutamate Transporter 1 Orchestrates Recruitment of Other Synaptic Vesicle Cargo Proteins during Synaptic Vesicle Recycling*

    PubMed Central

    Pan, Ping-Yue; Marrs, Julia; Ryan, Timothy A.

    2015-01-01

    A long standing question in synaptic physiology is how neurotransmitter-filled vesicles are rebuilt after exocytosis. Among the first steps in this process is the endocytic retrieval of the transmembrane proteins that are enriched in synaptic vesicles (SVs). At least six types of transmembrane proteins must be recovered, but the rules for how this multiple cargo selection is accomplished are poorly understood. Among these SV cargos is the vesicular glutamate transporter (vGlut). We show here that vGlut1 has a strong influence on the kinetics of retrieval of half of the known SV cargos and that specifically impairing the endocytosis of vGlut1 in turn slows down other SV cargos, demonstrating that cargo retrieval is a collective cargo-driven process. Finally, we demonstrate that different cargos can be retrieved in the same synapse with different kinetics, suggesting that additional post-endocytic sorting steps likely occur in the nerve terminal. PMID:26224632

  1. Self-propelling vesicles define glycolysis as the minimal energy machinery for neuronal transport

    PubMed Central

    Hinckelmann, María-Victoria; Virlogeux, Amandine; Niehage, Christian; Poujol, Christel; Choquet, Daniel; Hoflack, Bernard; Zala, Diana; Saudou, Frédéric

    2016-01-01

    The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) facilitates fast axonal transport in neurons. However, given that GAPDH does not produce ATP, it is unclear whether glycolysis per se is sufficient to propel vesicles. Although many proteins regulating transport have been identified, the molecular composition of transported vesicles in neurons has yet to be fully elucidated. Here we selectively enrich motile vesicles and perform quantitative proteomic analysis. In addition to the expected molecular motors and vesicular proteins, we find an enrichment of all the glycolytic enzymes. Using biochemical approaches and super-resolution microscopy, we observe that most glycolytic enzymes are selectively associated with vesicles and facilitate transport of vesicles in neurons. Finally, we provide evidence that mouse brain vesicles produce ATP from ADP and glucose, and display movement in a reconstituted in vitro transport assay of native vesicles. We conclude that transport of vesicles along microtubules can be autonomous. PMID:27775035

  2. Phospholipid flippases: building asymmetric membranes and transport vesicles

    PubMed Central

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

    2012-01-01

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

  3. Mechanisms of calcium transport in human colonic basolateral membrane vesicles.

    PubMed

    Saksena, Seema; Ammar, Mohammad S; Tyagi, Sangeeta; Elsharydah, Ahmed; Gill, Ravinder K; Ramaswamy, Krishnamurthy; Dudeja, Pradeep K

    2002-10-01

    Human colon has been suggested to play an important role in calcium absorption especially after extensive disease or resection of the small intestine. We have previously demonstrated the presence of a carrier-mediated calcium uptake mechanism in the human colonic luminal membrane vesicles. Current studies were, therefore, undertaken to investigate the mechanism(s) of calcium exit across the basolateral membrane domain of the human colon. Human colonic basolateral membrane vesicles (BLMVs) were isolated and purified from mucosal scrapings of organ donor colons, utilizing a technique developed in our laboratory. 45Ca uptake was measured by a rapid filtration technique. 45Ca uptake represented transport into the intravesicular space as evidenced by an osmolarity study and by the demonstration of Ca2' efflux from calcium preloaded vesicles by Ca2+ ionophore A23187. Calcium uptake was stimulated by Mg2+ ATP. The kinetic parameters for ATP-dependent Ca2+ uptake revealed saturation kinetics with Michaelis constant (Km) of 0.22 +/- 0.04 microM and a maximum rate of uptake (Vmax) of 0.38 +/- 0.12 nmol/mg protein/min. The Km of ATP concentration required for half maximal Ca2+ uptake was 0.39 +/- 0.04 mM. ATP-stimulated calcium uptake into these vesicles was further stimulated in the presence of calmodulin and was inhibited by calmodulin antagonist, trifluoperazine. Uptake of 45Ca into BLMVs was markedly inhibited by cis-Na+ but was significantly stimulated by trans-Na+ (40-50% stimulation). Our results demonstrate the presence of a Mg2+/ATP-dependent calmodulin-regulated Ca2+ transport system and a Na+-Ca2+ exchange process in the human colonic basolateral membranes.

  4. Rab proteins: The key regulators of intracellular vesicle transport

    SciTech Connect

    Bhuin, Tanmay; Roy, Jagat Kumar

    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 by 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.

  5. Rab proteins: the key regulators of intracellular vesicle transport.

    PubMed

    Bhuin, Tanmay; Roy, Jagat Kumar

    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 by 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.

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

    SciTech Connect

    Goldsmith, M.H.M.

    1989-01-01

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

  7. Astrocyte VAMP3 vesicles undergo Ca2+-independent cycling and modulate glutamate transporter trafficking

    PubMed Central

    Li, Dongdong; Hérault, Karine; Zylbersztejn, Kathleen; Lauterbach, Marcel A; Guillon, Marc; Oheim, Martin; Ropert, Nicole

    2015-01-01

    Key points Mouse cortical astrocytes express VAMP3 but not VAMP2. VAMP3 vesicles undergo Ca2+-independent exo- and endocytotic cycling at the plasma membrane. VAMP3 vesicle traffic regulates the recycling of plasma membrane glutamate transporters. cAMP modulates VAMP3 vesicle cycling and glutamate uptake. Abstract Previous studies suggest that small synaptic-like vesicles in astrocytes carry vesicle-associated vSNARE proteins, VAMP3 (cellubrevin) and VAMP2 (synaptobrevin 2), both contributing to the Ca2+-regulated exocytosis of gliotransmitters, thereby modulating brain information processing. Here, using cortical astrocytes taken from VAMP2 and VAMP3 knock-out mice, we find that astrocytes express only VAMP3. The morphology and function of VAMP3 vesicles were studied in cultured astrocytes at single vesicle level with stimulated emission depletion (STED) and total internal reflection fluorescence (TIRF) microscopies. We show that VAMP3 antibodies label small diameter (∼80 nm) vesicles and that VAMP3 vesicles undergo Ca2+-independent exo-endocytosis. We also show that this pathway modulates the surface expression of plasma membrane glutamate transporters and the glutamate uptake by astrocytes. Finally, using pharmacological and optogenetic tools, we provide evidence suggesting that the cytosolic cAMP level influences astrocytic VAMP3 vesicle trafficking and glutamate transport. Our results suggest a new role for VAMP3 vesicles in astrocytes. PMID:25864578

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

    SciTech Connect

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

    1987-09-01

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

  9. AP-3 regulates PAR1 ubiquitin-independent MVB/lysosomal sorting via an ALIX-mediated pathway

    PubMed Central

    Dores, Michael R.; Paing, May M.; Lin, Huilan; Montagne, William A.; Marchese, Adriano; Trejo, JoAnn

    2012-01-01

    The sorting of signaling receptors within the endocytic system is important for appropriate cellular responses. After activation, receptors are trafficked to early endosomes and either recycled or sorted to lysosomes and degraded. Most receptors trafficked to lysosomes are modified with ubiquitin and recruited into an endosomal subdomain enriched in hepatocyte growth factor–regulated tyrosine kinase substrate (HRS), a ubiquitin-binding component of the endosomal-sorting complex required for transport (ESCRT) machinery, and then sorted into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs)/lysosomes. However, not all receptors use ubiquitin or the canonical ESCRT machinery to sort to MVBs/lysosomes. This is exemplified by protease-activated receptor-1 (PAR1), a G protein–coupled receptor for thrombin, which sorts to lysosomes independent of ubiquitination and HRS. We recently showed that the adaptor protein ALIX binds to PAR1, recruits ESCRT-III, and mediates receptor sorting to ILVs of MVBs. However, the mechanism that initiates PAR1 sorting at the early endosome is not known. We now report that the adaptor protein complex-3 (AP-3) regulates PAR1 ubiquitin-independent sorting to MVBs through an ALIX-dependent pathway. AP-3 binds to a PAR1 cytoplasmic tail–localized tyrosine-based motif and mediates PAR1 lysosomal degradation independent of ubiquitination. Moreover, AP-3 facilitates PAR1 interaction with ALIX, suggesting that AP-3 functions before PAR1 engagement of ALIX and MVB/lysosomal sorting. PMID:22833563

  10. 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

  11. Vesicle Photonics

    SciTech Connect

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

    2013-04-03

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

  12. Calcium uptake by intestinal brush border membrane vesicles. Comparison with in vivo calcium transport.

    PubMed Central

    Schedl, H P; Wilson, H D

    1985-01-01

    In prior studies, we examined kinetics of steady state in vivo transepithelial calcium transport in rat and hamster. The present studies related calcium uptake by the brush border to in vivo transport. We measured calcium uptake by brush border membrane vesicles from the two species. In the rat, our prior in vivo studies had shown that (a) calcium transport was mediated, (b) no nonmediated component was detectable, and (c) Vmax was 2.5 times greater in proximal than distal small intestine. In brush border membrane vesicles from the rat, Vmax for the saturable component of calcium uptake was again 2.5 times greater in proximal than distal intestine. Contrasting with in vivo studies, a major nonsaturable component was present in vesicles from proximal and distal small intestine. In the hamster, our previous in vivo studies had shown (1) both mediated and nonmediated components of calcium transport, (2) greater nonmediated transport in proximal than distal small intestines, and (3) Vmax for calcium transport twice as great in distal as in proximal small intestine. In the present study with brush border membrane vesicles from hamster, Vmax for saturable calcium transport was again twice as great in distal as in proximal small intestine. However, nonsaturable calcium transport rates relative to saturable rates were much greater with vesicles than in in vivo studies, and were greater in vesicles from distal than proximal small intestine. Since rates of saturable calcium uptake by brush border membrane vesicles parallel corresponding in vivo mediated transport rates, we conclude that the segmental rates of calcium transport in rat and hamster could be determined by brush border function. PMID:2997294

  13. A Novel Pulse-Chase Paradigm to Visualize the Trafficking of Transport Vesicles in Neurons

    NASA Astrophysics Data System (ADS)

    Al-Bassam, Sarmad

    In neurons transmembrane proteins are targeted to dendrites in vesicles that traffic solely within the somatodendritic compartment. How these vesicles are retained within the somatodendritic domain is unknown. Here we adapt a novel pulse chase system that allows synchronous release of exogenous transmembrane proteins from the endoplasmic reticulum using FKBP12 and Rapamycin. We demonstrate proof-of-concept and establish protein trafficking controls in incremental steps. We demonstrate the utility of this approach in studying protein trafficking and establish parameters for analysis of time-lapse images. We implement this novel pulse-chase strategy to track the movements of post-Golgi transport vesicles. Surprisingly, we found that post-Golgi vesicles carrying dendritic proteins were equally likely to enter axons and dendrites. However, once such vesicles entered the axon they very rarely moved beyond the axon initial segment, but instead either halted or reversed direction in an actin and Myosin Va-dependent manner. In contrast, vesicles carrying either an axonal or a nonspecifically localized protein only rarely halted or reversed and instead generally proceeded to the distal axon. Thus, our results are consistent with the axon initial segment behaving as a vesicle filter that mediates the differential trafficking of transport vesicles.

  14. Visualization and quantification of transmembrane ion transport into giant unilamellar vesicles.

    PubMed

    Valkenier, Hennie; López Mora, Néstor; Kros, Alexander; Davis, Anthony P

    2015-02-09

    Transmembrane ion transporters (ionophores) are widely investigated as supramolecular agents with potential for biological activity. Tests are usually performed in synthetic membranes that are assembled into large unilamellar vesicles (LUVs). However transport must be followed through bulk properties of the vesicle suspension, because LUVs are too small for individual study. An alternative approach is described whereby ion transport can be revealed and quantified through direct observation. The method employs giant unilamellar vesicles (GUVs), which are 20-60 μm in diameter and readily imaged by light microscopy. This allows characterization of individual GUVs containing transporter molecules, followed by studies of transport through fluorescence emission from encapsulated indicators. The method provides new levels of certainty and relevance, given that the GUVs are similar in size to living cells. It has been demonstrated using a highly active anion carrier, and should aid the development of compounds for treating channelopathies such as cystic fibrosis.

  15. Active transport of vesicles in neurons is modulated by mechanical tension

    NASA Astrophysics Data System (ADS)

    Ahmed, Wylie W.; Saif, Taher A.

    2014-03-01

    Effective intracellular transport of proteins and organelles is critical in cells, and is especially important for ensuring proper neuron functionality. In neurons, most proteins are synthesized in the cell body and must be transported through thin structures over long distances where normal diffusion is insufficient. Neurons transport subcellular cargo along axons and neurites through a stochastic interplay of active and passive transport. Mechanical tension is critical in maintaining proper function in neurons, but its role in transport is not well understood. To this end, we investigate the active and passive transport of vesicles in Aplysia neurons while changing neurite tension via applied strain, and quantify the resulting dynamics. We found that tension in neurons modulates active transport of vesicles by increasing the probability of active motion, effective diffusivity, and induces a retrograde bias. We show that mechanical tension modulates active transport processes in neurons and that external forces can couple to internal (subcellular) forces and change the overall transport dynamics.

  16. Transport Vesicle Tethering at the Trans Golgi Network: Coiled Coil Proteins in Action

    PubMed Central

    Cheung, Pak-yan P.; Pfeffer, Suzanne R.

    2016-01-01

    The Golgi complex is decorated with so-called Golgin proteins that share a common feature: a large proportion of their amino acid sequences are predicted to form coiled-coil structures. The possible presence of extensive coiled coils implies that these proteins are highly elongated molecules that can extend a significant distance from the Golgi surface. This property would help them to capture or trap inbound transport vesicles and to tether Golgi mini-stacks together. This review will summarize our current understanding of coiled coil tethers that are needed for the receipt of transport vesicles at the trans Golgi network (TGN). How do long tethering proteins actually catch vesicles? Golgi-associated, coiled coil tethers contain numerous binding sites for small GTPases, SNARE proteins, and vesicle coat proteins. How are these interactions coordinated and are any or all of them important for the tethering process? Progress toward understanding these questions and remaining, unresolved mysteries will be discussed. PMID:27014693

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

    SciTech Connect

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

    1982-01-01

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

  18. The role of vesicles in the transport of ferritin through frog endothelium.

    PubMed Central

    Clough, G; Michel, C C

    1981-01-01

    1. The transport of ferritin molecules by endothelial cell vesicles has been quantitatively investigated by electron microscopy. Single mesenteric capillaries of pithed frogs were perfused with solutions containing 6.7 g ferritin 100 ml.-1 for known periods before fixation in situ with osmium tetroxide. 2. Two series of experiments were carried out: in the first series the perfusate contained bovine serum albumin (1.0 g 100 ml.-1); in the second series the perfusate contained no protein other than the ferritin. To assess the molecular radius of ferritin in solution, the free diffusion coefficient of ferritin was measured in the presence and absence of albumin. 3. The free diffusion coefficient of ferritin in saline solution (110 m-mole 1.-1) was found to be 0.35 X 10(-6) cm2 sec-1 at 21 degrees C and was not affected by the presence of bovine serum albumin. This indicates that there is no significant binding of albumin to ferritin in solution and yields a value for the Stokes-Einstein radius of ferritin of 6.1 nm. 4. In all perfusion experiments the percentage of luminal vesicles containing ferritin exceeded the percentage of labelled cytoplasmic vesicles, which in turn exceeded the percentage of labelled abluminal vesicles. 5. Labelling of all vesicle populations was seen after perfusions lasting less than 1 sec. At this time luminal vesicles were more heavily labelled in the absence of albumin. 6. The labelling of luminal vesicles increased with lengthening perfusion times up to 30-40 sec, after which steady levels of labelling were achieved. The rate of rise in luminal labelling and the steady-state levels reached were both greater in the absence of albumin. By contrast cytoplasmic labelling increased above its initial value only after perfusions of longer than 10 sec. 7. In the steady state, labelled cytoplasmic vesicles contained, on average, fewer ferritin molecules than labelled luminal vesicles. This finding is inconsistent with translocation of labelled

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

    SciTech Connect

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

    1986-11-01

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

  20. Light-activated amino acid transport in Halobacterium halobium envelope vesicles

    NASA Technical Reports Server (NTRS)

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

    1977-01-01

    Vesicles prepared from Halobacterium halobium cell envelopes accumulate amino acids in response to light-induced electrical and chemical gradients. Nineteen of 20 commonly occurring amino acids have been shown to be actively accumulated by these vesicles in response to illumination or in response to an artificially created Na+ gradient. On the basis of shared common carriers the transport systems can be divided into eight classes, each responsible for the transport of one or several amino acids: arginine, lysine, histidine; asparagine, glutamine; alanine, glycine, threonine, serine; leucine, valine, isoleucine, methionine; phenylalanine, tyrosine, tryptophan; aspartate; glutamate; proline. Available evidence suggests that these carriers are symmetrical in that amino acids can be transported equally well in both directions across the vesicle membranes. A tentative working model to account for these observations is presented.

  1. Chloride ion transport into pig jejunal brush-border membrane vesicles.

    PubMed Central

    Forsyth, G W; Gabriel, S E

    1988-01-01

    1. This study was carried out to determine the types and activities of carrier proteins which transport the chloride ion in pig jejunal brush-border membranes, with an emphasis on studying the properties of chloride conductance activity in vesicles prepared from these membranes. 2. Sodium-chloride co-transport activity was not detected in this tissue. A sodium-proton antiport with typical amiloride sensitivity was present. An anion exchanger linking chloride to hydroxyl or bicarbonate ions was also found in the pig jejunal brush-border membrane vesicles. 3. Chloride conductance activity in this system was specifically dependent on the buffering agents used for vesicle preparation. Conductance activity could not be demonstrated in vesicles prepared in imidazolium acetate or in HEPES-Tris buffers. HEPES-tetramethylammonium buffering of vesicles in the chloride uptake system produced a significant conductance response to a potassium gradient plus valinomycin. 4. Chloride conductance showed saturable kinetics with respect to substrate concentration, with a Michaelis-Menten constant (Km) of approximately 116 mM and a maximum velocity (Vmax) of 132 nmol (mg protein)-1 min-1. 5. Preliminary screening of potential inhibitors of chloride conductance showed only minimal inhibitor effects of SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-sulphonic acid), anthracene-9-carboxylate, N-phenylanthranilate and piretanide. 6. The conductance activity in pig jejunal vesicles appears to have stringent buffer requirements, and to be relatively insensitive to the effects of reported conductance inhibitors. PMID:2466986

  2. Plastocyanin stimulation of whole chain and photosystem I electron transport in inside-out thylakoid vesicles

    SciTech Connect

    Atta-Asafo-Adjei, E.; Dilley, R.A.

    1985-12-01

    Inside-out and right-side-out thylakoid vesicles were isolated from spinach chloroplasts by aqueous-polymer two-phase (dextran/polyethylene glycol) partitioning. Externally added plastocyanin stimulated the whole-chain and PSI electron transport rates in the inside-out thylakoid vesicles by about 500 and 350%, respectively, compared to about 50% stimulation for both assays in the fraction enriched in right-side-out vesicles. No apparent stimulation by plastocyanin was observed in unbroken Class II thylakoids. The electron transport between PSII and PSI in inside-out thylakoid vesicles appears to be interrupted due to plastocyanin release from the thylakoids by the Yeda press treatment, but it was restored by externally added plastocyanin. The P700 content of the inside-out membrane preparations, measured by chemical and photochemical methods, was 1 P700 per 1100 to 1500 chlorophylls while it was about 1 P700 per 500 chlorophylls for the right-side-out vesicles. The data presented support the concept of lateral heterogeneity of PS I and II in thylakoid membranes, but does not support a virtual or total absence of PSI in the appressed grana partitions. Further, the heterogeneity does not appear to be as extreme as suggested earlier. Although PSI is somewhat depleted in the appressed grana membrane region, there is adequate photochemically active P700, when sufficient plastocyanin is available, to effectively couple PSI electron transfer with the preponderant PSII in linear electron transport.

  3. Direct measurement of calcium transport across chloroplast inner-envelope vesicles

    SciTech Connect

    Roh, M.H.; Shingles, R.; Cleveland, M.J.; McCarty, R.E.

    1998-12-01

    The initial rate of Ca{sup 2+} movement across the inner-envelope membrane of pea (Pisum sativum L.) chloroplasts was directly measured by stopped-flow spectrofluorometry using membrane vesicles loaded with the Ca{sup 2+}-sensitive fluorophore fura-2. Calibration of fura-2 fluorescence was achieved by combining a ratiometric method with Ca{sup 2+}-selective minielectrodes to determine pCa values. The initial rate of Ca{sup 2+} influx in predominantly right-side-out inner-envelope membrane vesicles was greater than that in largely inside-out vesicles. Ca{sup 2+} movement was stimulated by an inwardly directed electrochemical proton gradient across the membrane vesicles, an effect that was diminished by the addition of valinomycin in the presence of K{sup +}. In addition, Ca{sup 2+} was shown to move across the membrane vesicles in the presence of K{sup +} diffusion potential gradient. The potential-stimulated rate of Ca{sup 2+} transport was slightly inhibited by diltiazem and greatly inhibited by ruthenium red. Other pharmacological agents such as LaCl{sub 3}, verapamil, and nifedipine had little or no effect. These results indicate that Ca{sup 2+} transport across the chloroplast inner envelope can occur by a potential-stimulated uniport mechanism.

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

    SciTech Connect

    Atta-Asafo-Adjei, E.

    1987-01-01

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

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

    SciTech Connect

    Slone, J.H.

    1985-01-01

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

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

    PubMed Central

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

    1987-01-01

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

  7. Auxin transport inhibitors impair vesicle motility and actin cytoskeleton dynamics in diverse eukaryotes

    PubMed Central

    Dhonukshe, Pankaj; Grigoriev, Ilya; Fischer, Rainer; Tominaga, Motoki; Robinson, David G.; Hašek, Jiří; Paciorek, Tomasz; Petrášek, Jan; Seifertová, Daniela; Tejos, Ricardo; Meisel, Lee A.; Zažímalová, Eva; Gadella, Theodorus W. J.; Stierhof, York-Dieter; Ueda, Takashi; Oiwa, Kazuhiro; Akhmanova, Anna; Brock, Roland; Spang, Anne; Friml, Jiří

    2008-01-01

    Many aspects of plant development, including patterning and tropisms, are largely dependent on the asymmetric distribution of the plant signaling molecule auxin. Auxin transport inhibitors (ATIs), which interfere with directional auxin transport, have been essential tools in formulating this concept. However, despite the use of ATIs in plant research for many decades, the mechanism of ATI action has remained largely elusive. Using real-time live-cell microscopy, we show here that prominent ATIs such as 2,3,5-triiodobenzoic acid (TIBA) and 2-(1-pyrenoyl) benzoic acid (PBA) inhibit vesicle trafficking in plant, yeast, and mammalian cells. Effects on micropinocytosis, rab5-labeled endosomal motility at the periphery of HeLa cells and on fibroblast mobility indicate that ATIs influence actin cytoskeleton. Visualization of actin cytoskeleton dynamics in plants, yeast, and mammalian cells show that ATIs stabilize actin. Conversely, stabilizing actin by chemical or genetic means interferes with endocytosis, vesicle motility, auxin transport, and plant development, including auxin transport-dependent processes. Our results show that a class of ATIs act as actin stabilizers and advocate that actin-dependent trafficking of auxin transport components participates in the mechanism of auxin transport. These studies also provide an example of how the common eukaryotic process of actin-based vesicle motility can fulfill a plant-specific physiological role. PMID:18337510

  8. The complexity of vesicle transport factors in plants examined by orthology search.

    PubMed

    Paul, Puneet; Simm, Stefan; Mirus, Oliver; Scharf, Klaus-Dieter; Fragkostefanakis, Sotirios; Schleiff, Enrico

    2014-01-01

    Vesicle transport is a central process to ensure protein and lipid distribution in eukaryotic cells. The current knowledge on the molecular components and mechanisms of this process is majorly based on studies in Saccharomyces cerevisiae and Arabidopsis thaliana, which revealed 240 different proteinaceous factors either experimentally proven or predicted to be involved in vesicle transport. In here, we performed an orthologue search using two different algorithms to identify the components of the secretory pathway in yeast and 14 plant genomes by using the 'core-set' of 240 factors as bait. We identified 4021 orthologues and (co-)orthologues in the discussed plant species accounting for components of COP-II, COP-I, Clathrin Coated Vesicles, Retromers and ESCRTs, Rab GTPases, Tethering factors and SNAREs. In plants, we observed a significantly higher number of (co-)orthologues than yeast, while only 8 tethering factors from yeast seem to be absent in the analyzed plant genomes. To link the identified (co-)orthologues to vesicle transport, the domain architecture of the proteins from yeast, genetic model plant A. thaliana and agriculturally relevant crop Solanum lycopersicum has been inspected. For the orthologous groups containing (co-)orthologues from yeast, A. thaliana and S. lycopersicum, we observed the same domain architecture for 79% (416/527) of the (co-)orthologues, which documents a very high conservation of this process. Further, publically available tissue-specific expression profiles for a subset of (co-)orthologues found in A. thaliana and S. lycopersicum suggest that some (co-)orthologues are involved in tissue-specific functions. Inspection of localization of the (co-)orthologues based on available proteome data or localization predictions lead to the assignment of plastid- as well as mitochondrial localized (co-)orthologues of vesicle transport factors and the relevance of this is discussed.

  9. Individual synaptic vesicles from the electroplaque of Torpedo californica, a classic cholinergic synapse, also contain transporters for glutamate and ATP

    PubMed Central

    Li, Huinan; Harlow, Mark L.

    2014-01-01

    Abstract The type of neurotransmitter secreted by a neuron is a product of the vesicular transporters present on its synaptic vesicle membranes and the available transmitters in the local cytosolic environment where the synaptic vesicles reside. Synaptic vesicles isolated from electroplaques of the marine ray, Torpedo californica, have served as model vesicles for cholinergic neurotransmission. Many lines of evidence support the idea that in addition to acetylcholine, additional neurotransmitters and/or neuromodulators are also released from cholinergic synapses. We identified the types of vesicular neurotransmitter transporters present at the electroplaque using immunoblot and immunofluoresence techniques with antibodies against the vesicle acetylcholine transporter (VAChT), the vesicular glutamate transporters (VGLUT1, 2, and 3), and the vesicular nucleotide transporter (VNUT). We found that VAChT, VNUT, VGLUT 1 and 2, but not 3 were present by immunoblot, and confirmed that the antibodies were specific to proteins of the axons and terminals of the electroplaque. We used a single‐vesicle imaging technique to determine whether these neurotransmitter transporters were present on the same or different populations of synaptic vesicles. We found that greater than 85% of vesicles that labeled for VAChT colabeled with VGLUT1 or VGLUT2, and approximately 70% colabeled with VNUT. Based upon confidence intervals, at least 52% of cholinergic vesicles isolated are likely to contain all four transporters. The presence of multiple types of neurotransmitter transporters – and potentially neurotransmitters – in individual synaptic vesicles raises fundamental questions about the role of cotransmitter release and neurotransmitter synergy at cholinergic synapses. PMID:24744885

  10. Characterization of Ca2+ Transport in Purified Endoplasmic Reticulum Membrane Vesicles from Lepidium sativum L. Roots

    PubMed Central

    Buckhout, Thomas J.

    1984-01-01

    The characteristics of Ca2+ transport into endoplasmic reticulum vesicles isolated from roots of Lepidium sativum L. cv Krause have been investigated. The concentration of free Ca2+ and ATP needed for half-maximal activity were 2.5 and 73 micromolar, respectively, and the enzyme obeyed Michaelis-Menten-like kinetics. The pH maximum occurred at 7.5 and the activity was greatly reduced at either pH 7.0 or 8.0. The Ca2+-dependent modulation protein, calmodulin, was tested for its effect on Ca2+ transport into endoplasmic reticulum vesicles. Although the phenothiazine inhibitors chlorpromazine, fluphenazine, and trifluoperazine all inhibited Ca2+ transport activity with a half-maximal effect at approximately 35 micromolar, authentic bovine brain calmodulin did not alter the activity at concentrations of 0.5 to 8 micrograms per milliliter. Calmodulin also showed no influence on the time-dependent accumulation of Ca2+ into vesicles. The membranes did not contain endogenously bound calmodulin since washing with (ethylenebis[oxyethylenenitrile])tetraacetic acid or fluphenazine, treatments which disrupt calmodulin binding, did not alter Ca2+ transport activity. The inhibition of Ca2+ transport by phenothiazine drugs was likely related to their nonspecific interaction with the membrane. Thus, there was no indication that calmodulin regulated Ca2+ uptake into root endoplasmic reticulum. PMID:16663981

  11. Identification of an Axonal Kinesin-3 Motor for Fast Anterograde Vesicle Transport that Facilitates Retrograde Transport of Neuropeptides

    PubMed Central

    Barkus, Rosemarie V.; Klyachko, Olga; Horiuchi, Dai; Dickson, Barry J.

    2008-01-01

    A screen for genes required in Drosophila eye development identified an UNC-104/Kif1 related kinesin-3 microtubule motor. Analysis of mutants suggested that Drosophila Unc-104 has neuronal functions that are distinct from those of the classic anterograde axonal motor, kinesin-1. In particular, unc-104 mutations did not cause the distal paralysis and focal axonal swellings characteristic of kinesin-1 (Khc) mutations. However, like Khc mutations, unc-104 mutations caused motoneuron terminal atrophy. The distributions and transport behaviors of green fluorescent protein-tagged organelles in motor axons indicate that Unc-104 is a major contributor to the anterograde fast transport of neuropeptide-filled vesicles, that it also contributes to anterograde transport of synaptotagmin-bearing vesicles, and that it contributes little or nothing to anterograde transport of mitochondria, which are transported primarily by Khc. Remarkably, unc-104 mutations inhibited retrograde runs by neurosecretory vesicles but not by the other two organelles. This suggests that Unc-104, a member of an anterograde kinesin subfamily, contributes to an organelle-specific dynein-driven retrograde transport mechanism. PMID:17989365

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

    PubMed Central

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

    1997-01-01

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

  13. Role of phosphatidylserine in phospholipid flippase-mediated vesicle transport in Saccharomyces cerevisiae.

    PubMed

    Takeda, Miyoko; Yamagami, Kanako; Tanaka, Kazuma

    2014-03-01

    Phospholipid flippases translocate phospholipids from the exoplasmic to the cytoplasmic leaflet of cell membranes to generate and maintain phospholipid asymmetry. The genome of budding yeast encodes four heteromeric flippases (Drs2p, Dnf1p, Dnf2p, and Dnf3p), which associate with the Cdc50 family noncatalytic subunit, and one monomeric flippase Neo1p. Flippases have been implicated in the formation of transport vesicles, but the underlying mechanisms are largely unknown. We show here that overexpression of the phosphatidylserine synthase gene CHO1 suppresses defects in the endocytic recycling pathway in flippase mutants. This suppression seems to be mediated by increased cellular phosphatidylserine. Two models can be envisioned for the suppression mechanism: (i) phosphatidylserine in the cytoplasmic leaflet recruits proteins for vesicle formation with its negative charge, and (ii) phosphatidylserine flipping to the cytoplasmic leaflet induces membrane curvature that supports vesicle formation. In a mutant depleted for flippases, a phosphatidylserine probe GFP-Lact-C2 was still localized to endosomal membranes, suggesting that the mere presence of phosphatidylserine in the cytoplasmic leaflet is not enough for vesicle formation. The CHO1 overexpression did not suppress the growth defect in a mutant depleted or mutated for all flippases, suggesting that the suppression was dependent on flippase-mediated phospholipid flipping. Endocytic recycling was not blocked in a mutant lacking phosphatidylserine or depleted in phosphatidylethanolamine, suggesting that a specific phospholipid is not required for vesicle formation. These results suggest that flippase-dependent vesicle formation is mediated by phospholipid flipping, not by flipped phospholipids.

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

    PubMed Central

    Said, H M; Redha, R

    1987-01-01

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

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

    PubMed Central

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

    1973-01-01

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

  16. Effect of diet on insulin binding and glucose transport in rat sarcolemmal vesicles

    SciTech Connect

    Grimditch, G.K.; Barnard, R.J.; Sternlicht, E.; Whitson, R.H.; Kaplan, S.A.

    1987-03-01

    The purpose of this study was to compare the effects of a high-fat, high-sucrose diet (HFS) and a low-fat, high-complex carbohydrate diet (LFC) on glucose tolerance, insulin binding, and glucose transport in rat skeletal muscle. During the intravenous glucose tolerance test, peak glucose values at 5 min were significantly higher in the HFS group; 0-, 20-, and 60-min values were similar. Insulin values were significantly higher in the HFS group at all time points (except 60 min), indicating whole-body insulin resistance. Skeletal muscle was responsible, in part, for this insulin resistance, because specific D-glucose transport in isolated sarcolemmal (SL) vesicles under basal conditions was similar between LFC and HFS rats, despite the higher plasma insulin levels. Scatchard analyses of insulin binding curves to sarcolemmal vesicles revealed that the K/sub a/ of the high-affinity binding sites was significantly reduced by the HFS diet; no other binding changes were noted. Specific D-glucose transport in SL vesicles after maximum insulin stimulation (1 U/kg) was significantly depressed in the HFS group, indicating that HFS feeding also caused a postbinding defect. These results indicate that the insulin resistance in skeletal muscle associated with a HFS diet is due to both a decrease in the K/sub a/ of the high-affinity insulin receptors and a postbinding defect.

  17. Proteins affecting thylakoid morphology - the key to understanding vesicle transport in chloroplasts?

    PubMed

    Lindquist, Emelie; Aronsson, Henrik

    2014-01-01

    We recently showed that a Rab protein, CPRabA5e (CP = chloroplast localized), is located in chloroplasts of Arabidopsis thaliana where it is involved in various processes, such as thylakoid biogenesis and vesicle transport. Using a yeast two-hybrid method, CPRabA5e was shown to interact with a number of chloroplast proteins, including the CURVATURE THYLAKOID 1A (CURT1A) protein and the light-harvesting chlorophyll a/b binding protein (LHCB1.5). CURT1A has recently been shown to modify thylakoid architecture by inducing membrane curvature in grana, whereas LHCB1.5 is a protein of PSII (Photosystem II) facilitating light capture. LHCB1.5 is imported to chloroplasts and transported to thylakoid membranes using the post-translational Signal Recognition Particle (SRP) pathway. With this information as starting point, we here discuss their subsequent protein-protein interactions, given by the literature and Interactome 3D. CURT1A itself and several of the proteins interacting with CURT1A and LHCB1.5 have relations to vesicle transport and thylakoid morphology, which are also characteristics of cprabA5e mutants. This highlights the previous hypothesis of an alternative thylakoid targeting pathway for LHC proteins using vesicles, in addition to the SRP pathway.

  18. Carrier-mediated concentrative urate transport in rat renal membrane vesicles.

    PubMed

    Abramson, R G; Lipkowitz, M S

    1985-04-01

    [2-14C]Urate uptake and efflux were studied in brush border and basolateral membrane vesicles of rat renal cortex that were exposed to 20 microM copper chloride. In the presence of inwardly directed NaCl gradients urate uptake was maintained at levels in excess of chemical equilibrium. Comparison of glucose and chloride uptakes revealed that equilibrium glucose uptake was not affected by copper, but chloride failed to reach equilibrium in copper-exposed vesicles. It is suggested that the persistence of an electrolyte gradient could provide a driving force to raise the concentration of free intravesicular urate above that in the media. Preincubation of vesicles with unlabeled urate failed to diminish uptake of added urate; rather, urate uptake was trans stimulated. Uptake of labeled urate was also significantly accelerated when an outward gradient for unlabeled urate was created. Pyrazinoic and oxonic acids also trans stimulated urate uptake. The demonstration of accelerated homeo- and heteroexchange diffusion indicates that transport is carrier mediated in both brush border and basolateral vesicles. Outwardly directed hydroxyl gradients failed to influence urate uptake in either the presence or absence of copper or NaCl. Thus, this carrier, which is active only in the presence of trace amounts of copper, is distinct from a urate/anion exchanger.

  19. Differential recognition of a dileucine-based sorting signal by AP-1 and AP-3 reveals a requirement for both BLOC-1 and AP-3 in delivery of OCA2 to melanosomes

    PubMed Central

    Sitaram, Anand; Dennis, Megan K.; Chaudhuri, Rittik; De Jesus-Rojas, Wilfredo; Tenza, Danièle; Setty, Subba Rao Gangi; Wood, Christopher S.; Sviderskaya, Elena V.; Bennett, Dorothy C.; Raposo, Graça; Bonifacino, Juan S.; Marks, Michael S.

    2012-01-01

    Cell types that generate unique lysosome-related organelles (LROs), such as melanosomes in melanocytes, populate nascent LROs with cargoes that are diverted from endosomes. Cargo sorting toward melanosomes correlates with binding via cytoplasmically exposed sorting signals to either heterotetrameric adaptor AP-1 or AP-3. Some cargoes bind both adaptors, but the relative contribution of each adaptor to cargo recognition and their functional interactions with other effectors during transport to melanosomes are not clear. Here we exploit targeted mutagenesis of the acidic dileucine–based sorting signal in the pigment cell–specific protein OCA2 to dissect the relative roles of AP-1 and AP-3 in transport to melanosomes. We show that binding to AP-1 or AP-3 depends on the primary sequence of the signal and not its position within the cytoplasmic domain. Mutants that preferentially bound either AP-1 or AP-3 each trafficked toward melanosomes and functionally complemented OCA2 deficiency, but AP-3 binding was necessary for steady-state melanosome localization. Unlike tyrosinase, which also engages AP-3 for optimal melanosomal delivery, both AP-1– and AP-3–favoring OCA2 variants required BLOC-1 for melanosomal transport. These data provide evidence for distinct roles of AP-1 and AP-3 in OCA2 transport to melanosomes and indicate that BLOC-1 can cooperate with either adaptor during cargo sorting to LROs. PMID:22718909

  20. Differential recognition of a dileucine-based sorting signal by AP-1 and AP-3 reveals a requirement for both BLOC-1 and AP-3 in delivery of OCA2 to melanosomes.

    PubMed

    Sitaram, Anand; Dennis, Megan K; Chaudhuri, Rittik; De Jesus-Rojas, Wilfredo; Tenza, Danièle; Setty, Subba Rao Gangi; Wood, Christopher S; Sviderskaya, Elena V; Bennett, Dorothy C; Raposo, Graça; Bonifacino, Juan S; Marks, Michael S

    2012-08-01

    Cell types that generate unique lysosome-related organelles (LROs), such as melanosomes in melanocytes, populate nascent LROs with cargoes that are diverted from endosomes. Cargo sorting toward melanosomes correlates with binding via cytoplasmically exposed sorting signals to either heterotetrameric adaptor AP-1 or AP-3. Some cargoes bind both adaptors, but the relative contribution of each adaptor to cargo recognition and their functional interactions with other effectors during transport to melanosomes are not clear. Here we exploit targeted mutagenesis of the acidic dileucine-based sorting signal in the pigment cell-specific protein OCA2 to dissect the relative roles of AP-1 and AP-3 in transport to melanosomes. We show that binding to AP-1 or AP-3 depends on the primary sequence of the signal and not its position within the cytoplasmic domain. Mutants that preferentially bound either AP-1 or AP-3 each trafficked toward melanosomes and functionally complemented OCA2 deficiency, but AP-3 binding was necessary for steady-state melanosome localization. Unlike tyrosinase, which also engages AP-3 for optimal melanosomal delivery, both AP-1- and AP-3-favoring OCA2 variants required BLOC-1 for melanosomal transport. These data provide evidence for distinct roles of AP-1 and AP-3 in OCA2 transport to melanosomes and indicate that BLOC-1 can cooperate with either adaptor during cargo sorting to LROs.

  1. 65Zn2+ transport by lobster hepato-pancreatic baso-lateral membrane vesicles.

    PubMed

    Capo, J A; Mandal, P K; Eyyunni, S; Ahearn, G A

    2005-01-01

    The lobster (Homarus americanus) hepato-pancreatic epithelial baso-lateral cell membrane possesses three transport proteins that transfer calcium between the cytoplasm and hemolymph: an ATP-dependent calcium ATPase, a sodium-calcium exchanger, and a verapamil-sensitive cation channel. We used standard centrifugation methods to prepare purified hepato-pancreatic baso-lateral membrane vesicles and a rapid filtration procedure to investigate whether (65)Zn(2+) transfer across this epithelial cell border occurs by any of these previously described transporters for calcium. Baso-lateral membrane vesicles were osmotically reactive and exhibited a time course of uptake that was linear for 10-15 s and approached equilibrium by 120 s. In the absence of sodium, (65)Zn(2+) influx was a hyperbolic function of external zinc concentration and followed the Michaelis-Menten equation for carrier transport. This carrier transport was stimulated by the addition of 150 microM ATP (increase in K(m) and J(max)) and inhibited by the simultaneous presence of 150 micromol l(-1) ATP+250 micromol l(-1) vanadate (decrease in both K(m) and J(max)). In the absence of ATP, (65)Zn(2+) influx was a sigmoidal function of preloaded vesicular sodium concentration (0, 5, 10, 20, 30, 45, and 75 mmol l(-1)) and exhibited a Hill Coefficient of 4.03+/-1.14, consistent with the exchange of 3 Na(+)/1Zn(2+). Using Dixon analysis, calcium was shown to be a competitive inhibitor of baso-lateral membrane vesicle (65)Zn(2+) influx by both the ATP-dependent (K(i)=205 nmol l(-1) Ca(2+)) and sodium-dependent (K(i)=2.47 micromol l(-1) Ca(2+)) transport processes. These results suggest that zinc transport across the lobster hepato-pancreatic baso-lateral membrane largely occurred by the ATP-dependent calcium ATPase and sodium-calcium exchanger carrier proteins.

  2. Calcium transport in tonoplast and endoplasmic reticulum vesicles isolated from cultured carrot cells. [Daucus carota Danvers

    SciTech Connect

    Bush, D.R.; Sze, H.

    1986-02-01

    Two active calcium (Ca/sup 2 +/) transport systems have been identified and partially characterized in membrane vesicles isolated from cultured carrot cells (Daucus carota Danvers). Both transport systems required MgATP for activity and were enhanced by 10 millimolar oxalate. Ca/sup 2 +/ transport in membrane vesicles derived from isolated vacuoles equilibrated at 1.10 grams per cubic centimeter and comigrated with Cl/sup -/-stimulated, NO/sub 3//sup -/-inhibited ATPase activity on sucrose density gradients. Ca/sup 2 +/ transport in this system was insensitive to vanadate, but was inhibited by nitrate, carbonyl cyanide-m-chlorophenylhydrazone (CCCP), N,N'-dicyclohexylcarbodiimide (DCCD), and 4,4-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS). The K/sub m/ for MgATP and Ca/sup 2 +/ were 0.1 mM and 21 micromolar, respectively. The predominant Ca/sup 2 +/ transport system detectable in microsomal membrane preparations equilibrated at a density of 1.13 grams per cubic centimeter and comigrated with the endoplasmic reticulum (ER) marker, antimycin A-insensitive NADH-dependent cytochrome c reductase. Ca/sup 2 +/ transport activity and the ER marker also shifted in parallel in ER shifting experiments. This transport system was inhibited by vanadate (I/sub 50/ = 12 micromolar) and was insensitive to nitrate, CCCP, DCCD, and DIDS. Transport exhibited cooperative MgATP dependent kinetics. Ca/sup 2 +/ dependent kinetics were complex with an apparent K/sub m/ ranging from 0.7 to 2 micromolar. We conclude that the vacuolar-derived system is a Ca/sup 2 +//H/sup +/ antiport located on the tonoplast and that the microsomal transport system is a Ca,Mg-ATPase enriched on the ER. These two Ca/sup 2 +/ transport systems are proposed to restore and maintain cytoplasmic Ca/sup 2 +/ homeostasis under changing cellular and environmental conditions.

  3. ATP regulation of calcium transport in back-inhibited sarcoplasmic reticulum vesicles.

    PubMed

    de Meis, L; Sorenson, M M

    1989-09-18

    At high concentrations of ATP, ATP hydrolysis and Ca2+ transport by the (Ca2+ + MG2+)-ATPase of intact sarcoplasmic reticulum vesicles exhibit a secondary activation that varies with the extent of back-inhibition by Ca2+ accumulated within the vesicles. When the internal ionized Ca2+ is clamped at low and intermediate levels by the use of Ca-precipitating anions, the apparent Km values for activation by ATP are lower than in fully back-inhibited vesicles (high internal Ca2+). In leaky vesicles unable to accumulate Ca2+, raising Ca2+ in the assay medium from 20-30 microM to 5 mM abolishes the activation of hydrolysis by high concentrations of ATP. The level of [32P]phosphoenzyme formed during ATP hydrolysis from [32P]phosphate added to the medium also varies with the extent of back-inhibition; it is highest when Ca2+ is raised to a level that saturates the internal, low-affinity Ca2+ binding sites. In intact vesicles, increasing the ATP concentration from 10 to 400 microM competitively inhibits the reaction of inorganic phosphate with the enzyme but does not change the rate of hydrolysis. In a previous report (De Meis, L., Gomez-Puyou, M.T. and Gomez-Puyou, A. (1988) Eur. J. Biochem. 171, 343-349), it has been shown that the hydrophobic molecules trifluoperazine and iron bathophenanthroline compete for the catalytic site of the Pi-reactive form of the enzyme. Here it is shown that inhibition of ATP hydrolysis by these compounds is reduced or abolished when Ca2+ binds to the low-affinity Ca2+ binding sites of the enzyme. Since inhibition by these agents is indifferent to activation of hydrolysis by high concentrations of ATP, it is suggested that the second Km for ATP and the inhibition by hydrophobic molecules involve two different Ca-free forms of the enzyme.

  4. Cimetidine transport in rabbit renal cortical brush-border membrane vesicles

    SciTech Connect

    McKinney, T.D.; Kunnemann, M.E.

    1987-03-01

    Cimetidine is an organic cation and commonly prescribed drug that is eliminated primarily by proximal renal tubular secretion. The present studies evaluated cimetidine transport in rabbit renal cortical brush-border membrane vesicles (BBMV). (/sup 3/H)Cimetidine uptake varied inversely with media osmolarity and was stimulated with uphill transport above equilibrium values (overshoot) produced by an initial proton gradient directed from the vesicle interior outwardly. Uphill transport occurred earlier and was of greater magnitude at 25/sup 0/C than at 5/sup 0/C. pH-stimulated (/sup 3/H)cimetidine uptake was inhibited by excess nonradiolabeled cimetidine, quinidine, and procainamide but was affected little by probenecid. Tetraethylammonium inhibited cimetidine uptake in the presence and absence of an initial proton gradient, indicating that nonionic diffusion and simple diffusion cannot totally account for cimetidine transport in BBMV. Preloading BBMV with an excess of procainamide enhanced cimetidine uptake. These results are consistent with the hypothesis that cimetidine is transported across BBMV by organic cation-proton exchange.

  5. Aboral changes in D-glucose transport by human intestinal brush-border membrane vesicles.

    PubMed Central

    Bluett, M K; Abumrad, N N; Arab, N; Ghishan, F K

    1986-01-01

    D-Glucose transport was investigated in isolated brush-border membrane vesicles from human small intestine. Characteristics of D-glucose transport from the jejunum were compared with that in the mid and terminal ileum. Jejunal and mid-ileal D-glucose transport was Na+-dependent and electrogenic. The transient overshoot of jejunal D-glucose transport was significantly greater than corresponding values in mid-ileum. The terminal ileum did not exhibit Na+-dependent D-glucose transport, but did exhibit Na+-dependent taurocholate transport. Na+-glucose co-transport activity as measured by tracer-exchange experiments was greatest in the jejunum, and diminished aborally. We conclude that D-glucose transport in man is Na+-dependent and electrogenic in the proximal intestine and directly related to the activity of D-glucose-Na+ transporters present in the brush-border membranes. D-Glucose transport in the terminal ileum resembles colonic transport of D-glucose. PMID:3800877

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

    SciTech Connect

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

    1987-12-01

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

  7. Cimetidine transport in isolated brush border membrane vesicles from bovine choroid plexus

    SciTech Connect

    Whittico, M.T.; Gang, Y.A.; Giacomini, K.M. )

    1990-11-01

    The purpose of this study was to elucidate the mechanisms involved in the transport of cimetidine across the brush border membrane of choroid plexus epithelium. Brush border membrane vesicles were prepared from bovine choroid plexus and the uptake of (3H)cimetidine was studied using the methods of rapid vacuum filtration and scintillation counting. Cimetidine accumulated in the vesicles with time reaching equilibrium within 2 hr. The amount of cimetidine taken up by the vesicles at equilibrium decreased with increasing extravesicular media osmolarity suggesting that cimetidine accumulates in an osmotically reactive intravesicular space. Binding of cimetidine to the membrane was estimated to be less than 18%. Michaelis-Menten studies demonstrated that cimetidine transport involved both a saturable and a nonsaturable component. The Vmax and Km (mean +/- S.E.) were 16.7 +/- 5.9 pmol/sec/mg protein and 58.1 +/- 3.1 microM, respectively, suggesting that cimetidine is transported across the choroid plexus brush border membrane with a lower affinity and a higher capacity than across the renal brush border membrane. The organic cation, quinidine (0.1 mM), and the amino acid, histidine (20 mM), both significantly reduced the initial, but not the equilibrium, uptake of cimetidine. However, high concentrations (5 mM) of more polar organic cations including tetraethylammonium, as well as of several organic anions including salicylate did not inhibit cimetidine transport. Studies with unlabeled cimetidine revealed a countertransport phenomenon. Attempts to drive the concentrative uptake of cimetidine with various ion gradients were unsuccessful. Of note was the fact that an outwardly directed proton gradient could significantly accelerate the uptake of cimetidine.

  8. Bicaudal-D binds clathrin heavy chain to promote its transport and augments synaptic vesicle recycling

    PubMed Central

    Li, Xuan; Kuromi, Hiroshi; Briggs, Laura; Green, David B; Rocha, João J; Sweeney, Sean T; Bullock, Simon L

    2010-01-01

    Cargo transport by microtubule-based motors is essential for cell organisation and function. The Bicaudal-D (BicD) protein participates in the transport of a subset of cargoes by the minus-end-directed motor dynein, although the full extent of its functions is unclear. In this study, we report that in Drosophila zygotic BicD function is only obligatory in the nervous system. Clathrin heavy chain (Chc), a major constituent of coated pits and vesicles, is the most abundant protein co-precipitated with BicD from head extracts. BicD binds Chc directly and interacts genetically with components of the pathway for clathrin-mediated membrane trafficking. Directed transport and subcellular localisation of Chc is strongly perturbed in BicD mutant presynaptic boutons. Functional assays show that BicD and dynein are essential for the maintenance of normal levels of neurotransmission specifically during high-frequency electrical stimulation and that this is associated with a reduced rate of recycling of internalised synaptic membrane. Our results implicate BicD as a new player in clathrin-associated trafficking processes and show a novel requirement for microtubule-based motor transport in the synaptic vesicle cycle. PMID:20111007

  9. Biophysics of active vesicle transport, an intermediate step that couples excitation and exocytosis of serotonin in the neuronal soma.

    PubMed

    De-Miguel, Francisco F; Santamaría-Holek, Iván; Noguez, Paula; Bustos, Carlos; Hernández-Lemus, Enrique; Rubí, J Miguel

    2012-01-01

    Transmitter exocytosis from the neuronal soma is evoked by brief trains of high frequency electrical activity and continues for several minutes. Here we studied how active vesicle transport towards the plasma membrane contributes to this slow phenomenon in serotonergic leech Retzius neurons, by combining electron microscopy, the kinetics of exocytosis obtained from FM1-43 dye fluorescence as vesicles fuse with the plasma membrane, and a diffusion equation incorporating the forces of local confinement and molecular motors. Electron micrographs of neurons at rest or after stimulation with 1 Hz trains showed cytoplasmic clusters of dense core vesicles at 1.5±0.2 and 3.7±0.3 µm distances from the plasma membrane, to which they were bound through microtubule bundles. By contrast, after 20 Hz stimulation vesicle clusters were apposed to the plasma membrane, suggesting that transport was induced by electrical stimulation. Consistently, 20 Hz stimulation of cultured neurons induced spotted FM1-43 fluorescence increases with one or two slow sigmoidal kinetics, suggesting exocytosis from an equal number of vesicle clusters. These fluorescence increases were prevented by colchicine, which suggested microtubule-dependent vesicle transport. Model fitting to the fluorescence kinetics predicted that 52-951 vesicles/cluster were transported along 0.60-6.18 µm distances at average 11-95 nms(-1) velocities. The ATP cost per vesicle fused (0.4-72.0), calculated from the ratio of the ΔG(process)/ΔG(ATP), depended on the ratio of the traveling velocity and the number of vesicles in the cluster. Interestingly, the distance-dependence of the ATP cost per vesicle was bistable, with low energy values at 1.4 and 3.3 µm, similar to the average resting distances of the vesicle clusters, and a high energy barrier at 1.6-2.0 µm. Our study confirms that active vesicle transport is an intermediate step for somatic serotonin exocytosis by Retzius neurons and provides a quantitative method

  10. Biophysics of Active Vesicle Transport, an Intermediate Step That Couples Excitation and Exocytosis of Serotonin in the Neuronal Soma

    PubMed Central

    De-Miguel, Francisco F.; Santamaría-Holek, Iván; Noguez, Paula; Bustos, Carlos; Hernández-Lemus, Enrique; Rubí, J. Miguel

    2012-01-01

    Transmitter exocytosis from the neuronal soma is evoked by brief trains of high frequency electrical activity and continues for several minutes. Here we studied how active vesicle transport towards the plasma membrane contributes to this slow phenomenon in serotonergic leech Retzius neurons, by combining electron microscopy, the kinetics of exocytosis obtained from FM1-43 dye fluorescence as vesicles fuse with the plasma membrane, and a diffusion equation incorporating the forces of local confinement and molecular motors. Electron micrographs of neurons at rest or after stimulation with 1 Hz trains showed cytoplasmic clusters of dense core vesicles at 1.5±0.2 and 3.7±0.3 µm distances from the plasma membrane, to which they were bound through microtubule bundles. By contrast, after 20 Hz stimulation vesicle clusters were apposed to the plasma membrane, suggesting that transport was induced by electrical stimulation. Consistently, 20 Hz stimulation of cultured neurons induced spotted FM1-43 fluorescence increases with one or two slow sigmoidal kinetics, suggesting exocytosis from an equal number of vesicle clusters. These fluorescence increases were prevented by colchicine, which suggested microtubule-dependent vesicle transport. Model fitting to the fluorescence kinetics predicted that 52–951 vesicles/cluster were transported along 0.60–6.18 µm distances at average 11–95 nms−1 velocities. The ATP cost per vesicle fused (0.4–72.0), calculated from the ratio of the ΔGprocess/ΔGATP, depended on the ratio of the traveling velocity and the number of vesicles in the cluster. Interestingly, the distance-dependence of the ATP cost per vesicle was bistable, with low energy values at 1.4 and 3.3 µm, similar to the average resting distances of the vesicle clusters, and a high energy barrier at 1.6–2.0 µm. Our study confirms that active vesicle transport is an intermediate step for somatic serotonin exocytosis by Retzius neurons and provides a quantitative

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

    PubMed Central

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

    2013-01-01

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

  12. Release of kinesin from vesicles by hsc70 and regulation of fast axonal transport

    NASA Technical Reports Server (NTRS)

    Tsai, M. Y.; Morfini, G.; Szebenyi, G.; Brady, S. T.

    2000-01-01

    The nature of kinesin interactions with membrane-bound organelles and mechanisms for regulation of kinesin-based motility have both been surprisingly difficult to define. Most kinesin is recovered in supernatants with standard protocols for purification of motor proteins, but kinesin recovered on membrane-bound organelles is tightly bound. Partitioning of kinesin between vesicle and cytosolic fractions is highly sensitive to buffer composition. Addition of either N-ethylmaleimide or EDTA to homogenization buffers significantly increased the fraction of kinesin bound to organelles. Given that an antibody against kinesin light chain tandem repeats also releases kinesin from vesicles, these observations indicated that specific cytoplasmic factors may regulate kinesin release from membranes. Kinesin light tandem repeats contain DnaJ-like motifs, so the effects of hsp70 chaperones were evaluated. Hsc70 released kinesin from vesicles in an MgATP-dependent and N-ethylmaleimide-sensitive manner. Recombinant kinesin light chains inhibited kinesin release by hsc70 and stimulated the hsc70 ATPase. Hsc70 actions may provide a mechanism to regulate kinesin function by releasing kinesin from cargo in specific subcellular domains, thereby effecting delivery of axonally transported materials.

  13. Inhibition by Levorphanol and Related Drugs of Amino Acid Transport by Isolated Membrane Vesicles from Escherichia coli

    PubMed Central

    Holland, Mary J. C.; Simon, Eric J.

    1975-01-01

    Levorphanol inhibits the transport of the amino acids proline and lysine by cytoplasmic membrane vesicles derived from Escherichia coli. The degree of inhibition increases with increasing levorphanol concentration and ranges from 26% at 10−6 M levorphanol to 92% at 10−3 M levorphanol. The effect is independent of the energy source, since levorphanol inhibits proline uptake to the same extent in the presence of 20 mM d-lactate or 20 mM succinate and in the absence of an exogenous energy source. Levorphanol does not irreversibly alter the ability of membrane vesicles to transport proline, since incubation of membrane vesicles for 15 min in the presence of 0.25 mM levorphanol, a concentration which inhibits proline transport by more than 75%, has no effect on the rate of proline transport by these vesicles once the drug is removed. Both the maximum velocity and the Km of proline transport are modified by levorphanol, hence, the type of inhibition produced by levorphanol is mixed. The inhibitor constant (Ki) for levorphanol inhibition of proline transport is approximately 3 × 10−4 M. Membrane vesicles incubated in the presence of levorphanol accumulate much less proline at the steady state than do control vesicles. Furthermore, the addition of levorphanol to membrane vesicles preloaded to the steady state with proline produces a marked net efflux of proline. Levorphanol does not block either temperature-induced efflux or exchange of external proline with [14C]proline present in the intravesicular pool. Dextrorphan, the enantiomorph of levorphanol, and levallorphan, the N-allyl analogue of levorphanol, inhibit proline and lysine transport in a similar manner. Possible mechanisms of the effects of these drugs on cell membranes are discussed. PMID:1096802

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

    SciTech Connect

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

    1990-02-01

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

  15. The human synaptic vesicle protein, SV2A, functions as a galactose transporter in Saccharomyces cerevisiae.

    PubMed

    Madeo, Marianna; Kovács, Attila D; Pearce, David A

    2014-11-28

    SV2A is a synaptic vesicle membrane protein expressed in neurons and endocrine cells and involved in the regulation of neurotransmitter release. Although the exact function of SV2A still remains elusive, it was identified as the specific binding site for levetiracetam, a second generation antiepileptic drug. Our sequence analysis demonstrates that SV2A has significant homology with several yeast transport proteins belonging to the major facilitator superfamily (MFS). Many of these transporters are involved in sugar transport into yeast cells. Here we present evidence showing, for the first time, that SV2A is a galactose transporter. We expressed human SV2A in hexose transport-deficient EBY.VW4000 yeast cells and demonstrated that these cells are able to grow on galactose-containing medium but not on other fermentable carbon sources. Furthermore, the addition of the SV2A-binding antiepileptic drug levetiracetam to the medium inhibited the galactose-dependent growth of hexose transport-deficient EBY.VW4000 yeast cells expressing human SV2A. Most importantly, direct measurement of galactose uptake in the same strain verified that SV2A is able to transport extracellular galactose inside the cells. The newly identified galactose transport capability of SV2A may have an important role in regulating/modulating synaptic function.

  16. Huntingtin differentially regulates the axonal transport of a sub-set of Rab-containing vesicles in vivo

    PubMed Central

    White, Joseph A.; Anderson, Eric; Zimmerman, Katherine; Zheng, Kan Hong; Rouhani, Roza; Gunawardena, Shermali

    2015-01-01

    Loss of huntingtin (HTT), the Huntington's disease (HD) protein, was previously shown to cause axonal transport defects. Within axons, HTT can associate with kinesin-1 and dynein motors either directly or via accessory proteins for bi-directional movement. However, the composition of the vesicle-motor complex that contains HTT during axonal transport is unknown. Here we analyze the in vivo movement of 16 Rab GTPases within Drosophila larval axons and show that HTT differentially influences the movement of a particular sub-set of these Rab-containing vesicles. While reduction of HTT perturbed the bi-directional motility of Rab3 and Rab19-containing vesicles, only the retrograde motility of Rab7-containing vesicles was disrupted with reduction of HTT. Interestingly, reduction of HTT stimulated the anterograde motility of Rab2-containing vesicles. Simultaneous dual-view imaging revealed that HTT and Rab2, 7 or 19 move together during axonal transport. Collectively, our findings indicate that HTT likely influences the motility of different Rab-containing vesicles and Rab-mediated functions. These findings have important implications for our understanding of the complex role HTT plays within neurons normally, which when disrupted may lead to neuronal death and disease. PMID:26450517

  17. Alpha-Synuclein affects neurite morphology, autophagy, vesicle transport and axonal degeneration in CNS neurons

    PubMed Central

    Koch, J C; Bitow, F; Haack, J; d'Hedouville, Z; Zhang, J-N; Tönges, L; Michel, U; Oliveira, L M A; Jovin, T M; Liman, J; Tatenhorst, L; Bähr, M; Lingor, P

    2015-01-01

    Many neuropathological and experimental studies suggest that the degeneration of dopaminergic terminals and axons precedes the demise of dopaminergic neurons in the substantia nigra, which finally results in the clinical symptoms of Parkinson disease (PD). The mechanisms underlying this early axonal degeneration are, however, still poorly understood. Here, we examined the effects of overexpression of human wildtype alpha-synuclein (αSyn-WT), a protein associated with PD, and its mutant variants αSyn-A30P and -A53T on neurite morphology and functional parameters in rat primary midbrain neurons (PMN). Moreover, axonal degeneration after overexpression of αSyn-WT and -A30P was analyzed by live imaging in the rat optic nerve in vivo. We found that overexpression of αSyn-WT and of its mutants A30P and A53T impaired neurite outgrowth of PMN and affected neurite branching assessed by Sholl analysis in a variant-dependent manner. Surprisingly, the number of primary neurites per neuron was increased in neurons transfected with αSyn. Axonal vesicle transport was examined by live imaging of PMN co-transfected with EGFP-labeled synaptophysin. Overexpression of all αSyn variants significantly decreased the number of motile vesicles and decelerated vesicle transport compared with control. Macroautophagic flux in PMN was enhanced by αSyn-WT and -A53T but not by αSyn-A30P. Correspondingly, colocalization of αSyn and the autophagy marker LC3 was reduced for αSyn-A30P compared with the other αSyn variants. The number of mitochondria colocalizing with LC3 as a marker for mitophagy did not differ among the groups. In the rat optic nerve, both αSyn-WT and -A30P accelerated kinetics of acute axonal degeneration following crush lesion as analyzed by in vivo live imaging. We conclude that αSyn overexpression impairs neurite outgrowth and augments axonal degeneration, whereas axonal vesicle transport and autophagy are severely altered. PMID:26158517

  18. [Vesicular intracellular transport in the digestive organs. Membrane vesicle--the universal mechanism of the functional transport].

    PubMed

    Morozov, I A

    2014-01-01

    On the basis of long-term research of the morpho-functional characteristics of the cells of the stomach, small intestine and gallbladder the mechanism and function of membrane vesicles in the implementation of the main functions of these organs sets out in this article: the secretion of hydrochloric acid by parietal cells, the absorption of nutrients in the small intestine and the fluid at a concentration of bile epitheliocytes of gallbladder. Proofs of the intracellular formation of hydrochloric acid in tubulovesicles of the parietal cells and turnover of its secretory membranes in the process of secretory cycle, that has ensured the re-use and explained the extraordinary life of these unique cells are presented. The credible mechanism of HCl output oppression by H(+)-K(+)-ATPase activity blockers has set out on this basis. The article provides detailed endocytosis mechanism of the ions and nutrients absorption by enterocytes. The mechanism of participation of the apical contractile complex of brush border of epithelial cells in the initiation of endocytosis and cytoplasmic microtubules in transport of membrane vesicles in the cytoplasm was analyzed. Based on our research and numerous of the world scientific proceedings the conclusion was done about the existence of two energy dependent types of transport in the absorptive epithelium of the digestive--transmembrane (ionic and nutritive) homeostatic type which is realized by the ATP-system of the basal plasmalemma, and vesicular (endocytosis) type which is impltmented by apical contractile complex of brush border and cytoplasmic microtubules. Both types of transport are interrelated and are under constant cellular control. This observation is relevant to the majority of cells, including those involved in the secretion of various substances: hydrochloric acid by parietal cells, enzymes by main cells of the gastric glands and exocrinocytes of the pancreas, hormone by endocrine cells of the APUD system and, finally

  19. Calcium transport in vesicles from carrot cells: Stimulation by calmodulin and phosphatidylserine. [Daucus carota cv. Danvers

    SciTech Connect

    Wenling Hsieh; Sze, Heven )

    1991-05-01

    The transport properties of Ca-pumping ATPases from carrot (Daucus carota cv. Danvers) tissue culture cells were studied. ATP dependent Ca transport in vesicles that comigrated with an ER marker, was stimulated 3-4 fold by calmodulin. Cyclopiazonic acid (a specific inhibitor of the sarcoplasmic/endoplasmic reticulum Ca-ATPase) partially inhibited oxalate-stimulated Ca transport activity; however, it had little or not effect on calmodulin-stimulated Ca uptake. The results suggested the presence of two types of Ca ATPases, and ER- and a plasma membrane-type. Incubation of membranes with (gamma{sup 32}P)ATP resulted in the formation of a single acyl ({sup 32}P) phosphoprotein of 120 kDa. Formation of this phosphoprotein was dependent on Ca, and enhanced by La {sup 3+}, characteristic of the plasma membrane CaATPase. Acidic phospholipids, like phosphatidylserine, stimulated Ca transport, similar to their effect on the erythrocyte plasma membrane CaATPase. These results would indicate that the calmodulin-stimulated Ca transport originated in large part from a plasma membrane-type Ca pump of 120 kDa.

  20. β-COP as a Component of Transport Vesicles for HDL Apolipoprotein-Mediated Cholesterol Exocytosis

    PubMed Central

    Ma, Weilie; Lin, Margarita; Ding, Hang; Lin, Guorong; Zhang, Zhizhen

    2016-01-01

    Objective HDL and its apolipoproteins protect against atherosclerotic disease partly by removing excess cholesterol from macrophage foam cells. But the underlying mechanisms of cholesterol clearance are still not well defined. We investigated roles of vesicle trafficking of coatomer β-COP in delivering cholesterol to the cell surface during apoA-1 and apoE-mediated lipid efflux from fibroblasts and THP-1 macrophages. Methods shRNA knockout, confocal and electron microscopy and biochemical analysis were used to investigate the roles of β-COP in apolipoprotein-mediated cholesterol efflux in fibroblasts and THP-1 macrophages. Results We showed that β-COP knockdown by lentiviral shRNA resulted in reduced apoA-1-mediated cholesterol efflux, while increased cholesterol accumulation and formation of larger vesicles were observed in THP-1 macrophages by laser scanning confocal microscopy. Immunogold electron microscopy showed that β-COP appeared on the membrane protrusion complexes and colocalized with apoA-1 or apoE during cholesterol efflux. This was associated with releasing heterogeneous sizes of small particles into the culture media of THP-1 macrophage. Western blotting also showed that apoA-1 promotes β-COP translocation to the cell membrane and secretion into culture media, in which a total of 17 proteins were identified by proteomics. Moreover, β-COP exclusively associated with human plasma HDL fractions. Conclusion ApoA-1 and apoE promoted transport vesicles consisting of β-COP and other candidate proteins to exocytose cholesterol, forming the protrusion complexes on cell surface, which were then released from the cell membrane as small particles to media. PMID:26986486

  1. Reserpine-induced reduction in norepinephrine transporter function requires catecholamine storage vesicles.

    PubMed

    Mandela, Prashant; Chandley, Michelle; Xu, Yao-Yu; Zhu, Meng-Yang; Ordway, Gregory A

    2010-01-01

    Treatment of rats with reserpine, an inhibitor of the vesicular monoamine transporter (VMAT), depletes norepinephrine (NE) and regulates NE transporter (NET) expression. The present study examined the molecular mechanisms involved in regulation of the NET by reserpine using cultured cells. Exposure of rat PC12 cells to reserpine for a period as short as 5min decreased [(3)H]NE uptake capacity, an effect characterized by a robust decrease in the V(max) of the transport of [(3)H]NE. As expected, reserpine did not displace the binding of [(3)H]nisoxetine from the NET in membrane homogenates. The potency of reserpine for reducing [(3)H]NE uptake was dramatically lower in SK-N-SH cells that have reduced storage capacity for catecholamines. Reserpine had no effect on [(3)H]NE uptake in HEK-293 cells transfected with the rat NET (293-hNET), cells that lack catecholamine storage vesicles. NET regulation by reserpine was independent of trafficking of the NET from the cell surface. Pre-exposure of cells to inhibitors of several intracellular signaling cascades known to regulate the NET, including Ca(2+)/Ca(2+)-calmodulin dependent kinase and protein kinases A, C and G, did not affect the ability of reserpine to reduce [(3)H]NE uptake. Treatment of PC12 cells with the catecholamine depleting agent, alpha-methyl-p-tyrosine, increased [(3)H]NE uptake and eliminated the inhibitory effects of reserpine on [(3)H]NE uptake. Reserpine non-competitively inhibits NET activity through a Ca(2+)-independent process that requires catecholamine storage vesicles, revealing a novel pharmacological method to modify NET function. Further characterization of the molecular nature of reserpine's action could lead to the development of alternative therapeutic strategies for treating disorders known to be benefitted by treatment with traditional competitive NET inhibitors.

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

    SciTech Connect

    Goldsmith, M.H.M.

    1989-12-31

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

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

    SciTech Connect

    Goldsmith, M.H.M.

    1986-01-01

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

  4. Effect of barium ion on p-aminohippurate transport in basolateral membrane vesicles isolated from rat kidney cortex.

    PubMed

    Hori, M; Gemba, M

    1985-06-01

    To clarify the cause of the stimulation of p-aminohippurate (PAH) accumulation in rat kidney cortical slices by barium, an experiment was carried out with basolateral membrane vesicles isolated from rat kidney cortex. The effect of barium on PAH uptake by the membrane vesicles was compared with that of verapamil which also stimulated PAH accumulation in the slices. The enzyme marker for basolateral membrane, (Na+ + K+)- ATPase, was enriched 15-fold and the brushborder enzyme marker, alkaline phosphatase, was 1.3-fold in our membrane preparation. Contamination in this preparation by lysosomes, mitochondria and cytosol was also low but that by endoplasmic reticulum was slightly high as judged by the enzyme markers. PAH uptake by the membrane vesicles possessed the usual characteristics, i.e., sodium-dependence and probenecid-sensitivity. PAH uptake by the membrane vesicles was enhanced by barium, but not by verapamil. On the other hand, barium did not affect tetraethylammonium (TEA) uptake by the vesicles, and verapamil strongly inhibited it. Manganese also stimulated PAH uptake to the same extent as did barium, but calcium and strontium did not affect the uptake. Barium did not act on sodium transport in the membrane vesicles. An 'anion-sensitively transported lipophilic cation', triphenylmethylphosphonium iodide (TPMP), uptake was depressed by barium. These results suggest that barium stimulates selectively PAH uptake in basolateral membrane vesicles. Its stimulatory action may contribute at least partly to an increase in PAH accumulation in rat kidney cortical slices by this ion and may prove useful in an analysis of the mechanism of PAH transport system in renal basolateral membranes.

  5. Substituted quinolines as inhibitors of L-glutamate transport into synaptic vesicles.

    PubMed

    Bartlett, R D; Esslinger, C S; Thompson, C M; Bridges, R J

    1998-07-01

    This study investigated the structure-activity relationships and kinetic properties of a library of kynurenate analogues as inhibitors of 3H-L-glutamate transport into rat forebrain synaptic vesicles. The lack of inhibitory activity observed with the majority of the monocyclic pyridine derivatives suggested that the second aromatic ring of the quinoline-based compounds played a significant role in binding to the transporter. A total of two kynurenate derivatives, xanthurenate and 7-chloro-kynurenate, differing only in the carbocyclic ring substituents, were identified as potent competitive inhibitors, exhibiting Ki values of 0.19 and 0.59 mM, respectively. The Km value for L-glutamate was found to be 2.46 mM. Parallel experiments demonstrated that while none of the kynurenate analogues tested effectively inhibited the synaptosomal transport of 3H-D-aspartate, some cross-reactivity was observed with the EAA ionotropic receptors. Molecular modeling studies were carried out with the identified inhibitors and glutamate in an attempt to preliminarily define the pharmacophore of the vesicular transporter. It is hypothesized that the ability of the kynurenate analogues to bind to the transporter may be tied to the capacity of the quinoline carbocyclic ring to mimic the negative charge of the gamma-carboxylate of glutamate. A total of two low energy solution conformers of glutamate were identified that exhibited marked functional group overlap with the most potent inhibitor, xanthurenate. These results help to further refine the pharmacological specificity of the glutamate binding site on the vesicular transporter and identify a series of inhibitors with which to investigate transporter function.

  6. Pep7p provides a novel protein that functions in vesicle-mediated transport between the yeast Golgi and endosome.

    PubMed Central

    Webb, G C; Zhang, J; Garlow, S J; Wesp, A; Riezman, H; Jones, E W

    1997-01-01

    Saccharomyces cerevisiae pep7 mutants are defective in transport of soluble vacuolar hydrolases to the lysosome-like vacuole. PEP7 is a nonessential gene that encodes a hydrophilic protein of 515 amino acids. A cysteine-rich tripartite motif in the N-terminal half of the polypeptide shows striking similarity to sequences found in many other eukaryotic proteins. Several of these proteins are thought to function in the vacuolar/lysosomal pathway. Mutations that change highly conserved cysteine residues in this motif lead to a loss of Pep7p function. Kinetic studies demonstrate that Pep7p function is required for the transport of the Golgi-precursors of the soluble hydrolases carboxypeptidase Y, proteinase A, and proteinase B to the endosome. Integral membrane hydrolase alkaline phosphatase is transported to the vacuole by a parallel intracellular pathway that does not require Pep7p function. pep7 mutants accumulate a 40-60-nm vesicle population, suggesting that Pep7p functions in a vesicle consumption step in vesicle-mediated transport of soluble hydrolases to the endosome. Whereas pep7 mutants demonstrate no defects in endocytic uptake at the plasma membrane, the mutants demonstrate defects in transport of receptor-mediated macromolecules through the endocytic pathway. Localization studies indicate that Pep7p is found both as a soluble cytoplasmic protein and associated with particulate fractions. We conclude that Pep7p functions as a novel regulator of vesicle docking and/or fusion at the endosome. Images PMID:9168472

  7. Effects of uricosuric and antiuricosuric agents on urate transport in human brush-border membrane vesicles.

    PubMed

    Roch-Ramel, F; Guisan, B; Diezi, J

    1997-02-01

    Inhibition of [14C]-urate uptake by uricosuric and antiuricosuric agents was investigated in human brush-border membrane vesicles, urate being transported either by anion exchange mechanisms or by voltage sensitive pathway. The IC50 for drugs on [14C]-urate uptake in vesicles loaded with 1 mM cold urate or with 5 mM lactate was, respectively: 0.7 and 0.3 microM for benzbromarone; 6 and 4 microM for salicylate; 133 and 13 microM for losartan; 520 and 190 microM for sulfinpyrazone and 807 and 150 microM, for probenecid. The IC50 ratio for [14C]-urate uptake in exchange for cold urate or for lactate varied from about 1 for salicylate to 10 for losartan, supporting the hypothesis that two distinct anion exchangers are involved in urate transport. Application of Hill equation revealed that urate/anion exchangers have more than one binding site, possibly two binding sites with high cooperativity, for benzbromarone and sulfinpyrazone, but only one for probenecid, salicylate and losartan. The uricosuric diuretic, tienilic acid was 10 to 50 times more potent than hydrochlorothiazide, chlorothiazide and furosemide, for inhibiting [14C]-urate uptake in exchange for cold urate. This higher potency is the reason of its uricosuric properties. All uricosuric agents, as well as the antiuricosuric agents, pyrazinoate and ethambutol, had a much lower potency for inhibiting [14C]-urate uptake through the voltage sensitive pathway (apical secretory step) than through the urate/anion exchangers. This suggests that antiuricosuria, induced by pyrazinoate and ethambutol, as well as by low concentrations of uricosuric agents, does not result from an inhibition of the apical voltage sensitive pathway.

  8. Analysis of COPII vesicles indicates a role for the Emp47-Ssp120 complex in transport of cell surface glycoproteins

    PubMed Central

    Margulis, Neil G.; Wilson, Joshua D.; Bentivoglio, Christine M.; Dhungel, Nripesh; Gitler, Aaron D.; Barlowe, Charles

    2015-01-01

    Coat protein complex II (COPII) vesicle formation at the endoplasmic reticulum (ER) transports nascent secretory proteins forward to the Golgi complex. To further define the machinery that packages secretory cargo and targets vesicles to Golgi membranes, we performed a comprehensive proteomic analysis of purified COPII vesicles. In addition to previously known proteins, we identified new vesicle proteins including Coy1, Sly41 and Ssp120, which were efficiently packaged into COPII vesicles for trafficking between the ER and Golgi compartments. Further characterization of the putative calcium-binding Ssp120 protein revealed a tight association with Emp47 and in emp47Δ cells Ssp120 was mislocalized and secreted. Genetic analyses demonstrated that EMP47 and SSP120 display identical synthetic positive interactions with IRE1 and synthetic negative interactions with genes involved in cell wall assembly. Our findings support a model in which the Emp47-Ssp120 complex functions in transport of plasma membrane glycoproteins through the early secretory pathway. PMID:26650540

  9. Molecular characterization of a trafficking organelle: dissecting the axonal paths of calsyntenin-1 transport vesicles.

    PubMed

    Steuble, Martin; Gerrits, Bertran; Ludwig, Alexander; Mateos, José María; Diep, Tu-My; Tagaya, Mitsuo; Stephan, Alexander; Schätzle, Philipp; Kunz, Beat; Streit, Peter; Sonderegger, Peter

    2010-11-01

    Kinesin motors play crucial roles in the delivery of membranous cargo to its destination and thus for the establishment and maintenance of cellular polarization. Recently, calsyntenin-1 was identified as a cargo-docking protein for Kinesin-1-mediated axonal transport of tubulovesicular organelles along axons of central nervous system neurons. To further define the function of calsyntenin-1, we immunoisolated calsyntenin-1 organelles from murine brain homogenates and determined their proteome by MS. We found that calsyntenin-1 organelles are endowed with components of the endosomal trafficking machinery and contained the β-amyloid precursor protein (APP). Detailed biochemical analyses of calsyntenin-1 immunoisolates in conjunction with immunocytochemical colocalization studies with cultured hippocampal neurons, using endosomal marker proteins for distinct subcompartments of the endosomal pathways, indicated that neuronal axons contain at least two distinct, nonoverlapping calsyntenin-1-containing transport packages: one characterized as early-endosomal, APP positive, the other as recycling-endosomal, APP negative. We postulate that calsyntenin-1 acts as a general mediator of anterograde axonal transportation of endosomal vesicles. In this role, calsyntenin-1 may actively contribute to axonal growth and pathfinding in the developing as well as to the maintenance of neuronal polarity in the adult nervous system; further, it may actively contribute to the stabilization of APP during its anterograde axonal trajectory.

  10. Cercospora beticola Toxin Inhibits Vanadate-Sensitive H+ Transport in Corn Root Membrane Vesicles

    PubMed Central

    Blein, Jean-Pierre; Bourdil, Isabelle; Rossignol, Michel; Scalla, René

    1988-01-01

    The effect of Cercospora beticola toxin on the transport of protons by vanadate-sensitive ATPase was studied with corn (Zea mays) root microsomal vesicles prepared by differential centrifugation, sedimentation through a sucrose cushion, and washing with Triton X-100 plus KBr. In these preparations, addition of ATP induced intravesicular H+-accumulation as evidenced by a rapid quenching of the fluorescence of 9-amino-6-chloro-2-methoxy acridine. This quenching was relatively unaffected by inhibitors of mitochondrial and tonoplast-type ATPases, but was strongly reduced by inhibitors of plasma membrane H+-ATPase. C. beticola toxin markedly inhibited ATP dependent H+-transport, and this effect increased with the length of preincubation with the toxin. The same observations were made concerning ATPase activity. Inhibition of H+-transport was greater at pH 7.3 than at pH 5.7. Lineweaver-Burk plot analysis showed that inhibition kinetics were competitive with respect to ATP. These data suggest a direct effect of C. beticola toxin on vanadate-sensitive ATPase presumed to be associated with the plasma membrane. PMID:16666321

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

    PubMed

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

    2015-01-01

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

  12. Nuclear transportation of exogenous epidermal growth factor receptor and androgen receptor via extracellular vesicles.

    PubMed

    Read, Jolene; Ingram, Alistair; Al Saleh, Hassan A; Platko, Khrystyna; Gabriel, Kathleen; Kapoor, Anil; Pinthus, Jehonathan; Majeed, Fadwa; Qureshi, Talha; Al-Nedawi, Khalid

    2017-01-01

    Epidermal growth factor receptor (EGFR) plays a central role in the progression of several human malignancies. Although EGFR is a membrane receptor, it undergoes nuclear translocation, where it has a distinct signalling pathway. Herein, we report a novel mechanism by which cancer cells can directly transport EGFR to the nucleus of other cells via extracellular vesicles (EVs). The transported receptor is active and stimulates the nuclear EGFR pathways. Interestingly, the translocation of EGFR via EVs occurs independently of the nuclear localisation sequence that is required for nuclear translocation of endogenous EGFR. Also, we found that the mutant receptor EGFRvIII could be transported to the nucleus of other cells via EVs. To assess the role of EVs in the regulation of an actual nuclear receptor, we studied the regulation of androgen receptor (AR). We found that full-length AR and mutant variant ARv7 are secreted in EVs derived from prostate cancer cell lines and could be transported to the nucleus of AR-null cells. The EV-derived AR was able to bind the androgen-responsive promoter region of prostate specific antigen, and recruit RNA Pol II, an indication of active transcription. The nuclear-translocated AR via EVs enhanced the proliferation of acceptor cells in the absence of androgen. Finally, we provide evidence that nuclear localisation of AR could occur in vivo via orthotopically-injected EVs in male SCID mice prostate glands. To our knowledge, this is the first study showing the nuclear translocation of nuclear receptors via EVs, which significantly extends the role of EVs as paracrine transcriptional regulators.

  13. VAN3 ARF-GAP-mediated vesicle transport is involved in leaf vascular network formation.

    PubMed

    Koizumi, Koji; Naramoto, Satoshi; Sawa, Shinichiro; Yahara, Natsuko; Ueda, Takashi; Nakano, Akihiko; Sugiyama, Munetaka; Fukuda, Hiroo

    2005-04-01

    Within the leaf of an angiosperm, the vascular system is constructed in a complex network pattern called venation. The formation of this vein pattern has been widely studied as a paradigm of tissue pattern formation in plants. To elucidate the molecular mechanism controlling the vein patterning process, we previously isolated Arabidopsis mutants van1 to van7, which show a discontinuous vein pattern. Here we report the phenotypic analysis of the van3 mutant in relation to auxin signaling and polar transport, and the molecular characterization of the VAN3 gene and protein. Double mutant analyses with pin1, emb30-7/gn and mp, and physiological analyses using the auxin-inducible marker DR5::GUS and an auxin transport inhibitor indicated that VAN3 may be involved in auxin signal transduction, but not in polar auxin transport. Positional cloning identified VAN3 as a gene that encodes an adenosine diphosphate (ADP)-ribosylation factor-guanosine triphosphatase (GTPase) activating protein (ARF-GAP). It resembles animal ACAPs and contains four domains: a BAR (BIN/amphiphysin/RVS) domain, a pleckstrin homology (PH) domain, an ARF-GAP domain and an ankyrin (ANK)-repeat domain. Recombinant VAN3 protein showed GTPase-activating activity and a specific affinity for phosphatidylinositols. This protein can self-associate through the N-terminal BAR domain in the yeast two-hybrid system. Subcellular localization analysis by double staining for Venus-tagged VAN3 and several green-fluorescent-protein-tagged intracellular markers indicated that VAN3 is located in a subpopulation of the trans-Golgi network (TGN). Our results indicate that the expression of this gene is induced by auxin and positively regulated by VAN3 itself, and that a specific ACAP type of ARF-GAP functions in vein pattern formation by regulating auxin signaling via a TGN-mediated vesicle transport system.

  14. Biogenesis of the crystalloid organelle in Plasmodium involves microtubule-dependent vesicle transport and assembly

    PubMed Central

    Saeed, Sadia; Tremp, Annie Z.; Dessens, Johannes T.

    2015-01-01

    Malaria parasites possess unique subcellular structures and organelles. One of these is the crystalloid, a multivesicular organelle that forms during the parasite’s development in vector mosquitoes. The formation and function of these organelles remain poorly understood. A family of six conserved and modular proteins named LCCL-lectin adhesive-like proteins (LAPs), which have essential roles in sporozoite transmission, localise to the crystalloids. In this study we analyse crystalloid formation using transgenic Plasmodium berghei parasites expressing GFP-tagged LAP3. We show that deletion of the LCCL domain from LAP3 causes retarded crystalloid development, while knockout of LAP3 prevents formation of the organelle. Our data reveal that the process of crystalloid formation involves active relocation of endoplasmic reticulum-derived vesicles to common assembly points via microtubule-dependent transport. Inhibition of microtubule-dependent cargo transport disrupts this process and replicates the LCCL domain deletion mutant phenotype in wildtype parasites. These findings provide the first clear insight into crystalloid biogenesis, demonstrating a fundamental role for the LAP family in this process, and identifying the crystalloid and its formation as potential targets for malaria transmission control. PMID:25900212

  15. Distinct stages in the recognition, sorting, and packaging of proTGFα into COPII-coated transport vesicles

    PubMed Central

    Zhang, Pengcheng; Schekman, Randy

    2016-01-01

    In addition to its role in forming vesicles from the endoplasmic reticulum (ER), the coat protein complex II (COPII) is also responsible for selecting specific cargo proteins to be packaged into COPII transport vesicles. Comparison of COPII vesicle formation in mammalian systems and in yeast suggested that the former uses more elaborate mechanisms for cargo recognition, presumably to cope with a significantly expanded repertoire of cargo that transits the secretory pathway. Using proTGFα, the transmembrane precursor of transforming growth factor α (TGFα), as a model cargo protein, we demonstrate in cell-free assays that at least one auxiliary cytosolic factor is specifically required for the efficient packaging of proTGFα into COPII vesicles. Using a knockout HeLa cell line generated by CRISPR/Cas9, we provide functional evidence showing that a transmembrane protein, Cornichon-1 (CNIH), acts as a cargo receptor of proTGFα. We show that both CNIH and the auxiliary cytosolic factor(s) are required for efficient recruitment of proTGFα to the COPII coat in vitro. Moreover, we provide evidence that the recruitment of cargo protein by the COPII coat precedes and may be distinct from subsequent cargo packaging into COPII vesicles. PMID:27122606

  16. Mechanisms of transport of nontransferrin-bound iron in basolateral and canalicular rat liver plasma membrane vesicles

    SciTech Connect

    Wright, T.L.; Lake, J.R. )

    1990-09-01

    Although most iron in plasma is bound to transferrin, recent evidence suggests that the nontransferrin-bound fraction contributes to hepatic iron loading and toxicity seen in iron-overload disorders. Our studies of isolated perfused rat liver previously demonstrated saturable uptake of nontransferrin-bound iron that continues despite hepatic iron overload. To further characterize the mechanism of transport of this form of iron, we measured binding of 55Fe-labeled ferrous ascorbate to rat liver plasma membrane vesicles under varying conditions. Binding of 5 mumol/L iron by both basolateral and canalicular membranes was time-dependent and linear for the first 5 sec. Initial rate of binding of ferrous ascorbate to basolateral membrane vesicles was temperature dependent and increased by calcium but, in contrast to the perfused rat liver, was not inhibited by other divalent cations. Binding velocities by basolateral membrane vesicles were saturable at increasing iron concentration (Km = 33 mumol/L, Vmax = 16 pmol/mg protein/sec). Ferrous iron binding by canalicular membrane vesicles was also temperature dependent, but initial association rates were not saturable over the concentration range studied (2 to 20 mumol/L). We conclude that nontransferrin-bound iron associates with basolateral liver plasma membrane vesicles by a saturable mechanism sensitive to temperature and calcium and consistent with a membrane carrier. Other divalent cations do not inhibit membrane association but may compete for a subsequent cytosolic binding site.

  17. An endosomal beta COP is involved in the pH-dependent formation of transport vesicles destined for late endosomes

    PubMed Central

    1996-01-01

    In this paper, we show that beta COP is present on endosomes and is required for the formation of vesicles which mediate transport from early to late endosomes. Both the association of beta COP to endosomal membranes as well as transport vesicle formation depend on the lumenal pH. We find that epsilon COP, but not gamma COP, is also associated to endosomes, and that this association is also lumenal pH dependent. Our data, thus, indicate that a subset of COPs is part of the mechanism regulating endosomal membrane transport, and that membrane association of these COPs is controlled by the acidic properties of early endosomes, presumably via a trans-membrane pH sensor. PMID:8601610

  18. Regulation of vesicle transport and cell motility by Golgi-localized Dbs

    PubMed Central

    Fitzpatrick, Ethan R; Hu, Tinghui; Ciccarelli, Bryan T; Whitehead, Ian P

    2014-01-01

    DBS/MCF2L has been recently identified as a risk locus for osteoarthritis. It encodes a guanine nucleotide exchange factor (Dbs) that has been shown to regulate both normal and tumor cell motility. In the current study, we have determined that endogenous Dbs is predominantly expressed as 2 isoforms, a 130 kDa form (Dbs-130) that is localized to the Golgi complex, and an 80 kDa form (Dbs-80) that is localized to the endoplasmic reticulum (ER). We have previously described an inhibitor that binds to the RhoGEF domain of Dbs and blocks its transforming activity. Here we show that the inhibitor localizes to the Golgi, where it specifically interacts with Dbs-130. Inhibition of endogenous Dbs-130 activity is associated with reduced levels of activated Cdc42, enlarged Golgi, and resistance to Brefeldin A-mediated Golgi dispersal, suggesting a role for Dbs in vesicle transport. Cells treated with the inhibitor exhibit normal protein transport from the ER to the Golgi, but are defective in transport from the Golgi to the plasma membrane. Inhibition of Dbs-130 in MDA-MB-231 human breast tumor cells limits motility in both transwell and wound healing assays, but appears to have no effect on the organization of the microtubule cytoskeleton. The reduced motility is associated with a failure to reorient the Golgi toward the leading edge. This is consistent with the Golgi localization, and suggests that the Dbs-130 regulates aspects of the secretory pathway that are required to support cell polarization during directed migration. PMID:25483302

  19. Dimethyltryptamine and other hallucinogenic tryptamines exhibit substrate behavior at the serotonin uptake transporter and the vesicle monoamine transporter.

    PubMed

    Cozzi, Nicholas V; Gopalakrishnan, Anupama; Anderson, Lyndsey L; Feih, Joel T; Shulgin, Alexander T; Daley, Paul F; Ruoho, Arnold E

    2009-12-01

    N,N-dimethyltryptamine (DMT) is a potent plant hallucinogen that has also been found in human tissues. When ingested, DMT and related N,N-dialkyltryptamines produce an intense hallucinogenic state. Behavioral effects are mediated through various neurochemical mechanisms including activity at sigma-1 and serotonin receptors, modification of monoamine uptake and release, and competition for metabolic enzymes. To further clarify the pharmacology of hallucinogenic tryptamines, we synthesized DMT, N-methyl-N-isopropyltryptamine (MIPT), N,N-dipropyltryptamine (DPT), and N,N-diisopropyltryptamine. We then tested the abilities of these N,N-dialkyltryptamines to inhibit [(3)H]5-HT uptake via the plasma membrane serotonin transporter (SERT) in human platelets and via the vesicle monoamine transporter (VMAT2) in Sf9 cells expressing the rat VMAT2. The tryptamines were also tested as inhibitors of [(3)H]paroxetine binding to the SERT and [(3)H]dihydrotetrabenazine binding to VMAT2. Our results show that DMT, MIPT, DPT, and DIPT inhibit [(3)H]5-HT transport at the SERT with K ( I ) values of 4.00 +/- 0.70, 8.88 +/- 4.7, 0.594 +/- 0.12, and 2.32 +/- 0.46 microM, respectively. At VMAT2, the tryptamines inhibited [(3)H]5-HT transport with K ( I ) values of 93 +/- 6.8, 20 +/- 4.3, 19 +/- 2.3, and 19 +/- 3.1 muM, respectively. On the other hand, the tryptamines were very poor inhibitors of [(3)H]paroxetine binding to SERT and of [(3)H]dihydrotetrabenazine binding to VMAT2, resulting in high binding-to-uptake ratios. High binding-to-uptake ratios support the hypothesis that the tryptamines are transporter substrates, not uptake blockers, at both SERT and VMAT2, and also indicate that there are separate substrate and inhibitor binding sites within these transporters. The transporters may allow the accumulation of tryptamines within neurons to reach relatively high levels for sigma-1 receptor activation and to function as releasable transmitters.

  20. Porewater methane transport within the gas vesicles of diurnally migrating Chaoborus spp.: An energetic advantage.

    PubMed

    McGinnis, Daniel F; Flury, Sabine; Tang, Kam W; Grossart, Hans-Peter

    2017-03-14

    Diurnally-migrating Chaoborus spp. reach populations of up to 130,000 individuals m(-2) in lakes up to 70 meters deep on all continents except Antarctica. Linked to eutrophication, migrating Chaoborus spp. dwell in the anoxic sediment during daytime and feed in the oxic surface layer at night. Our experiments show that by burrowing into the sediment, Chaoborus spp. utilize the high dissolved gas partial pressure of sediment methane to inflate their tracheal sacs. This mechanism provides a significant energetic advantage that allows the larvae to migrate via passive buoyancy rather than more energy-costly swimming. The Chaoborus spp. larvae, in addition to potentially releasing sediment methane bubbles twice a day by entering and leaving the sediment, also transport porewater methane within their gas vesicles into the water column, resulting in a flux of 0.01-2 mol m(-2) yr(-1) depending on population density and water depth. Chaoborus spp. emerging annually as flies also result in 0.1-6 mol m(-2) yr(-1) of carbon export from the system. Finding the tipping point in lake eutrophication enabling this methane-powered migration mechanism is crucial for ultimately reconstructing the geographical expansion of Chaoborus spp., and the corresponding shifts in the lake's biogeochemistry, carbon cycling and food web structure.

  1. Global gene expression analysis reveals a link between NDRG1 and vesicle transport.

    PubMed

    Askautrud, Hanne A; Gjernes, Elisabet; Gunnes, Gjermund; Sletten, Marit; Ross, Douglas T; Børresen-Dale, Anne Lise; Iversen, Nina; Tranulis, Michael A; Frengen, Eirik

    2014-01-01

    N-myc downstream-regulated gene 1 (NDRG1) is induced by cellular stress such as hypoxia and DNA damage, and in humans, germ line mutations cause Charcot-Marie-Tooth disease. However, the cellular roles of NDRG1 are not fully understood. Previously, NDRG1 was shown to mediate doxorubicin resistance under hypoxia, suggesting a role for NDRG1 in cell survival under these conditions. We found decreased apoptosis in doxorubicin-treated cells expressing NDRG1 shRNAs under normoxia, demonstrating a requirement for NDRG1 in apoptosis in breast epithelial cells under normal oxygen pressure. Also, different cellular stress regimens, such as hypoxia and doxorubicin treatment, induced NDRG1 through different stress signalling pathways. We further compared expression profiles in human breast epithelial cells ectopically over-expressing NDRG1 with cells expressing NDRG1 shRNAs in order to identify biological pathways where NDRG1 is involved. The results suggest that NDRG1 may have roles connected to vesicle transport.

  2. Extracellular vesicles from Paracoccidioides pathogenic species transport polysaccharide and expose ligands for DC-SIGN receptors

    DOE PAGES

    da Silva, Roberta Peres; Heiss, Christian; Black, Ian; ...

    2015-09-21

    Extracellular vesicles (EVs) mediate non-conventional transport of molecules across the fungal cell wall. We aimed at describing the carbohydrate composition and surface carbohydrate epitopes of EVs isolated from the pathogenic fungi Paracoccidioides brasiliensis and P. lutzii using standard procedures. Total EV carbohydrates were ethanol-precipitated from preparations depleted of lipids and proteins, then analyzed by chemical degradation, gas chromatography-mass spectrometry, nuclear magnetic resonance and size-exclusion chromatography. EV glycosyl residues of Glc, Man, and Gal comprised most probably two major components: a high molecular mass 4,6-α-glucan and a galactofuranosylmannan, possibly an oligomer, bearing a 2-α-Manp main chain linked to β-Galf (1,3) andmore » α-Manp (1,6) end units. The results also suggested the presence of small amounts of a (1→6)- Manp polymer, (1→3)-glucan and (1→6)-glucan. Glycan microarrays allowed identification of EV surface lectin(s), while plant lectin microarray profiling revealed terminal Man and GlcNAc residues exposed at the EVs surface. Mammalian lectin microarray profiling showed that DC-SIGN receptors recognized surface carbohydrate in Paracoccidioides EVs. Our results suggest that oligosaccharides, cytoplasmic storage, and cell wall polysaccharides can be exported in fungal EVs, which also expose surface PAMPs and lectins. As a result, the role of these newly identified components in the interaction with the host remains to be unraveled.« less

  3. Porewater methane transport within the gas vesicles of diurnally migrating Chaoborus spp.: An energetic advantage

    PubMed Central

    McGinnis, Daniel F.; Flury, Sabine; Tang, Kam W.; Grossart, Hans-Peter

    2017-01-01

    Diurnally-migrating Chaoborus spp. reach populations of up to 130,000 individuals m−2 in lakes up to 70 meters deep on all continents except Antarctica. Linked to eutrophication, migrating Chaoborus spp. dwell in the anoxic sediment during daytime and feed in the oxic surface layer at night. Our experiments show that by burrowing into the sediment, Chaoborus spp. utilize the high dissolved gas partial pressure of sediment methane to inflate their tracheal sacs. This mechanism provides a significant energetic advantage that allows the larvae to migrate via passive buoyancy rather than more energy-costly swimming. The Chaoborus spp. larvae, in addition to potentially releasing sediment methane bubbles twice a day by entering and leaving the sediment, also transport porewater methane within their gas vesicles into the water column, resulting in a flux of 0.01–2 mol m−2 yr−1 depending on population density and water depth. Chaoborus spp. emerging annually as flies also result in 0.1–6 mol m−2 yr−1 of carbon export from the system. Finding the tipping point in lake eutrophication enabling this methane-powered migration mechanism is crucial for ultimately reconstructing the geographical expansion of Chaoborus spp., and the corresponding shifts in the lake’s biogeochemistry, carbon cycling and food web structure. PMID:28290556

  4. Porewater methane transport within the gas vesicles of diurnally migrating Chaoborus spp.: An energetic advantage

    NASA Astrophysics Data System (ADS)

    McGinnis, Daniel F.; Flury, Sabine; Tang, Kam W.; Grossart, Hans-Peter

    2017-03-01

    Diurnally-migrating Chaoborus spp. reach populations of up to 130,000 individuals m‑2 in lakes up to 70 meters deep on all continents except Antarctica. Linked to eutrophication, migrating Chaoborus spp. dwell in the anoxic sediment during daytime and feed in the oxic surface layer at night. Our experiments show that by burrowing into the sediment, Chaoborus spp. utilize the high dissolved gas partial pressure of sediment methane to inflate their tracheal sacs. This mechanism provides a significant energetic advantage that allows the larvae to migrate via passive buoyancy rather than more energy-costly swimming. The Chaoborus spp. larvae, in addition to potentially releasing sediment methane bubbles twice a day by entering and leaving the sediment, also transport porewater methane within their gas vesicles into the water column, resulting in a flux of 0.01–2 mol m‑2 yr‑1 depending on population density and water depth. Chaoborus spp. emerging annually as flies also result in 0.1–6 mol m‑2 yr‑1 of carbon export from the system. Finding the tipping point in lake eutrophication enabling this methane-powered migration mechanism is crucial for ultimately reconstructing the geographical expansion of Chaoborus spp., and the corresponding shifts in the lake’s biogeochemistry, carbon cycling and food web structure.

  5. Aldosterone induction of electrogenic sodium transport in the apical membrane vesicles of rat distal colon

    SciTech Connect

    Rajendran, V.M.; Kashgarian, M.; Binder, H.J. )

    1989-11-05

    Na-H exchange is present in apical membrane vesicles (AMV) isolated from distal colon of normal rats. Because in intact tissue aldosterone both induces amiloride-sensitive electrogenic sodium transport and inhibits electroneutral sodium absorption, these studies with AMV were designed to establish the effect of aldosterone on sodium transport. An outward-directed proton gradient stimulated 22Na uptake in AMV isolated from distal colon of normal and dietary sodium depleted (with elevated aldosterone levels) experimental rats. Unlike normal AMV, proton gradient-dependent 22Na uptake in experimental AMV was inhibited when uptake was measured under voltage-clamped conditions. 10 microM amiloride inhibited the initial rate of proton gradient-dependent 22Na uptake in AMV of normal and experimental rats by 30 and 75%, respectively. In contrast, 1 mM amiloride produced comparable inhibition (90 and 80%) of 22Na uptake in normal and experimental AMV. Intravesicular-negative potential stimulated 22Na uptake in experimental but not in normal AMV. This increase was inhibited by 90% by 10 microM amiloride. An analogue of amiloride, 5-(N-ethylisopropyl) amiloride (1 microM), a potent inhibitor of electroneutral Na-H exchange in AMV of normal rat distal colon, did not alter potassium diffusion potential-dependent 22Na uptake. Increasing sodium concentration saturated proton gradient-dependent 22Na uptake in normal AMV. However, in experimental AMV, 22Na uptake stimulated by both proton gradient and potassium diffusion potential did not saturate as a function of increasing sodium concentration. We conclude from these results that an electrically sensitive conductive channel, not electroneutral Na-H exchange, mediates 22Na uptake in AMV isolated from the distal colon of aldosterone rats.

  6. Effect of chronic (4 weeks) ingestion of ethanol on transport of proline into intestinal brush border membrane vesicles

    SciTech Connect

    Beesley, R.C.; Jones, T.D.

    1986-03-01

    Hamsters were separated into two groups and fed liquid diets ad lib. Controls were given a diet similar to that described by DeCarli and Lieber while alcoholics received the same diet containing 5% ethanol isocalorically substituted for sucrose. The volume of diet consumed daily and the gain in body weights of alcoholics were not significantly different from those of controls. After four weeks the animals were sacrificed and the upper third of the small intestine was used to prepare brush border membrane vesicles. In the presence of a Na/sup +/ gradient, uptake of proline into vesicles prepared from both groups was rapid, reaching a maximum accumulation in 1 to 2 min and then decreasing to the equilibrium level. To normalize the results, the amount of proline take up at each time point was divided by the amount present at equilibrium. From the normalized data it was concluded that both the rate of uptake and the maximum accumulation of proline into brush border membrane vesicles isolated from alcoholics were significantly less than those obtained with vesicles from controls. These results suggest that chronic ingestion of ethanol results in a reduction in Na/sup +/-dependent transport of proline across the brush border membrane and, thus, may contribute to the malnutrition which is frequently associated with chronic alcoholism.

  7. GPI anchor attachment is required for Gas1p transport from the endoplasmic reticulum in COP II vesicles.

    PubMed Central

    Doering, T L; Schekman, R

    1996-01-01

    Inositol starvation of auxotrophic yeast interrupts glycolipid biosynthesis and prevents lipid modification of a normally glycosyl phosphatidylinositol (GPI)-linked protein, Gas1p. The unanchored Gas1p precursor undergoes progressive modification in the endoplasmic reticulum (ER), but is not modified by Golgi-specific glycosylation. Starvation-induced defects in anchor assembly and protein processing are rapid, and occur without altered maturation of other proteins. Cells remain competent to manufacture anchor components and to process Gas1p efficiently once inositol is restored. Newly synthesized Gas1p is packaged into vesicles formed in vitro from perforated yeast spheroplasts incubated with either yeast cytosol or the purified Sec proteins (COP II) required for vesicle budding from the ER. In vitro synthesized vesicles produced by inositol-starved membranes do not contain detectable Gas1p. These studies demonstrate that COP II components fulfill the soluble protein requirements for packaging a GPI-anchored protein into ER-derived transport vesicles. However, GPI anchor attachment is required for this packaging to occur. Images PMID:8598201

  8. Roles of BLOC-1 and Adaptor Protein-3 Complexes in Cargo Sorting to Synaptic Vesicles

    PubMed Central

    Newell-Litwa, Karen; Salazar, Gloria; Smith, Yoland

    2009-01-01

    Neuronal lysosomes and their biogenesis mechanisms are primarily thought to clear metabolites and proteins whose abnormal accumulation leads to neurodegenerative disease pathology. However, it remains unknown whether lysosomal sorting mechanisms regulate the levels of membrane proteins within synaptic vesicles. Using high-resolution deconvolution microscopy, we identified early endosomal compartments where both selected synaptic vesicle and lysosomal membrane proteins coexist with the adaptor protein complex 3 (AP-3) in neuronal cells. From these early endosomes, both synaptic vesicle membrane proteins and characteristic AP-3 lysosomal cargoes can be similarly sorted to brain synaptic vesicles and PC12 synaptic-like microvesicles. Mouse knockouts for two Hermansky–Pudlak complexes involved in lysosomal biogenesis from early endosomes, the ubiquitous isoform of AP-3 (Ap3b1−/−) and muted, defective in the biogenesis of lysosome-related organelles complex 1 (BLOC-1), increased the content of characteristic synaptic vesicle proteins and known AP-3 lysosomal proteins in isolated synaptic vesicle fractions. These phenotypes contrast with those of the mouse knockout for the neuronal AP-3 isoform involved in synaptic vesicle biogenesis (Ap3b2−/−), in which the content of select proteins was reduced in synaptic vesicles. Our results demonstrate that lysosomal and lysosome-related organelle biogenesis mechanisms regulate steady-state synaptic vesicle protein composition from shared early endosomes. PMID:19144828

  9. Sec31 encodes an essential component of the COPII coat required for transport vesicle budding from the endoplasmic reticulum.

    PubMed Central

    Salama, N R; Chuang, J S; Schekman, R W

    1997-01-01

    The COPII vesicle coat protein promotes the formation of endoplasmic reticulum- (ER) derived transport vesicles that carry secretory proteins to the Golgi complex in Saccharomyces cerevisiae. This coat protein consists of Sar1p, the Sec23p protein complex containing Sec23p and Sec24p, and the Sec13p protein complex containing Sec13p and a novel 150-kDa protein, p150. Here, we report the cloning and characterization of the p150 gene. p150 is encoded by an essential gene. Depletion of this protein in vivo blocks the exit of secretory proteins from the ER and causes an elaboration of ER membranes, indicating that p150 is encoded by a SEC gene. Additionally, overproduction of the p150 gene product compromises the growth of two ER to Golgi sec mutants: sec16-2 and sec23-1. p150 is encoded by SEC31, a gene isolated in a genetic screen for mutations that accumulate unprocessed forms of the secretory protein alpha-factor. The sec31-1 mutation was mapped by gap repair, and sequence analysis revealed an alanine to valine change at position 1239, near the carboxyl terminus. Sec31p is a phosphoprotein and treatment of the Sec31p-containing fraction with alkaline phosphatase results in a 50-75% inhibition of transport vesicle formation activity in an ER membrane budding assay. Images PMID:9190202

  10. GRIP1 interlinks N-cadherin and AMPA receptors at vesicles to promote combined cargo transport into dendrites

    PubMed Central

    Heisler, Frank F.; Lee, Han Kyu; Gromova, Kira V.; Pechmann, Yvonne; Schurek, Beate; Ruschkies, Laura; Schroeder, Markus; Schweizer, Michaela; Kneussel, Matthias

    2014-01-01

    The GluA2 subunit of AMPA-type glutamate receptors (AMPARs) regulates excitatory synaptic transmission in neurons. In addition, the transsynaptic cell adhesion molecule N-cadherin controls excitatory synapse function and stabilizes dendritic spine structures. At postsynaptic membranes, GluA2 physically binds N-cadherin, underlying spine growth and synaptic modulation. We report that N-cadherin binds to PSD-95/SAP90/DLG/ZO-1 (PDZ) domain 2 of the glutamate receptor interacting protein 1 (GRIP1) through its intracellular C terminus. N-cadherin and GluA2-containing AMPARs are presorted to identical transport vesicles for dendrite delivery, and live imaging reveals cotransport of both proteins. The kinesin KIF5 powers GluA2/N-cadherin codelivery by using GRIP1 as a multilink interface. Notably, GluA2 and N-cadherin use different PDZ domains on GRIP1 to simultaneously bind the transport complex, and interference with either binding motif impairs the turnover of both synaptic cargoes. Depolymerization of microtubules, deletion of the KIF5 motor domain, or specific blockade of AMPAR exocytosis affects delivery of GluA2/N-cadherin vesicles. At the functional level, interference with this cotransport reduces the number of spine protrusions and excitatory synapses. Our data suggest the concept that the multi-PDZ-domain adaptor protein GRIP1 can act as a scaffold at trafficking vesicles in the combined delivery of AMPARs and N-cadherin into dendrites. PMID:24639525

  11. Effects of internal and external pH on amiloride-blockable Na transport across toad urinary bladder vesicles

    SciTech Connect

    Garty, H.; Civan, E.D.; Civan, M.M.

    1985-01-01

    The authors have examined the effect of internal and external pH on Na+ transport across toad bladder membrane vesicles. Of the total SSNa uptake measured 0.5-2.0 min after introducing tracer, 80 +/- 4% (mean +/- SE, n = 9) is blocked by the diuretic with a KI of 2 X 10(-8) M. Thus, this amiloride-sensitive flux is mediated by the apical sodium-selective channels. Varying the internal (cytosolic) pH over the physiologic range 7.0-8.0 had no effect on sodium transport; this result suggests that variation of intracellular pH in vivo has no direct apical effect on modulating sodium uptake. On the other hand, SSNa was directly and monotonically dependent on external pH. External acidification also reduced the amiloride-sensitive efflux across the walls of the vesicles. This inhibition of 22Na efflux was noted at external Na concentrations of both 0.2 microM and 53 mM. These results are different from those reported with whole toad bladder. A number of possible bases for these differences are considered and discussed. They suggest that the natriferic response induced by mucosal acidification of whole toad urinary bladder appears to operate indirectly through one or more factors, presumably cytosolic, present in whole cells and absent from the vesicles.

  12. Relationship between proton motive force and potassium ion transport in Halobacterium halobium envelope vesicles

    NASA Technical Reports Server (NTRS)

    Lanyi, J. K.; Helgerson, S. L.; Silverman, M. P.

    1979-01-01

    The permeability of Halobacterium halobium vesicle membranes to potassium ions was investigated, and possible mechanisms for the regulation of the gradient of protons by the transmembrane movement of these ions were studied. The lack of a potassium ion diffusion potential in the absence of valinomycin, light-induced electrical potentials in excess of the chemical potential difference for potassium ions, and direct measurements of potassium ion influx during illumination show that the membranes are relatively impermeable to these ions. As a result of sodium ion extrusion during illumination, chlorine ions and water must be lost and the vesicles collapse. The light-induced collapse of vesicles is diminished only if the influx of potassium ions is increased.

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

    SciTech Connect

    Goldsmith, M.H.

    1985-01-01

    The accumulation of indoleacetic acid (IAA) inside microsomal vesicles depends on the presence of a pH gradient and is reversible when the ..delta..pH is collapsed by ionosphores. Accumulation is stimulated by either napthylphthalamic acid or TIBA. The accumulation of IAA by the vesicles can be saturated. At concentrations of 1 ..mu..M or less, IAA, synthetic auxins, or auxin antagonists do not affect the pH gradient, but decrease the accumulation of /sup 3/H-IAA, and therefore compete specifically for uptake. Concentrations of 10 ..mu..M and above, uptake of either the auxins or weak acids is sufficient to overcome the buffering capacity of the solution within the vesicles. The collapse of the pH gradient by such high concentrations affects uptake of either /sup 3/H-IAA or /sup 14/C-BA to similar extents and thus is nonspecific. 3 refs.

  14. A vacuolar-type proton pump in a vesicle fraction enriched with potassium transporting plasma membranes from tobacco hornworm midgut

    SciTech Connect

    Wieczorek, H.; Weerth, S.; Schindlbeck, M.; Klein, U.

    1989-07-05

    Mg-ATP dependent electrogenic proton transport, monitored with fluorescent acridine orange, 9-aminoacridine, and oxonol V, was investigated in a fraction enriched with potassium transporting goblet cell apical membranes of Manduca sexta larval midgut. Proton transport and the ATPase activity from the goblet cell apical membrane exhibited similar substrate specificity and inhibitor sensitivity. ATP and GTP were far better substrates than UTP, CTP, ADP, and AMP. Azide and vanadate did not inhibit proton transport, whereas 100 microM N,N'-dicyclohexylcarbodiimide and 30 microM N-ethylmaleimide were inhibitors. The pH gradient generated by ATP and limiting its hydrolysis was 2-3 pH units. Unlike the ATPase activity, proton transport was not stimulated by KCl. In the presence of 20 mM KCl, a proton gradient could not be developed or was dissipated. Monovalent cations counteracted the proton gradient in an order of efficacy like that for stimulation of the membrane-bound ATPase activity: K+ = Rb+ much greater than Li+ greater than Na+ greater than choline (chloride salts). Like proton transport, the generation of an ATP dependent and azide- and vanadate-insensitive membrane potential (vesicle interior positive) was prevented largely by 100 microM N,N'-dicyclohexylcarbodiimide and 30 microM N-ethylmaleimide. Unlike proton transport, the membrane potential was not affected by 20 mM KCl. In the presence of 150 mM choline chloride, the generation of a membrane potential was suppressed, whereas the pH gradient increased 40%, indicating an anion conductance in the vesicle membrane. Altogether, the results led to the following new hypothesis of electrogenic potassium transport in the lepidopteran midgut. A vacuolar-type electrogenic ATPase pumps protons across the apical membrane of the goblet cell, thus energizing electroneutral proton/potassium antiport. The result is a net active and electrogenic potassium flux.

  15. Role of Tetanus Neurotoxin Insensitive Vesicle-Associated Membrane Protein (Ti-Vamp) in Vesicular Transport Mediating Neurite Outgrowth

    PubMed Central

    Martinez-Arca, Sonia; Alberts, Philipp; Zahraoui, Ahmed; Louvard, Daniel; Galli, Thierry

    2000-01-01

    How vesicular transport participates in neurite outgrowth is still poorly understood. Neurite outgrowth is not sensitive to tetanus neurotoxin thus does not involve synaptobrevin-mediated vesicular transport to the plasma membrane of neurons. Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is a vesicle-SNARE (soluble N-ethylmaleimide-sensitive fusion protein [NSF] attachment protein [SNAP] receptor), involved in transport to the apical plasma membrane in epithelial cells, a tetanus neurotoxin-resistant pathway. Here we show that TI-VAMP is essential for vesicular transport-mediating neurite outgrowth in staurosporine-differentiated PC12 cells. The NH2-terminal domain, which precedes the SNARE motif of TI-VAMP, inhibits the association of TI-VAMP with synaptosome-associated protein of 25 kD (SNAP25). Expression of this domain inhibits neurite outgrowth as potently as Botulinum neurotoxin E, which cleaves SNAP25. In contrast, expression of the NH2-terminal deletion mutant of TI-VAMP increases SNARE complex formation and strongly stimulates neurite outgrowth. These results provide the first functional evidence for the role of TI-VAMP in neurite outgrowth and point to its NH2-terminal domain as a key regulator in this process. PMID:10811829

  16. Electron transport and electrochemical proton gradient in membrane vesicles of Clostridium thermoautotrophicum.

    PubMed Central

    Hugenholtz, J; Ljungdahl, L G

    1989-01-01

    Membrane vesicles of Clostridium thermoautotrophicum containing carbon monoxide dehydrogenase generated a proton motive force when exposed to CO. This proton motive force, with a value of -140 mV, consisted of only an electrical potential at pH 7.5 and above and of an electrical potential and pH gradient at a lower pH. The proton motive force drove the uptake of L-alanine by the vesicles to a concentration of 300 times that of the medium. PMID:2708323

  17. Septin6 and Septin7 GTP Binding Proteins Regulate AP-3- and ESCRT-Dependent Multivesicular Body Biogenesis

    PubMed Central

    Traikov, Sofia; Stange, Christoph; Wassmer, Thomas; Paul-Gilloteaux, Perrine; Salamero, Jean; Raposo, Graça; Hoflack, Bernard

    2014-01-01

    Septins (SEPTs) form a family of GTP-binding proteins implicated in cytoskeleton and membrane organization, cell division and host/pathogen interactions. The precise function of many family members remains elusive. We show that SEPT6 and SEPT7 complexes bound to F-actin regulate protein sorting during multivesicular body (MVB) biogenesis. These complexes bind AP-3, an adapter complex sorting cargos destined to remain in outer membranes of maturing endosomes, modulate AP-3 membrane interactions and the motility of AP-3-positive endosomes. These SEPT-AP interactions also influence the membrane interaction of ESCRT (endosomal-sorting complex required for transport)-I, which selects ubiquitinated cargos for degradation inside MVBs. Whereas our findings demonstrate that SEPT6 and SEPT7 function in the spatial, temporal organization of AP-3- and ESCRT-coated membrane domains, they uncover an unsuspected coordination of these sorting machineries during MVB biogenesis. This requires the E3 ubiquitin ligase LRSAM1, an AP-3 interactor regulating ESCRT-I sorting activity and whose mutations are linked with Charcot-Marie-Tooth neuropathies. PMID:25380047

  18. Light-Activated Amino Acid Transport Systems in Halobacterium halobium Envelope Vesicles: Role of Chemical and Electrical Gradients

    NASA Technical Reports Server (NTRS)

    MacDonald, Russell E.; Greene, Richard V.; Lanyi, Janos K.

    1977-01-01

    The accumulation of 20 commonly occurring L-amino acids by cell envelope vesicles of Halobacterium halobium, in response to light-induced membrane potential and an artificially created sodium gradient, has been studied. Nineteen of these amino acids are actively accumulated under either or both of these conditions. Glutamate is unique in that its uptake is driven only by a chemical gradient for sodium. Amino acid concentrations at half-maximal uptake rates (Km) and maximal transport rates (V(sub max) have been determined for the uptake of all 19 amino acids. The transport systems have been partially characterized with respect to groups of amino acids transported by common carriers, cation effects, and relative response to the electrical and chemical components of the sodium gradient, the driving forces for uptake. The data presented clearly show that the carrier systems, which are responsible for uptake of individual amino acids, are as variable in their properties as those found in other organisms, i. e., some are highly specific for individual amino acids, some transport several amino acids competitively, some are activated by a chemical gradient of sodium only, and some function also in the complete absence of such a gradient. For all amino acids, Na(+) and K(+) are both required for maximal rate of uptake. The carriers for L-leucine and L-histidine are symmetrical in that these amino acids are transported in both directions across the vesicle membrane. It is suggested that coupling of substrate transport to metabolic energy via transient ionic gradients may be a general phenomenon in procaryotes.

  19. Vesicles versus Tubes: Is Endoplasmic Reticulum-Golgi Transport in Plants Fundamentally Different from Other Eukaryotes?1

    PubMed Central

    Robinson, David G.; Brandizzi, Federica; Nakano, Akihiko

    2015-01-01

    The endoplasmic reticulum (ER) is the gateway to the secretory pathway in all eukaryotic cells. Its products subsequently pass through the Golgi apparatus on the way to the cell surface (true secretion) or to the lytic compartment of the cell (vacuolar protein transport). In animal cells, the Golgi apparatus is present as a stationary larger order complex near the nucleus, and transport between the cortical ER and the Golgi complex occurs via an intermediate compartment which is transported on microtubules. By contrast, higher plant cells have discrete mobile Golgi stacks that move along the cortical ER, and the intermediate compartment is absent. Although many of the major molecular players involved in ER-Golgi trafficking in mammalian and yeast (Saccharomyces cerevisiae) cells have homologs in higher plants, the narrow interface (less than 500 nm) between the Golgi and the ER, together with the motility factor, makes the identification of the transport vectors responsible for bidirectional traffic between these two organelles much more difficult. Over the years, a controversy has arisen over the two major possibilities by which transfer can occur: through vesicles or direct tubular connections. In this article, four leading plant cell biologists attempted to resolve this issue. Unfortunately, their opinions are so divergent and often opposing that it was not possible to reach a consensus. Thus, we decided to let each tell his or her version individually. The review begins with an article by Federica Brandizzi that provides the necessary molecular background on coat protein complexes in relation to the so-called secretory units model for ER-Golgi transport in highly vacuolated plant cells. The second article, written by Chris Hawes, presents the evidence in favor of tubules. It is followed by an article from David Robinson defending the classical notion that transport occurs via vesicles. The last article, by Akihiko Nakano, introduces the reader to possible

  20. Taurocholate transport by brush-border membrane vesicles from the developing rabbit ileum: Structure/function relationships

    SciTech Connect

    Schwarz, S.M.; Watkins, J.B.; Ling, S.C. )

    1990-05-01

    To examine the ontogenesis of bile acid transport in the rabbit ileum, brush-border membrane vesicles (12- to 20-fold purified) were prepared from 14- to 49-day-old animals. Taurocholate uptake was characterized by the emergence of secondary active, Na(+)-dependent transport at the start of weaning (21 days). Transient intravesicular accumulation (overshoot) of taurocholate occurred at 5-10 s of incubation, and the overshoot maximum increased significantly from 21 days (349.2 +/- 22.4 nmol/mg protein) to 35 days (569.0 +/- 84.3 nmol/mg protein; p less than 0.001), without further increase at maturity (49 days, not equal to 607.6 +/- 136.7 nmol/mg protein). No significant taurocholate active uptake component was noted at 14 days; however, ileal vesicles from sucklings showed carrier-mediated, Na+ D-glucose cotransport. In greater than or equal to 35-day-old rabbits, osmolarity studies at 20 s of incubation showed that only approximately 12% of (14C)taurocholate uptake was secondary to bile acid-to-membrane binding. Conversely, at 20 min, greater than 95% of radiolabel incorporation represented solute bound to the external and/or internal membrane surface. Arrhenius plots establish brush-border membrane taurocholate uptake as an intrinsic, lipid-dependent process, with a slope discontinuity between 24 and 28 degrees C, similar to the membrane lipid thermotropic transition region. Steady-state fluorescence polarization studies (1,6-diphenyl-1,3,5-hexatriene) demonstrate a temporal association between the maturation of taurocholate uptake and age-related decreases in ileal brush-border membrane fluidity. These data indicate that maturation of bile acid secondary active transport in the rabbit ileum may be regulated, at least in part, by changes in brush-border membrane lipid dynamics.

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

    PubMed Central

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

    1985-01-01

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

  2. Vesicle miR-195 derived from Endothelial Cells Inhibits Expression of Serotonin Transporter in Vessel Smooth Muscle Cells.

    PubMed

    Gu, Junzhong; Zhang, Huiyuan; Ji, Bingyang; Jiang, Hui; Zhao, Tao; Jiang, Rongcai; Zhang, Zhiren; Tan, Shengjiang; Ahmed, Asif; Gu, Yuchun

    2017-03-08

    Serotonin or 5-hydroxytryptamine (5-HT) has been shown to be essential in lots of physiological and pathological processes. It is well known that 5-HT and 5-HT transporter (5-HTT) play important roles in the pulmonary artery in pulmonary hypertension. However, little is known about the function of 5-HTT in other arteries. In this study we found that the expression of 5-HTT was elevated in injured carotid arteries and over-expression of 5-HTT induced proliferation of smooth muscle cells (SMCs); however, this phenotype could be reversed by knocking-down of 5-HTT or endothelial cells conditional medium (EC-CM). A 5-HTT inhibitor, fluoxetine, treated animals also exhibited reduced restenosis after injury. We identified that miR-195 was packaged in the extracellular vesicles from EC-CM. We further confirmed that extracellular vesicles could transfer miR-195 from ECs to SMCs to inhibit the expression of 5-HTT in SMCs and the proliferation of SMCs. These results provide the first evidence that ECs communicate with SMCs via micro-RNA195 in the regulation of the proliferation of SMCs through 5-HTT, which will contribute to a better understanding of communications between ECs and SMCs via micro-RNA. Our findings suggest a potential target for the control of vessel restenosis.

  3. Indirect coupling of urate and p-aminohippurate transport to sodium in human brush-border membrane vesicles.

    PubMed

    Roch-Ramel, F; Guisan, B; Schild, L

    1996-01-01

    [14C]urate and p-[14C]aminohippurate (PAH) uptake by human brush-border membrane vesicles (BBMV) were measured in the presence of an inwardly oriented sodium gradient. No direct sodium cotransport was observed. Indirect [14C]urate coupling to sodium transport was demonstrated by cis-stimulation of [14C]urate with nicotinate or pyrazinoate (PZA) in the extravesicular medium but not by adding lactate, alpha-ketoglutarate, or beta-hydroxybutyrate. Indirect sodium coupling of [14C]PAH uptake was observed only when alpha-ketoglutarate was added to the extravesicular medium, a mechanism similar to that of basolateral membranes. The ability for PZA (and nicotinate) to cis-stimulate urate uptake was correlated with a high apparent affinity for the urate/anion exchanger. In urate-loaded vesicles, for identical medium concentrations, [14C]PZA uptake via the urateanion exchanger was 10 times higher than [14C]lactate uptake. Such high PZA affinity for the urate exchanger, working in parallel with PZA sodium cotransport can account for the stimulation of urate reabsorption by PZA in vivo.

  4. Vesicle miR-195 derived from Endothelial Cells Inhibits Expression of Serotonin Transporter in Vessel Smooth Muscle Cells

    PubMed Central

    Gu, Junzhong; Zhang, Huiyuan; Ji, Bingyang; Jiang, Hui; Zhao, Tao; Jiang, Rongcai; Zhang, Zhiren; Tan, Shengjiang; Ahmed, Asif; Gu, Yuchun

    2017-01-01

    Serotonin or 5-hydroxytryptamine (5-HT) has been shown to be essential in lots of physiological and pathological processes. It is well known that 5-HT and 5-HT transporter (5-HTT) play important roles in the pulmonary artery in pulmonary hypertension. However, little is known about the function of 5-HTT in other arteries. In this study we found that the expression of 5-HTT was elevated in injured carotid arteries and over-expression of 5-HTT induced proliferation of smooth muscle cells (SMCs); however, this phenotype could be reversed by knocking-down of 5-HTT or endothelial cells conditional medium (EC-CM). A 5-HTT inhibitor, fluoxetine, treated animals also exhibited reduced restenosis after injury. We identified that miR-195 was packaged in the extracellular vesicles from EC-CM. We further confirmed that extracellular vesicles could transfer miR-195 from ECs to SMCs to inhibit the expression of 5-HTT in SMCs and the proliferation of SMCs. These results provide the first evidence that ECs communicate with SMCs via micro-RNA195 in the regulation of the proliferation of SMCs through 5-HTT, which will contribute to a better understanding of communications between ECs and SMCs via micro-RNA. Our findings suggest a potential target for the control of vessel restenosis. PMID:28272473

  5. Diacylglycerol Is Required for the Formation of COPI Vesicles in the Golgi-to-ER Transport Pathway

    PubMed Central

    Fernández-Ulibarri, Inés; Vilella, Montserrat; Lázaro-Diéguez, Francisco; Sarri, Elisabet; Martínez, Susana E.; Jiménez, Nuria; Claro, Enrique; Mérida, Isabel; Burger, Koert N.J.

    2007-01-01

    Diacylglycerol is necessary for trans-Golgi network (TGN) to cell surface transport, but its functional relevance in the early secretory pathway is unclear. Although depletion of diacylglycerol did not affect ER-to-Golgi transport, it led to a redistribution of the KDEL receptor to the Golgi, indicating that Golgi-to-ER transport was perturbed. Electron microscopy revealed an accumulation of COPI-coated membrane profiles close to the Golgi cisternae. Electron tomography showed that the majority of these membrane profiles originate from coated buds, indicating a block in membrane fission. Under these conditions the Golgi-associated pool of ARFGAP1 was reduced, but there was no effect on the binding of coatomer or the membrane fission protein CtBP3/BARS to the Golgi. The addition of 1,2-dioctanoyl-sn-glycerol or the diacylglycerol analogue phorbol 12,13-dibutyrate reversed the effects of endogenous diacylglycerol depletion. Our findings implicate diacylglycerol in the retrograde transport of proteins from Golgi to the ER and suggest that it plays a critical role at a late stage of COPI vesicle formation. PMID:17567948

  6. X Linkage of AP3A, a Homolog of the Y-Linked MADS-Box Gene AP3Y in Silene latifolia and S. dioica

    PubMed Central

    Penny, Rebecca H.; Montgomery, Benjamin R.; Delph, Lynda F.

    2011-01-01

    Background The duplication of autosomal genes onto the Y chromosome may be an important element in the evolution of sexual dimorphism.A previous cytological study reported on a putative example of such a duplication event in a dioecious tribe of Silene (Caryophyllaceae): it was inferred that the Y-linked MADS-box gene AP3Y originated from a duplication of the reportedly autosomal orthologAP3A. However, a recent study, also using cytological methods, indicated that AP3A is X-linked in Silenelatifolia. Methodology/Principal Findings In this study, we hybridized S. latifolia and S. dioicato investigate whether the pattern of X linkage is consistent among distinct populations, occurs in both species, and is robust to genetic methods. We found inheritance patterns indicative of X linkage of AP3A in widely distributed populations of both species. Conclusions/Significance X linkage ofAP3A and Y linkage of AP3Yin both species indicates that the genes' ancestral progenitor resided on the autosomes that gave rise to the sex chromosomesand that neither gene has moved between chromosomes since species divergence.Consequently, our results do not support the contention that inter-chromosomal gene transfer occurred in the evolution of SlAP3Y from SlAP3A. PMID:21533056

  7. Profilin I attached to the Golgi is required for the formation of constitutive transport vesicles at the trans-Golgi network.

    PubMed

    Dong, J; Radau, B; Otto, A; Müller, E; Lindschau, C; Westermann, P

    2000-07-21

    Profilin I was identified, by mass spectrometric sequencing and immunoblotting, as a component of purified Golgi cisternae from HepG2 cells. Binding to the Golgi was verified by indirect immunofluorescence in MT-1 cells showing that a fraction of profilin I colocalizes with TGN38, a marker of the trans-Golgi network (TGN). Studying the formation of constitutive exocytic vesicles at the TGN in a cell-free system demonstrated that cytosolic profilin I has no effect, while incubation of Golgi cisternae with a profilin I-specific antibody reduced vesicle formation by about 50%. Notably, the antibody displaces a fraction of the Golgi-bound dynamin II indicating that profilin I may indirectly promote vesicle formation by supporting the binding of dynamin II to the Golgi membrane. The impact of dynamin II on vesicle formation is demonstrated by incubating the Golgi with the proline-rich domain of dynamin II which concomitantly displaces dynamin II and inhibits vesicle formation. The data provide evidence that profilin I attaches to the Golgi apparatus and is required for the formation of constitutive transport vesicles.

  8. K⁺-dependent ³H-D-glucose transport by hepatopancreatic brush border membrane vesicles of a marine shrimp.

    PubMed

    Obi, Ijeoma E; Sterling, Kenneth M; Ahearn, Gregory A

    2013-01-01

    The effects of sodium, potassium, sugar inhibitors, and membrane potential on ³H-D-glucose uptake by hepatopancreatic epithelial brush border membrane vesicles (BBMV) of the Atlantic marine shrimp, Litopenaeus setiferus, were investigated. Brush border membrane vesicles were prepared using a MgCl₂/EGTA precipitation method and uptake experiments were conducted using a high speed filtration technique. ³H-D-Glucose uptake was stimulated by both sodium and potassium and these transport rates were almost doubled in the presence of an inside-negative-induced membrane potential. Kinetics of ³H-D-glucose influx were hyperbolic functions of both external Na⁺ or K⁺, and an induced membrane potential increased influx J(max) and lowered K(m) in both salts. ³H-D-Glucose influx versus [glucose] in both Na⁺ or K⁺ media also displayed Michaelis-Menten properties that were only slightly affected by induced membrane potential. Phloridzin was a poor inhibitor of 0.5 mM ³H-D-glucose influx, requiring at least 5 mM in NaCl and 10 mM in KCl to significantly reduce hexose transport. Several sugars (D-galactose, α-methyl-D-gluco-pyranoside, unlabeled D-glucose, D-fructose, and D-mannose) were used at 75 mM as potential inhibitors of 0.1 mM ³H-D-glucose influx. Only unlabeled D-glucose, D-fructose, and D-mannose significantly (p < 0.05) reduced labeled glucose transport. An additional experiment using increasing concentrations of D-mannose (0, 10, 25, 75, and 100 mM) showed this hexose to be an effective inhibitor of 0.1 mM ³H-D-glucose uptake at concentrations of 75 mM and higher. As a whole these results suggest that ³H-D-glucose transport by hepatopancreatic BBMV occurs by a carrier system that is able to use both Na⁺ and K⁺ as drivers, is enhanced by membrane potential, is relatively refractory to phloridzin, and is only inhibited by itself, D-fructose, and D-mannose. These properties are similar to those exhibited by the mammalian SLC5A9/SGLT4 transporter

  9. Visualization of the vesicular acetylcholine transporter in cholinergic nerve terminals and its targeting to a specific population of small synaptic vesicles.

    PubMed Central

    Weihe, E; Tao-Cheng, J H; Schäfer, M K; Erickson, J D; Eiden, L E

    1996-01-01

    Immunohistochemical visualization of the rat vesicular acetylcholine transporter (VAChT) in cholinergic neurons and nerve terminals has been compared to that for choline acetyltransferase (ChAT), heretofore the most specific marker for cholinergic neurons. VAChT-positive cell bodies were visualized in cerebral cortex, basal forebrain, medial habenula, striatum, brain stem, and spinal cord by using a polyclonal anti-VAChT antiserum. VAChT-immuno-reactive fibers and terminals were also visualized in these regions and in hippocampus, at neuromuscular junctions within skeletal muscle, and in sympathetic and parasympathetic autonomic ganglia and target tissues. Cholinergic nerve terminals contain more VAChT than ChAT immunoreactivity after routine fixation, consistent with a concentration of VAChT within terminal neuronal arborizations in which secretory vesicles are clustered. These include VAChT-positive terminals of the median eminence or the hypothalamus, not observed with ChAT antiserum after routine fixation. Subcellular localization of VAChT in specific organelles in neuronal cells was examined by immunoelectron microscopy in a rat neuronal cell line (PC 12-c4) expressing VAChT as well as the endocrine and neuronal forms of the vesicular monoamine transporters (VMAT1 and VMAT2). VAChT is targeted to small synaptic vesicles, while VMAT1 is found mainly but not exclusively on large dense-core vesicles. VMAT2 is found on large dense-core vesicles but not on the small synaptic vesicles that contain VAChT in PC12-c4 cells, despite the presence of VMAT2 immunoreactivity in central and peripheral nerve terminals known to contain monoamines in small synaptic vesicles. Thus, VAChT and VMAT2 may be specific markers for "cholinergic" and "adrenergic" small synaptic vesicles, with the latter not expressed in nonstimulated neuronally differentiated PC12-c4 cells. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8622973

  10. Transmembrane electron transport in sealed and NAD(P)H-loaded right-side-out plasma membrane vesicles isolated from maize (Zea mays L.) roots.

    PubMed

    Menckhoff, Mathias; Lüthje, Sabine

    2004-06-01

    Electron transport across plasma membranes has been observed in vivo in several plant species and tissues after the application of ferricyanide (hexacyanoferrate III, HCF III). In the present work, a transmembrane electron flow was demonstrated in sealed and NAD(P)H-loaded right-side-out (apoplastic-side-out) plasma membrane vesicles isolated from maize (Zea mays L.) roots. HCF III was reduced at a rate of up to 126 nmol min(-1) mg(-1) protein by NADPH-loaded vesicles, while reduction rates with NADH-loaded vesicles were several-fold lower. Coincident with the reduction of HCF III, NAD(P)H oxidation was observed inside the vesicles. The dependence of reduction on K+ indicated an electrogenic transmembrane electron flow. Application of 100 microM calcium decreased HCF III reduction up to 66%, while pre-incubation with 200 microM warfarin or diphenylene iodonium inhibited transmembrane electron transport only weakly. Fe(3+)-EDTA was not reduced significantly by NADPH-loaded plasma membrane vesicles, whereas XTT was reduced at a rate of 765 pmol min(-1) mg(-1) protein. The results suggested a major function for NADPH in transmembrane electron flow and were discussed in conjunction with in vivo experiments.

  11. Functional reconstitution of the purified phosphoenolpyruvate-dependent mannitol-specific transport system of Escherichia coli in phospholipid vesicles: coupling between transport and phosphorylation.

    PubMed Central

    Elferink, M G; Driessen, A J; Robillard, G T

    1990-01-01

    Purified mannitol-specific enzyme II (EII) from Escherichia coli was reconstituted into phospholipid vesicles with the aid of a detergent-dialysis procedure followed by a freeze-thaw sonication step. The orientation of EII in the proteoliposomes was random. The cytoplasmic moiety of the inverted EII could be removed with trypsin without effecting the integrity of the liposomal membrane. This enabled us to study the two different EII orientations independently. The population of inverted EII molecules was monitored by measuring active extrusion of mannitol after the addition of phosphoenolpyruvate, EI, and histidine-containing phosphocarrier protein (HPr) at the outside of the vesicles. The population of correctly oriented EII molecules was monitored by measuring active uptake of mannitol with internal phosphoenolpyruvate, EI, and HPr. A low rate of facilitated diffusion of mannitol via the unphosphorylated carrier could be measured. On the other hand, a high phosphorylation activity without translocation was observed at the outside of the liposomes. The kinetics of the phosphoenolpyruvate-dependent transport reaction and the nonvectorial phosphorylation reaction were compared. Transport of mannitol into the liposomes via the correctly oriented EII molecules occurred with a high affinity (Km, lower than 10 microM) and with a relatively low Vmax. Phosphorylation at the outside of the liposomes catalyzed by the inverted EII molecules occurred with a low affinity (Km of about 66 microM), while the maximal velocity was about 10 times faster than the transport reaction. The latter observation is kinetic proof for the lack of strict coupling between transport and phosphorylation in these enzymes. Images PMID:2123863

  12. Ca sup 2+ transport in membrane vesicles from pinto bean leaves and its alteration after ozone exposure. [Phaseolus vulgaris

    SciTech Connect

    Castillo, F.J.; Heath, R.L. )

    1990-10-01

    The influence of ozone on Ca{sup 2+} transport in plant membranes from pinto bean (Phaseolus vulgaris L. var Pinto) leaves was investigated in vitro by means of a filtration method using purified vesicles. Two transport mechanisms located at the plasma membrane are involved in a response to ozone: (a) passive Ca{sup 2+} influx into the cell and (b) active Ca{sup 2+} efflux driven by an ATP-dependent system, which has two components: a primary Ca{sup 2+} transport directly linked to ATP which is partially activated by calmodulin and a H{sup +}/Ca{sup 2+} antiport coupled to activity of a H{sup +}-ATPase. The passive Ca{sup 2+} permeability is increased by ozone. A triangular pulse of ozone stimulates a higher influx of Ca{sup 2+} than does a square wave, even though the total dose with the same (0.6 microliter per liter {times} hour). Leaves exposed to a square wave did not exhibit visible injury and were still able to recover from oxidant stress by activation of calmodulin-dependent Ca{sup 2+} extrusion mechanisms. On the other hand, leaves exposed to a triangular wave of ozone, exhibit visible injury and lost the ability of extruding Ca{sup 2+} out of the cell.

  13. A novel chloroplast localized Rab GTPase protein CPRabA5e is involved in stress, development, thylakoid biogenesis and vesicle transport in Arabidopsis.

    PubMed

    Karim, Sazzad; Alezzawi, Mohamed; Garcia-Petit, Christel; Solymosi, Katalin; Khan, Nadir Zaman; Lindquist, Emelie; Dahl, Peter; Hohmann, Stefan; Aronsson, Henrik

    2014-04-01

    A novel Rab GTPase protein in Arabidopsis thaliana, CPRabA5e (CP = chloroplast localized) is located in chloroplasts and has a role in transport. Transient expression of CPRabA5e:EGFP fusion protein in tobacco (Nicotiana tabacum) leaves, and immunoblotting using Arabidopsis showed localization of CPRabA5e in chloroplasts (stroma and thylakoids). Ypt31/32 in the yeast Saccharomyces cerevisiae are involved in regulating vesicle transport, and CPRabA5e a close homolog of Ypt31/32, restores the growth of the ypt31Δ ypt32(ts) mutant at 37 °C in yeast complementation. Knockout mutants of CPRabA5e displayed delayed seed germination and growth arrest during oxidative stress. Ultrastructural studies revealed that after preincubation at 4 °C mutant chloroplasts contained larger plastoglobules, lower grana, and more vesicles close to the envelopes compared to wild type, and vesicle formation being enhanced under oxidative stress. This indicated altered thylakoid development and organization of the mutants. A yeast-two-hybrid screen with CPRabA5e as bait revealed 13 interacting partner proteins, mainly located in thylakoids and plastoglobules. These proteins are known or predicted to be involved in development, stress responses, and photosynthesis related processes, consistent with the stress phenotypes observed. The results observed suggest a role of CPRabA5e in transport to and from thylakoids, similar to cytosolic Rab proteins involved in vesicle transport.

  14. Directed transport of neutrophil-derived extracellular vesicles enables platelet-mediated innate immune response

    PubMed Central

    Rossaint, Jan; Kühne, Katharina; Skupski, Jennifer; Van Aken, Hugo; Looney, Mark R.; Hidalgo, Andres; Zarbock, Alexander

    2016-01-01

    The innate immune response to bacterial infections requires the interaction of neutrophils and platelets. Here, we show that a multistep reciprocal crosstalk exists between these two cell types, ultimately facilitating neutrophil influx into the lung to eliminate infections. Activated platelets adhere to intravascular neutrophils through P-selectin/P-selectin glycoprotein ligand-1 (PSGL-1)-mediated binding, a primary interaction that allows platelets glycoprotein Ibα (GPIbα)-induced generation of neutrophil-derived extracellular vesicles (EV). EV production is directed by exocytosis and allows shuttling of arachidonic acid into platelets. EVs are then specifically internalized into platelets in a Mac1-dependent fashion, and relocated into intracellular compartments enriched in cyclooxygenase1 (Cox1), an enzyme processing arachidonic acid to synthesize thromboxane A2 (TxA2). Finally, platelet-derived-TxA2 elicits a full neutrophil response by inducing the endothelial expression of ICAM-1, intravascular crawling, and extravasation. We conclude that critical substrate–enzyme pairs are compartmentalized in neutrophils and platelets during steady state limiting non-specific inflammation, but bacterial infection triggers regulated EV shuttling resulting in robust inflammation and pathogen clearance. PMID:27845343

  15. Effect of alpha interferon on glucose and alanine transport by rat renal brush border membrane vesicles

    SciTech Connect

    Batuman, V.; Chadha, I. New Jersey Medical School, Newark )

    1990-01-01

    To investigate the pathogenetic mechanisms of interferon nephrotoxicity, we studied the effect of recombinant interferon alfa-2b on the uptake of {sup 14}C-D-glucose and {sup 14}C-L-alanine by rat renal brush-border-membrane vesicles. Interferon significantly inhibited 20 sec. sodium-dependent and 5 and 10 min. equilibrium uptake of both glucose and alanine. The inhibitory effect was dose dependent with maximum effect achieved at interferon concentration of 5 {times} 10{sup {minus}8}M in the uptake media. The half-maximal inhibitory concentrations, IC{sub 50}, of interferon on glucose uptake was 1.8 {times} 10{sup {minus}8}M, and 5.4 {times} 10{sup {minus}9}M on alanine uptake. Dixon plot analysis of uptake data was consistent with pure non-competitive inhibition. The inhibition constants, K{sub i}, 1.5 {times} 10{sup {minus}8}M for glucose uptake, and 7.3 {times} 10{sup {minus}9}M for alanine uptake, derived from Dixon plots were in close agreement with the IC{sub 50}s calculated from the semilog dose response curves. These observations reveal that direct interactions at the proximal tubule cell membrane are involved in the pathogenesis of interferon nephrotoxicity, and that its mechanism of nephrotoxicity is similar to that of other low molecular weight proteins.

  16. Active transport of. gamma. -aminobutyric acid and glycine into synaptic vesicles

    SciTech Connect

    Kish, P.E.; Fischer-Bovenkerk, C.; Ueda, T. )

    1989-05-01

    Although {gamma}-aminobutyric acid (GABA) and glycine are recognized as major amino acid inhibitory neurotransmitters in the central nervous system, their storage is poorly understood. In this study the authors have characterized vesicular GABA and glycine uptakes in the cerebrum and spinal cord, respectively. They present evidence that GABA and glycine are each taken up into isolated synaptic vesicles in an ATP-dependent manner and that the uptake is driven by an electrochemical proton gradient. Uptake for both amino acids exhibited kinetics with low affinity similar to a vesicular glutamate uptake. The ATP-dependent GABA uptake was not inhibited by the putative amino acid neurotransmitters glycine, taurine, glutamate, or aspartate or by GABA analogs, agonists, and antagonists. Similarly, ATP-dependent glycine uptake was hardly affected by GABA, taurine, glutamate, or aspartate or by glycine analogs or antagonists. The GABA uptake was not affected by chloride, which is in contrast to the uptake of the excitatory neurotransmitter glutamate, whereas the glycine uptake was slightly stimulated by low concentrations of chloride. Tissue distribution studies indicate that the vesicular uptake systems for GABA, glycine, and glutamate are distributed in different proportions in the cerebrum and spinal cord. These results suggest that the vesicular uptake systems for GABA, glycine, and glutamate are distinct from each other.

  17. 78 kDa receptor for Man6P-independent lysosomal enzyme targeting: Biosynthetic transport from endoplasmic reticulum to 'high-density vesicles'

    SciTech Connect

    Gonzalez-Noriega, Alfonso . E-mail: gonor@biomedicas.unam.mx; Ortega Cuellar, Daniel D.; Michalak, Colette

    2006-04-15

    Recent work has shown that the cation-independent mannose 6-phosphate and the 78 kDa receptors for lysosomal enzyme targeting are located in different cell compartments. While the mannose 6-phosphate receptor is enriched in the Percoll fractions that contain Golgi apparatus, most of the 78 kDa receptor is localized in a heavy fraction at the bottom of the Percoll gradient. This report presents the biosynthetic transport of the 78 kDa receptor. Newly synthesized 78 kDa receptor was transported to Golgi from endoplasmic reticulum with a half life of 5 min. From the Golgi apparatus, the receptor takes two routes; about 15-25% is transported to the plasma membrane, and the rest migrates to late endosomes, subsequently to prelysosomes and finally to the dense vesicles. The 78 kDa receptor starts appearing at the dense vesicles 120 min after biosynthesis and reaches a maximum of 40-50% of the total receptor. Treatment of cells with NH{sub 4}Cl causes depletion of the receptor from the dense vesicles and prelysosomes and corresponding augmentation in endosomes and plasma membrane. These results suggest that the 78 kDa receptor cycles between compartments and that the dense vesicles seem to represent the most distal compartment in the biosynthetic pathway of this receptor.

  18. Role of Golgi derived clathrin coated vesicles in the transport of calsequestrin to the sarcoplasmic reticulum of developing myotubes

    SciTech Connect

    Thomas, K.M.

    1988-01-01

    The sarcoplasmic reticulum (SR) is the major intracellular calcium sequestering organelle in skeletal and heart muscle. Calsequestrin (CSQ) is a glycoprotein with a molecular weight of 42,000. This protein is located in the lumen of the SR and it binds Ca/sup 2 +/ to maintain the concentration of this cation in the lumen at 10/sup /minus/3/M. Highly purified coated vesicles (CVs) were isolated from chick muscle and a Western blot using polyclonal anti-CSQ revealed the presence of CSQ within the CVs. Another major protein in the SR, the Ca/sup 2 +/-ATPase, was not contained in CVs suggesting different routes of insertion into the SR. Cultured chick myotubes were labelled with Trans/sup 35/S-label contain (/sup 35/S)-methionine and (/sup 35/S)-cysteine to follow the transport of CSQ. Labelled CSQ remained high in the CVs until after 45 minutes of chase, then declined. The amount of labelled CSQ in the SR continued to rise over the chase period. No CSQ was secreted. All the CSQ in CVs and SR was sensitive to the activity of endoglycosidase H, and a significant fraction also bound wheat germ agglutinin. A small amount of CSQ was also co-transported with the muscle protein acetylcholinesterase within CVs. Non-secreted forms of acetylcholinesterase had the same carbohydrate structure as CSQ and were shown to be degraded in the SR.

  19. A microRNA negative feedback loop downregulates vesicle transport and inhibits fear memory

    PubMed Central

    Mathew, Rebecca S; Tatarakis, Antonis; Rudenko, Andrii; Johnson-Venkatesh, Erin M; Yang, Yawei J; Murphy, Elisabeth A; Todd, Travis P; Schepers, Scott T; Siuti, Nertila; Martorell, Anthony J; Falls, William A; Hammack, Sayamwong E; Walsh, Christopher A; Tsai, Li-Huei; Umemori, Hisashi; Bouton, Mark E; Moazed, Danesh

    2016-01-01

    The SNARE-mediated vesicular transport pathway plays major roles in synaptic remodeling associated with formation of long-term memories, but the mechanisms that regulate this pathway during memory acquisition are not fully understood. Here we identify miRNAs that are up-regulated in the rodent hippocampus upon contextual fear-conditioning and identify the vesicular transport and synaptogenesis pathways as the major targets of the fear-induced miRNAs. We demonstrate that miR-153, a member of this group, inhibits the expression of key components of the vesicular transport machinery, and down-regulates Glutamate receptor A1 trafficking and neurotransmitter release. MiR-153 expression is specifically induced during LTP induction in hippocampal slices and its knockdown in the hippocampus of adult mice results in enhanced fear memory. Our results suggest that miR-153, and possibly other fear-induced miRNAs, act as components of a negative feedback loop that blocks neuronal hyperactivity at least partly through the inhibition of the vesicular transport pathway. DOI: http://dx.doi.org/10.7554/eLife.22467.001 PMID:28001126

  20. Polymer micelle assisted transport and delivery of model hydrophilic components inside a biological lipid vesicle: a coarse-grain simulation study.

    PubMed

    Srinivas, Goundla; Mohan, Ram V; Kelkar, Ajit D

    2013-10-10

    Understanding drug transportation and delivery mechanism from a molecular viewpoint is essential to find better treatment pathways. Despite the fact that many significant drugs such as anticancer doxorubicin and mitoxantrone are predominantly hydrophilic, an efficient methodology to deliver hydrophilic drug components is not well established. Here we explore this problem by studying "patchy" polymeric micelle assisted hydrophilic component transportation across a lipid membrane and delivery inside a biological lipid vesicle. Using the MARTINI force field as the basis, we study the interaction of polymeric micelle with DPPC lipid vesicles in detail. In order to facilitate hydrophilic drug transportation study, a primitive CG model for hydrophilic drug component is used. Extensive simulations carried out over hundreds of nanoseconds demonstrate successful encapsulation, transportation of hydrophilic components by patchy polymeric micelles. Results show the polymeric micelle releases a significant portion of hydrophilic contents inside the lipid vesicle. The present simulation study also reveals a possible mechanism for efficient hydrophilic component transportation and delivery. Insights from this study could potentially help the experimental community to design better delivery vehicles, especially for hydrophilic drug molecules.

  1. Comparison between calcium transport and adenosine triphosphatase activity in membrane vesicles derived from rabbit kidney proximal tubules.

    PubMed

    Vieyra, A; Nachbin, L; de Dios-Abad, E; Goldfeld, M; Meyer-Fernandes, J R; de Moraes, L

    1986-03-25

    Characteristics of Ca2+ uptake were studied in a vesicular preparation of proximal tubule plasma membranes from rabbit kidney and compared with the properties of both membrane-bound and solubilized Ca2+-ATPase activities. Calcium uptake required both ATP and MgCl2 and revealed two kinetic components with respect to Ca2+ concentration requirements, one with a high affinity for Ca2+ (1.8 microM), operative in the range of cytosolic Ca2+ activity, and one with a low affinity for Ca2+ (250 microM) which may become active only at abnormally high cytosolic Ca2+ concentrations. The high- and low-affinity components were stimulated to similar extents by phosphate, and required similar concentrations of ATP (0.6 mM) for half-maximal activity. The amount of membrane-bound phosphoenzyme formed from ATP in the presence of Ca2+ was the same regardless of whether only one or both sites were saturated, suggesting that occupancy of the second Ca2+ binding site accelerates the enzyme turnover. Inhibition of Ca2+ transport by Na+ was reversed by the addition of ouabain or an ATP-regenerating system, indicating that this inhibitory effect of Na+ on Ca2+ uptake may be due to the accumulation of ADP in the medium as a result of Na+ pump activity. Low concentrations of carbonyl cyanide p-trifluoromethoxyphenylhydrazone and valinomycin (2.5 and 1 microM, respectively) were without effect on Ca2+ uptake in the presence of phosphate, whereas higher concentrations of the ionophores (200 and 100 microM, respectively) reduced uptake by 60% or more. The calmodulin antagonist 48/80 also reduced Ca2+ uptake with half-maximal effectiveness at 100 micrograms/ml. None of these drugs affected either ATPase activity or the EGTA-induced Ca2+ efflux from preloaded vesicles. The Ca2+ dependence of ATP hydrolysis by the membrane-bound enzyme preparation was similar to that observed for Ca2+ uptake by the vesicles. However, with solubilized enzyme, concentrations of Ca2+ similar to that found in the

  2. NINL and DZANK1 Co-function in Vesicle Transport and Are Essential for Photoreceptor Development in Zebrafish.

    PubMed

    Dona, Margo; Bachmann-Gagescu, Ruxandra; Texier, Yves; Toedt, Grischa; Hetterschijt, Lisette; Tonnaer, Edith L; Peters, Theo A; van Beersum, Sylvia E C; Bergboer, Judith G M; Horn, Nicola; de Vrieze, Erik; Slijkerman, Ralph W N; van Reeuwijk, Jeroen; Flik, Gert; Keunen, Jan E; Ueffing, Marius; Gibson, Toby J; Roepman, Ronald; Boldt, Karsten; Kremer, Hannie; van Wijk, Erwin

    2015-10-01

    Ciliopathies are Mendelian disorders caused by dysfunction of cilia, ubiquitous organelles involved in fluid propulsion (motile cilia) or signal transduction (primary cilia). Retinal dystrophy is a common phenotypic characteristic of ciliopathies since photoreceptor outer segments are specialized primary cilia. These ciliary structures heavily rely on intracellular minus-end directed transport of cargo, mediated at least in part by the cytoplasmic dynein 1 motor complex, for their formation, maintenance and function. Ninein-like protein (NINL) is known to associate with this motor complex and is an important interaction partner of the ciliopathy-associated proteins lebercilin, USH2A and CC2D2A. Here, we scrutinize the function of NINL with combined proteomic and zebrafish in vivo approaches. We identify Double Zinc Ribbon and Ankyrin Repeat domains 1 (DZANK1) as a novel interaction partner of NINL and show that loss of Ninl, Dzank1 or both synergistically leads to dysmorphic photoreceptor outer segments, accumulation of trans-Golgi-derived vesicles and mislocalization of Rhodopsin and Ush2a in zebrafish. In addition, retrograde melanosome transport is severely impaired in zebrafish lacking Ninl or Dzank1. We further demonstrate that NINL and DZANK1 are essential for intracellular dynein-based transport by associating with complementary subunits of the cytoplasmic dynein 1 motor complex, thus shedding light on the structure and stoichiometry of this important motor complex. Altogether, our results support a model in which the NINL-DZANK1 protein module is involved in the proper assembly and folding of the cytoplasmic dynein 1 motor complex in photoreceptor cells, a process essential for outer segment formation and function.

  3. NINL and DZANK1 Co-function in Vesicle Transport and Are Essential for Photoreceptor Development in Zebrafish

    PubMed Central

    Texier, Yves; Toedt, Grischa; Hetterschijt, Lisette; Tonnaer, Edith L.; Peters, Theo A.; van Beersum, Sylvia E. C.; Bergboer, Judith G. M.; Horn, Nicola; de Vrieze, Erik; Slijkerman, Ralph W. N.; van Reeuwijk, Jeroen; Flik, Gert; Keunen, Jan E.; Ueffing, Marius; Gibson, Toby J.; Roepman, Ronald; Boldt, Karsten; Kremer, Hannie; van Wijk, Erwin

    2015-01-01

    Ciliopathies are Mendelian disorders caused by dysfunction of cilia, ubiquitous organelles involved in fluid propulsion (motile cilia) or signal transduction (primary cilia). Retinal dystrophy is a common phenotypic characteristic of ciliopathies since photoreceptor outer segments are specialized primary cilia. These ciliary structures heavily rely on intracellular minus-end directed transport of cargo, mediated at least in part by the cytoplasmic dynein 1 motor complex, for their formation, maintenance and function. Ninein-like protein (NINL) is known to associate with this motor complex and is an important interaction partner of the ciliopathy-associated proteins lebercilin, USH2A and CC2D2A. Here, we scrutinize the function of NINL with combined proteomic and zebrafish in vivo approaches. We identify Double Zinc Ribbon and Ankyrin Repeat domains 1 (DZANK1) as a novel interaction partner of NINL and show that loss of Ninl, Dzank1 or both synergistically leads to dysmorphic photoreceptor outer segments, accumulation of trans-Golgi-derived vesicles and mislocalization of Rhodopsin and Ush2a in zebrafish. In addition, retrograde melanosome transport is severely impaired in zebrafish lacking Ninl or Dzank1. We further demonstrate that NINL and DZANK1 are essential for intracellular dynein-based transport by associating with complementary subunits of the cytoplasmic dynein 1 motor complex, thus shedding light on the structure and stoichiometry of this important motor complex. Altogether, our results support a model in which the NINL-DZANK1 protein module is involved in the proper assembly and folding of the cytoplasmic dynein 1 motor complex in photoreceptor cells, a process essential for outer segment formation and function. PMID:26485514

  4. A Putative Small Solute Transporter Is Responsible for the Secretion of G377 and TRAP-Containing Secretory Vesicles during Plasmodium Gamete Egress and Sporozoite Motility

    PubMed Central

    Kehrer, Jessica; Singer, Mirko; Lemgruber, Leandro; Silva, Patricia A. G. C.; Frischknecht, Friedrich; Mair, Gunnar R.

    2016-01-01

    Regulated protein secretion is required for malaria parasite life cycle progression and transmission between the mammalian host and mosquito vector. During transmission from the host to the vector, exocytosis of highly specialised secretory vesicles, such as osmiophilic bodies, is key to the dissolution of the red blood cell and parasitophorous vacuole membranes enabling gamete egress. The positioning of adhesins from the TRAP family, from micronemes to the sporozoite surface, is essential for gliding motility of the parasite and transmission from mosquito to mammalian host. Here we identify a conserved role for the putative pantothenate transporter PAT in Plasmodium berghei in vesicle fusion of two distinct classes of vesicles in gametocytes and sporozoites. PAT is a membrane component of osmiophilic bodies in gametocytes and micronemes in sporozoites. Despite normal formation and trafficking of osmiophilic bodies to the cell surface upon activation, PAT-deficient gametes fail to discharge their contents, remain intraerythrocytic and unavailable for fertilisation and further development in the mosquito. Sporozoites lacking PAT fail to secrete TRAP, are immotile and thus unable to infect the subsequent rodent host. Thus, P. berghei PAT appears to regulate exocytosis in two distinct populations of vesicles in two different life cycle forms rather than acting as pantothenic transporter during parasite transmission. PMID:27427910

  5. A model for growth of a single fungal hypha based on well-mixed tanks in series: simulation of nutrient and vesicle transport in aerial reproductive hyphae.

    PubMed

    Balmant, Wellington; Sugai-Guérios, Maura Harumi; Coradin, Juliana Hey; Krieger, Nadia; Furigo Junior, Agenor; Mitchell, David Alexander

    2015-01-01

    Current models that describe the extension of fungal hyphae and development of a mycelium either do not describe the role of vesicles in hyphal extension or do not correctly describe the experimentally observed profile for distribution of vesicles along the hypha. The present work uses the n-tanks-in-series approach to develop a model for hyphal extension that describes the intracellular transport of nutrient to a sub-apical zone where vesicles are formed and then transported to the tip, where tip extension occurs. The model was calibrated using experimental data from the literature for the extension of reproductive aerial hyphae of three different fungi, and was able to describe different profiles involving acceleration and deceleration of the extension rate. A sensitivity analysis showed that the supply of nutrient to the sub-apical vesicle-producing zone is a key factor influencing the rate of extension of the hypha. Although this model was used to describe the extension of a single reproductive aerial hypha, the use of the n-tanks-in-series approach to representing the hypha means that the model has the flexibility to be extended to describe the growth of other types of hyphae and the branching of hyphae to form a complete mycelium.

  6. BNIP-2 binds phosphatidylserine, localizes to vesicles, and is transported by kinesin-1.

    PubMed

    Akamatsu, Rie; Ishida-Kitagawa, Norihiro; Aoyama, Takane; Oka, Chio; Kawaichi, Masashi

    2015-02-01

    BNIP-2 shows high homology with the Cayman ataxia protein, caytaxin, which functions as a kinesin-1 adapter bridging cargos and kinesin light chains (KLCs). BNIP-2 is known to induce cell shape changes when over-expressed in culture cells, but its physiological functions are mostly unknown. BNIP-2 interacts with KLC through the conserved WED motif in the N-terminal region of BNIP-2. Interaction with KLC and transportation by kinesin-1 are essential for over-expressed BNIP-2 to elongate cells and induce cellular processes. Endogenous BNIP-2 localizes to the Golgi apparatus, early and recycling endosomes and mitochondria, aligned with microtubules, and moves at a speed compatible with kinesin-1 transportation. The CRAL-TRIO domain of BNIP-2 specifically interacts with phosphatidylserine, and the vesicular localization of BNIP-2 requires interaction with this phospholipid. BNIP-2 mutants which do not bind phosphatidylserine do not induce morphological changes in cells. These data show that similar to caytaxin, BNIP-2 is a kinesin-1 adapter involved in vesicular transportation in the cytoplasm and that association with cargos depends on interaction of the CRAL-TRIO domain with membrane phosphatidylserine.

  7. [Effect of natural or synthetic detergents on the transport of D-glucose in the membranes of vesicles of the brush border of the intestine of the rabbit].

    PubMed

    Favilli, F; Iantomasi, T; Stio, M; Treves, C; Vanni, P; Vincenzini, M T

    1988-01-01

    We describe here the effects of natural and synthetic detergents on the D-glucose transport into brush-border membranes of vesicles of rabbit's intestine. Two synthetic detergents: Triton X-100 and dodecyltrimethylammonium bromide have been found very strong inhibitors (more than 50 p. 100 of inhibition of maximal D-glucose uptake). Kinetic studies showed that these detergents behaved as mixed type inhibitors. The Na+-dependent transport of amino acids (aspartic acid, lysine, phenylalanine) is only poorly affected by dodecyltrimethylammonium bromide, while Triton X-100 inhibits unspecifically all the transport studied.

  8. Extracellular vesicles from Paracoccidioides pathogenic species transport polysaccharide and expose ligands for DC-SIGN receptors

    SciTech Connect

    da Silva, Roberta Peres; Heiss, Christian; Black, Ian; Azadi, Parastoo; Gerlach, Jared Q.; Travassos, Luiz R.; Joshi, Lokesh; Kilcoyne, Michelle; Puccia, Rosana

    2015-09-21

    Extracellular vesicles (EVs) mediate non-conventional transport of molecules across the fungal cell wall. We aimed at describing the carbohydrate composition and surface carbohydrate epitopes of EVs isolated from the pathogenic fungi Paracoccidioides brasiliensis and P. lutzii using standard procedures. Total EV carbohydrates were ethanol-precipitated from preparations depleted of lipids and proteins, then analyzed by chemical degradation, gas chromatography-mass spectrometry, nuclear magnetic resonance and size-exclusion chromatography. EV glycosyl residues of Glc, Man, and Gal comprised most probably two major components: a high molecular mass 4,6-α-glucan and a galactofuranosylmannan, possibly an oligomer, bearing a 2-α-Manp main chain linked to β-Galf (1,3) and α-Manp (1,6) end units. The results also suggested the presence of small amounts of a (1→6)- Manp polymer, (1→3)-glucan and (1→6)-glucan. Glycan microarrays allowed identification of EV surface lectin(s), while plant lectin microarray profiling revealed terminal Man and GlcNAc residues exposed at the EVs surface. Mammalian lectin microarray profiling showed that DC-SIGN receptors recognized surface carbohydrate in Paracoccidioides EVs. Our results suggest that oligosaccharides, cytoplasmic storage, and cell wall polysaccharides can be exported in fungal EVs, which also expose surface PAMPs and lectins. As a result, the role of these newly identified components in the interaction with the host remains to be unraveled.

  9. Erv41p and Erv46p: new components of COPII vesicles involved in transport between the ER and Golgi complex.

    PubMed

    Otte, S; Belden, W J; Heidtman, M; Liu, J; Jensen, O N; Barlowe, C

    2001-02-05

    Proteins contained on purified COPII vesicles were analyzed by matrix-assisted laser desorption ionization mass spectrometry combined with database searching. We identified four known vesicle proteins (Erv14p, Bet1p, Emp24p, and Erv25p) and an additional nine species (Yip3p, Rer1p, Erp1p, Erp2p, Erv29p, Yif1p, Erv41p, Erv46p, and Emp47p) that had not been localized to ER vesicles. Using antibodies, we demonstrate that these proteins are selectively and efficiently packaged into COPII vesicles. Three of the newly identified vesicle proteins (Erv29p, Erv41p, and Erv46p) represent uncharacterized integral membrane proteins that are conserved across species. Erv41p and Erv46p were further characterized. These proteins colocalized to ER and Golgi membranes and exist in a detergent-soluble complex that was isolated by immunoprecipitation. Yeast strains lacking Erv41p and/or Erv46p are viable but display cold sensitivity. The expression levels of Erv41p and Erv46p are interdependent such that Erv46p was reduced in an erv41Delta strain, and Erv41p was not detected in an erv46Delta strain. When the erv41Delta or ev46Delta alleles were combined with other mutations in the early secretory pathway, altered growth phenotypes were observed in some of the double mutant strains. A cell-free assay that reproduces transport between the ER and Golgi indicates that deletion of the Erv41p-Erv46p complex influences the membrane fusion stage of transport.

  10. The prokaryotic V4R domain is the likely ancestor of a key component of the eukaryotic vesicle transport system.

    PubMed

    Podar, Mircea; Wall, Mark A; Makarova, Kira S; Koonin, Eugene V

    2008-01-25

    Intracellular vesicle traffic that enables delivery of proteins between the endoplasmic reticulum, Golgi and various endosomal subcompartments is one of the hallmarks of the eukaryotic cell. Its evolutionary history is not well understood but the process itself and the core vesicle traffic machinery are believed to be ancient. We show here that the 4-vinyl reductase (V4R) protein domain present in bacteria and archaea is homologous to the Bet3 subunit of the TRAPP1 vesicle-tethering complex that is conserved in all eukaryotes. This suggests, for the first time, a prokaryotic origin for one of the key eukaryotic trafficking proteins.

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

    PubMed Central

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

    1992-01-01

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

  12. 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

  13. CsAP3: A Cucumber Homolog to Arabidopsis APETALA3 with Novel Characteristics

    PubMed Central

    Sun, Jin-Jing; Li, Feng; Wang, Dong-Hui; Liu, Xiao-Feng; Li, Xia; Liu, Na; Gu, Hai-Tao; Zou, Cheng; Luo, Jing-Chu; He, Chao-Xing; Huang, San-Wen; Zhang, Xiao-Lan; Xu, Zhi-Hong; Bai, Shu-Nong

    2016-01-01

    In our previous efforts to understand the regulatory mechanisms of cucumber unisexual flower development, we observed a stamen-specific down-regulation of the ethylene receptor CsETR1 in stage 6 female flowers of cucumber (Cucumis sativus L.). This down-regulation is correlated with the primordial anther-specific DNA damage that characterizes inappropriate stamen development in cucumber female flowers. To understand how CsETR1 is down regulated in the stamen, we characterized a cucumber MADS box gene homologous to Arabidopsis AP3, CsAP3. We demonstrated that CsAP3 is functionally equivalent to the Arabidopsis B-class MADS gene AP3. However, three novel characteristics of CsAP3 were found. These include firstly, binding and activating CsETR1 promoter in vitro and in vivo; secondly, containing a GV repeat in its C-terminus, which is conserved in cucurbits and required for the transcription activation; and thirdly, decreased expression as the node number increases, which is similar to that found for CsETR1. These findings revealed not only the conserved function of CsAP3 as a B-class floral identity gene, but also its unique functions in regulation of female flower development in cucumber. PMID:27540391

  14. pH gradient effects on chloride transport across basolateral membrane vesicles from guinea-pig jejunum.

    PubMed Central

    Touzani, K; Alvarado, F; Vasseur, M

    1997-01-01

    1. The effects of alkaline-inside pH gradients on 36Cl- uptake were quantified by using brush-border membrane (BBM) and basolateral membrane (BLM) vesicles from guinea-pig jejunum. 2. With BBM vesicles, a pHo/pHi gradient of 5.0/7.5 yielded fast overshoots involving a random, non-obligatory Cl(-)-H+ symport, strongly inhibited by CCCP. In contrast, BLM vesicles responded to similar pH gradients with much smaller, delayed overshoots, unaffected by CCCP. 3. The initial Cl- entry rates into BLM vesicles were a function of each pHo, pHi and delta pH value. They were stimulated by valinomycin in the presence of inward-directed K+ gradients. Short-circuiting the membrane potential with equilibrated K+ and valinomycin inhibited pH gradient-dependent Cl- uptake, but only partially. 4. Taken together, these results indicate that guinea-pig jejunal BLM vesicles possess both Cl- conductance and Cl(-)-H+ symport activities. 5. Even when different, the BBM and the BLM symporters are mechanistically similar. Neither of them involves a Cl(-)-OH- antiport, nor a simultaneous Cl(-)-anion exchange mechanism. Rather, for each membrane, all of these activities (symport, anion exchange) can be explained in terms of a single mobile carrier acting as a random, non-obligatory Cl(-)-H+ symporter where exchange occurs simply by counterflow. Net Cl- translocation via either the ternary (Cl(-)-C-H+) or the binary (Cl(-)-C) complexes accounts, respectively, for the existence of two, operationally distinct, electroneutral and rheogenic components. 6. The BBM symporter appears to involve an AE2 protein, but the molecular identity of the BLM one remains to be established. PMID:9147326

  15. Functions of adaptor protein (AP)-3 and AP-1 in tyrosinase sorting from endosomes to melanosomes.

    PubMed

    Theos, Alexander C; Tenza, Danièle; Martina, José A; Hurbain, Ilse; Peden, Andrew A; Sviderskaya, Elena V; Stewart, Abigail; Robinson, Margaret S; Bennett, Dorothy C; Cutler, Daniel F; Bonifacino, Juan S; Marks, Michael S; Raposo, Graça

    2005-11-01

    Specialized cells exploit adaptor protein complexes for unique post-Golgi sorting events, providing a unique model system to specify adaptor function. Here, we show that AP-3 and AP-1 function independently in sorting of the melanocyte-specific protein tyrosinase from endosomes to the melanosome, a specialized lysosome-related organelle distinguishable from lysosomes. AP-3 and AP-1 localize in melanocytes primarily to clathrin-coated buds on tubular early endosomes near melanosomes. Both adaptors recognize the tyrosinase dileucine-based melanosome sorting signal, and tyrosinase largely colocalizes with each adaptor on endosomes. In AP-3-deficient melanocytes, tyrosinase accumulates inappropriately in vacuolar and multivesicular endosomes. Nevertheless, a substantial fraction still accumulates on melanosomes, concomitant with increased association with endosomal AP-1. Our data indicate that AP-3 and AP-1 function in partially redundant pathways to transfer tyrosinase from distinct endosomal subdomains to melanosomes and that the AP-3 pathway ensures that tyrosinase averts entrapment on internal membranes of forming multivesicular bodies.

  16. Compartmentation of malic acid in mesophyll cells of Kalanchoee daigremontiana: indications of a intracellular cytosolic vesicle transport mechanism

    SciTech Connect

    Balsamo, R.A.; Uribe, E.G.

    1987-04-01

    Leaf tissue was harvested over a 24hr period at one to three hour intervals. The malic acid levels in the tissue were assayed spectrophotometrically and the percent cell volume occupied by cytosolic vesicles in replicate samples was determined. The total volume of the cytosolic vesicles fluctuated throughout the photoperiod concommitantly with malic acid concentrations present in the tissue. An intact leaf tissue section (10.2cm/sup 2/) was radiolabeled with /sup 14/CO/sub 2/ seven hours into the dark period for thirty minutes. Two dimensional thin layer chromatography and electrophoresis of the tissue determined that 96% of the label was incorporated into malic acid. A freeze substitution procedure was initiated followed by microautoradiography (Fisher 1971) which allowed for the tracing of intracellular malic acid migration and compartmentation within the mesophyll cells. The results and interpretation of this experiment will be presented.

  17. Linking cytoplasmic dynein and transport of Rab8 vesicles to the midbody during cytokinesis by the doublecortin domain-containing 5 protein.

    PubMed

    Kaplan, Anna; Reiner, Orly

    2011-12-01

    Completion of mitosis requires microtubule-dependent transport of membranes to the midbody. Here, we identified a role in cytokinesis for doublecortin domain-containing protein 5 (DCDC5), a member of the doublecortin protein superfamily. DCDC5 is a microtubule-associated protein expressed in both specific and dynamic fashions during mitosis. We show that DCDC5 interacts with cytoplasmic dynein and Rab8 (also known as Ras-related protein Rab-8A), as well as with the Rab8 nucleotide exchange factor Rabin8 (also known as Rab-3A-interacting protein). Following DCDC5 knockdown, the durations of the metaphase to anaphase transition and cytokinesis, and the proportion of multinucleated cells increases, whereas cell viability decreases. Furthermore, knockdown of DCDC5 or addition of a dynein inhibitor impairs the entry of Golgi-complex-derived Rab8-positive vesicles to the midbody. These findings suggest that DCDC5 plays an important role in mediating dynein-dependent transport of Rab8-positive vesicles and in coordinating late cytokinesis.

  18. AP-3 and Rabip4’ Coordinately Regulate Spatial Distribution of Lysosomes

    PubMed Central

    Ivan, Viorica; Martinez-Sanchez, Emma; Sima, Livia E.; Oorschot, Viola; Klumperman, Judith; Petrescu, Stefana M.; van der Sluijs, Peter

    2012-01-01

    The RUN and FYVE domain proteins rabip4 and rabip4’ are encoded by RUFY1 and differ in a 108 amino acid N-terminal extension in rabip4’. Their identical C terminus binds rab5 and rab4, but the function of rabip4s is incompletely understood. We here found that silencing RUFY1 gene products promoted outgrowth of plasma membrane protrusions, and polarized distribution and clustering of lysosomes at their tips. An interactor screen for proteins that function together with rabip4’ yielded the adaptor protein complex AP-3, of which the hinge region in the β3 subunit bound directly to the FYVE domain of rabip4’. Rabip4’ colocalized with AP-3 on a tubular subdomain of early endosomes and the extent of colocalization was increased by a dominant negative rab4 mutant. Knock-down of AP-3 had an ever more dramatic effect and caused accumulation of lysosomes in protrusions at the plasma membrane. The most peripheral lysosomes were localized beyond microtubules, within the cortical actin network. Our results uncover a novel function for AP-3 and rabip4’ in regulating lysosome positioning through an interorganellar pathway. PMID:23144738

  19. Immunological Evidence for the Existence of a Carrier Protein for Sucrose Transport in Tonoplast Vesicles from Red Beet (Beta vulgaris L.) Root Storage Tissue.

    PubMed Central

    Getz, H. P.; Grosclaude, J.; Kurkdjian, A.; Lelievre, F.; Maretzki, A.; Guern, J.

    1993-01-01

    Monoclonal antibodies were raised in mice against a highly purified tonoplast fraction from isolated red beet (Beta vulgaris L. ssp. conditiva) root vacuoles. Positive hybridoma clones and sub-clones were identified by prescreening using an enzyme-linked immunosorbent assay (ELISA) and by postscreening using a functional assay. This functional assay consisted of testing the impact of hybridoma supernatants and antibody-containing ascites fluids on basal and ATP-stimulated sugar uptake in vacuoles, isolated from protoplasts, as well as in tonoplast vesicles, prepared from tissue homogenates of red beet roots. Antibodies from four clones were particularly positive in ELISAs and they inhibited sucrose uptake significantly. These antibodies were specific inhibitors of sucrose transport, but they exhibited relatively low membrane and species specificity since uptake into red beet root protoplasts and sugarcane tonoplast vesicles was inhibited as well. Fast protein liquid chromatography assisted size exclusion chromatography on Superose 6 columns yielded two major peaks in the 55 to 65-kD regions and in the 110- to 130-kD regions of solubilized proteins from red beet root tonoplasts, which reacted positively in immunoglobulin-M(IgM)-specific ELISAs with anti-sugarcane tonoplast monoclonal IgM antibodies. Only reconstituted proteoliposomes containing polypeptides from the 55- to 65-kD band took up [14C]-sucrose with linear rates for 2 min, suggesting that this fraction contains the tonoplast sucrose carrier. PMID:12231863

  20. Ca2+ transport and heat production in vesicles derived from the sarcoplasmic reticulum terminal cisternae: regulation by K+.

    PubMed

    Nigro, Mariana; Arruda, Ana Paula; de Meis, Leopoldo

    2009-07-01

    In this work, we compared the effect of K+ on vesicles derived from the longitudinal (LSR) and terminal cisternae (HSR) of rabbit white muscle. In HSR, K+ was found to inhibit both the Ca2+ accumulation and the heat released during ATP hydrolysis by the Ca2+-ATPase (SERCA1). This was not observed in LSR. Valinomycin abolished the HSR Ca2+-uptake inhibition promoted by physiological K+ concentrations, but it did not modify the thermogenic activity of the Ca2+ pump. The results with HSR are difficult to interpret, assuming that a single K+ is binding to either the ryanodine channel or to the Ca2+-ATPase. It is suggested that an increase of K+ in the assay medium alters the interactions among the various proteins found in HSR, thus modifying the properties of both the ryanodine channel and SERCA1.

  1. Controlled enlargement of the glycoprotein vesicle surrounding a volvox embryo requires the InvB nucleotide-sugar transporter and is required for normal morphogenesis.

    PubMed

    Ueki, Noriko; Nishii, Ichiro

    2009-04-01

    Here, we report our analysis of a mutant of Volvox carteri, InvB, whose embryos fail to execute inversion, the process in which each Volvox embryo normally turns itself inside-out at the end of embryogenesis, thereby achieving the adult configuration. The invB gene encodes a nucleotide-sugar transporter that exhibits GDP-mannose transport activity when expressed in yeast. In wild-type embryos, the invB transcript is maximally abundant before and during inversion. A mannoside probe (fluorescent concanavalin A) stains the glycoprotein-rich gonidial vesicle (GV) surrounding wild-type embryos much more strongly than it stains the GV surrounding InvB embryos. Direct measurements revealed that throughout embryogenesis the GV surrounding a wild-type embryo increases in size much more than the GV surrounding an InvB embryo does, and the fully cleaved InvB embryo is much more tightly packed within its GV than a wild-type embryo is. To test the hypothesis that the restraint imposed by a smaller than normal GV directly causes the inversion defect in the mutant, we released InvB embryos from their GVs microsurgically. The resulting embryos inverted normally, demonstrating that controlled enlargement of the GV, by a process in which requires the InvB nucleotide-sugar transporter, is essential to provide the embryo sufficient space to complete inversion.

  2. Nuclear transport of cancer extracellular vesicle-derived biomaterials through nuclear envelope invagination-associated late endosomes.

    PubMed

    Rappa, Germana; Santos, Mark F; Green, Toni M; Karbanová, Jana; Hassler, Justin; Bai, Yongsheng; Barsky, Sanford H; Corbeil, Denis; Lorico, Aurelio

    2017-02-28

    Extracellular membrane vesicles (EVs) function as vehicles of intercellular communication, but how the biomaterials they carry reach the target site in recipient cells is an open question. We report that subdomains of Rab7+ late endosomes and nuclear envelope invaginations come together to create a sub-nuclear compartment, where biomaterials associated with CD9+ EVs are delivered. EV-derived biomaterials were also found in the nuclei of host cells. The inhibition of nuclear import and export pathways abrogated the nuclear localization of EV-derived biomaterials or led to their accumulation therein, respectively, suggesting that their translocation is dependent on nuclear pores. Nuclear envelope invagination-associated late endosomes were observed in ex vivo biopsies in both breast carcinoma and associated stromal cells. The transcriptome of stromal cells exposed to cancer cell-derived CD9+ EVs revealed that the regulation of eleven genes, notably those involved in inflammation, relies on the nuclear translocation of EV-derived biomaterials. Our findings uncover a new cellular pathway used by EVs to reach nuclear compartment.

  3. Poking Vesicles

    NASA Astrophysics Data System (ADS)

    Powers, Thomas R.; Huber, Greg; Goldstein, Raymond E.

    2000-03-01

    Lipid vesicles can exhibit a variety of interesting shapes when subject to point forcing, as has been demonstrated with laser-tweezed beads and with growing microtubules. Using numerical and analytic techniques, we study the force vs. extension in two regimes. At low forces, the resistance to deformation is primarily entropic, with a tension given by the fluctuating bending modes. At high forces, the thermal wrinkles are smoothed out, and there is bending and stretching which balance in a thin tether, which we treat using boundary-layer techniques. This work was supported in part by the Harvard MRSEC and Army Research Office Grant DAA655-97-1-014 (TRP) and NSF DMR9812526 (GH & REG).

  4. Leucine transport is affected by Bacillus thuringiensis Cry1 toxins in brush border membrane vesicles from Ostrinia nubilalis Hb (Lepidoptera: Pyralidae) and Sesamia nonagrioides Lefebvre (Lepidoptera: Noctuidae) midgut.

    PubMed

    Leonardi, M Giovanna; Caccia, Silvia; González-Cabrera, Joel; Ferré, Juan; Giordana, Barbara

    2006-01-01

    The pore-forming activity of Cry1Ab, Cry1Fa and Cry1Ca toxins and their interaction with leucine transport mediated by the K(+)/leucine cotransporter were studied in brush border membrane vesicles (BBMVs) isolated from the midgut of Ostrinia nubilalis and Sesamia nonagrioides. In both species, as in other Lepidoptera, leucine uptake by BBMVs can take place in the absence of cations, but it can also be driven by a K(+) gradient. Experiments with the voltage-sensitive fluorescent dye 3,3'-diethylthiacarbocyanine iodide proved that Cry1Ab, a Bacillus thuringiensis toxin active in vivo, enhanced the membrane permeability to potassium in O. nubilalis BBMVs. This result is in agreement with similar effects observed in S. nonagrioides BBMV incubated with various Cry1 toxins active in vivo. The effect of the above toxins was tested on the initial rate of 0.1 mM: leucine influx. Instead of an increase in leucine influx, a reduction was observed with the Cry1 toxins active in vivo. Cry1Ab and Cry1Fa, but not the inactive toxin Cry1Da, inhibited in a dose-dependent manner leucine uptake both in the absence and in the presence of a K(+) gradient, a clear indication that their effect is independent of the channel formed by the toxins and that this effect is exerted directly on the amino acid transport system.

  5. Preparation of Membrane Vesicles Enriched in ATP-Dependent Proton Transport from Suspension Cultures of Tomato Cells

    PubMed Central

    Dupont, Frances M.; Zabala, Maria De Gracia

    1985-01-01

    Membranes enriched in ATP-dependent proton transport were prepared from suspension cultures of tomato cells (Lycopersicon esculentum Mill cv VF36). Suspension cultures were a source of large quantities of membranes from rapidly growing, undifferentiated cells. Proton transport activity was assayed as quench of acridine orange fluorescence. The activity of the proton translocating ATPase and of several other membrane enzymes was measured as a function of the cell culture cycle. The relative distribution of the enzymes between the 3,000, 10,000, and 100,000g pellets remained the same throughout the cell culture cycle, but yield of total activity and activity per gram fresh weight with time had a unique profile for each enzyme tested. Maximal yield of the proton translocating ATPase activity was obtained from cells in the middle logarithmic phase of growth, and from 50 to 90% of the activity was found in the 10,000g pellet. The proton translocating ATPase activity was separable from NADPH cytochrome c reductase and cytochrome c oxidase on a sucrose gradient. Proton transport activity had a broad pH optimum (7.0-8.0), was stimulated by KCl with a Km of 5 to 10 millimolar, stimulation being due to the anion, Cl−, and not the cation, K+, and was not inhibited by vanadate, but was inhibited by NO3−. The activity is tentatively identified as the tonoplast ATPase. PMID:16664030

  6. Mechanisms of COPI vesicle formation

    PubMed Central

    Hsu, Victor W.; Yang, Jia-Shu

    2009-01-01

    Coat Protein I (COPI) is one of the most intensely investigated coat complexes. Numerous studies have contributed to a general understanding of how coat proteins act to initiate intracellular vesicular transport. This review highlights key recent findings that have shaped our current understanding of how COPI vesicles are formed. PMID:19854177

  7. Vesicle-associated Membrane Protein 3 (VAMP3) Mediates Constitutive Trafficking of the Renal Co-transporter NKCC2 in Thick Ascending Limbs

    PubMed Central

    Caceres, Paulo S.; Mendez, Mariela; Haque, Mohammed Z.; Ortiz, Pablo A.

    2016-01-01

    Renal cells of the thick ascending limb (TAL) reabsorb NaCl via the apical Na+/K+/2Cl− co-transporter NKCC2. Trafficking of NKCC2 to the apical surface regulates NKCC2-mediated NaCl absorption and blood pressure. The molecular mechanisms by which NKCC2 reaches the apical surface and their role in renal function and maintenance of blood pressure are poorly characterized. Here we report that NKCC2 interacts with the vesicle fusion protein VAMP3, and they co-localize at the TAL apical surface. We observed that silencing VAMP3 in vivo blocks constitutive NKCC2 exocytic delivery, decreasing the amount of NKCC2 at the TAL apical surface. VAMP3 is not required for cAMP-stimulated NKCC2 exocytic delivery. Additionally, genetic deletion of VAMP3 in mice decreased total expression of NKCC2 in the TAL and lowered blood pressure. Consistent with these results, urinary excretion of water and electrolytes was higher in VAMP3 knock-out mice, which produced more diluted urine. We conclude that VAMP3 interacts with NKCC2 and mediates its constitutive exocytic delivery to the apical surface. Additionally, VAMP3 is required for normal NKCC2 expression, renal function, and blood pressure. PMID:27551042

  8. The transmembrane pH gradient drives uphill folate transport in rabbit jejunum. Direct evidence for folate/hydroxyl exchange in brush border membrane vesicles.

    PubMed Central

    Schron, C M; Washington, C; Blitzer, B L

    1985-01-01

    In rabbit jejunal, but not ileal brush border membrane vesicles, an outwardly directed OH- gradient (pH 7.7 inside, pH 5.5 outside) markedly stimulated the initial velocity of folate (0.1 microM) uptake compared with uptake in the absence of a pH gradient. Under pH gradient conditions, folate was transiently accumulated at a concentration four times that found at equilibrium (over-shoot), implying uphill transport of the vitamin. Equilibrium folate uptake was inversely proportional to medium osmolality, suggesting uptake into an osmotically sensitive space. pH gradient-stimulated folate uptake was markedly reduced by inhibitors of anion exchange (4,4'-diisothiocyano-2,2'-disulfonic acid stilbene; 4-acetamido-4-isothiocyanostilbene-2,2'-disulfonic acid; furosemide), and was saturable (folate Km = 0.19 +/- 0.02 microM; Vmax = 12.8 +/- 0.4 pmol X mg protein-1 X min-1). Imposition of an inside-positive electrical potential did not stimulate folate uptake, suggesting that stimulation by a pH gradient was not due to an induced electrical potential. In contrast, an inwardly directed Na+ or K+ gradient did not stimulate folate uptake. These findings provide evidence for a carrier on the jejunal brush border membrane that mediates folate/OH- exchange (or H+/folate co-transport), and are consonant with the known presence of an outwardly directed OH- gradient in vivo (brush border acid microclimate), an acidic pH optimum for intestinal folate uptake, and the primary role of the jejunum in folate absorption. PMID:4056063

  9. Role of calcium in phosphoinositide metabolism and inhibition of norepinephrine transport into synaptic vesicles by amphetamine analogs

    SciTech Connect

    Knepper, S.M.

    1985-01-01

    Norepinephrine-(NE) and calcium ionophore A23187-stimulated phosphoinositide (PIn) metabolism in rat brain slices was studied under varying calcium conditions. Tissue was labelled with /sup 3/H-myo-inositol and /sup 3/H-inositol phosphates (IPn), products of PIn metabolism were measured. In the absence of media calcium the response to NE was decreased while that to A23187 was little affected A23187 can release calcium from intracellular stores. Basal and stimulated accumulation of /sup 3/H-IPn was reversibly antagonized with EGTA by addition of calcium. Using calcium buffers, approximately 10/sup -7/ M free calcium was required to support hydrolysis. Free intracellular calcium is maintained at approximately this level. Thus calcium is required for PIn hydrolysis but appears to play a permissive role, basal levels being sufficient to support metabolism. Conformationally-defined (rigid) and -restricted (semi-rigid) analogs of the most stable conformations of amphetamine, antiperiplanar (exo) and gauche (endo), were utilized to probe the conformational requirements of vesicular NE transport. Analogs tested were 2-aminotetralin (2AT), 3-methyltetrahydroisoquinoline, anti- and syn-9-aminobenzobicyclo(2.2.1)heptene, and endo and exo conformers of 2-aminobenzobicyclo(2.2.1)heptene and 2-aminobenzobicyclo(2.2.2)octene.

  10. BLOC-1 Interacts with BLOC-2 and the AP-3 Complex to Facilitate Protein Trafficking on Endosomes

    PubMed Central

    Di Pietro, Santiago M.; Falcón-Pérez, Juan M.; Tenza, Danièle; Setty, Subba R.G.; Marks, Michael S.; Raposo, Graça

    2006-01-01

    The adaptor protein (AP)-3 complex is a component of the cellular machinery that controls protein sorting from endosomes to lysosomes and specialized related organelles such as melanosomes. Mutations in an AP-3 subunit underlie a form of Hermansky-Pudlak syndrome (HPS), a disorder characterized by abnormalities in lysosome-related organelles. HPS in humans can also be caused by mutations in genes encoding subunits of three complexes of unclear function, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2, and -3. Here, we report that BLOC-1 interacts physically and functionally with AP-3 to facilitate the trafficking of a known AP-3 cargo, CD63, and of tyrosinase-related protein 1 (Tyrp1), a melanosomal membrane protein previously thought to traffic only independently of AP-3. BLOC-1 also interacts with BLOC-2 to facilitate Tyrp1 trafficking by a mechanism apparently independent of AP-3 function. Both BLOC-1 and -2 localize mainly to early endosome-associated tubules as determined by immunoelectron microscopy. These findings support the idea that BLOC-1 and -2 represent hitherto unknown components of the endosomal protein trafficking machinery. PMID:16837549

  11. BLOC-1 interacts with BLOC-2 and the AP-3 complex to facilitate protein trafficking on endosomes.

    PubMed

    Di Pietro, Santiago M; Falcón-Pérez, Juan M; Tenza, Danièle; Setty, Subba R G; Marks, Michael S; Raposo, Graça; Dell'Angelica, Esteban C

    2006-09-01

    The adaptor protein (AP)-3 complex is a component of the cellular machinery that controls protein sorting from endosomes to lysosomes and specialized related organelles such as melanosomes. Mutations in an AP-3 subunit underlie a form of Hermansky-Pudlak syndrome (HPS), a disorder characterized by abnormalities in lysosome-related organelles. HPS in humans can also be caused by mutations in genes encoding subunits of three complexes of unclear function, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2, and -3. Here, we report that BLOC-1 interacts physically and functionally with AP-3 to facilitate the trafficking of a known AP-3 cargo, CD63, and of tyrosinase-related protein 1 (Tyrp1), a melanosomal membrane protein previously thought to traffic only independently of AP-3. BLOC-1 also interacts with BLOC-2 to facilitate Tyrp1 trafficking by a mechanism apparently independent of AP-3 function. Both BLOC-1 and -2 localize mainly to early endosome-associated tubules as determined by immunoelectron microscopy. These findings support the idea that BLOC-1 and -2 represent hitherto unknown components of the endosomal protein trafficking machinery.

  12. Improved Pharmacological and Structural Properties of HIV Fusion Inhibitor AP3 over Enfuvirtide: Highlighting Advantages of Artificial Peptide Strategy

    DOE PAGES

    Zhu, Xiaojie; Zhu, Yun; Ye, Sheng; ...

    2015-08-19

    Enfuvirtide (T20), is the first HIV fusion inhibitor approved for treatment of HIV/AIDS patients who fail to respond to the current antiretroviral drugs. However, its clinical application is limited because of short half-life, drug resistance and cross-reactivity with the preexisting antibodies in HIV-infected patients. Using an artificial peptide strategy, we designed a peptide with non-native protein sequence, AP3, which exhibited potent antiviral activity against a broad spectrum of HIV-1 strains, including those resistant to T20, and had remarkably longer in vivo half-life than T20. While the preexisting antibodies in HIV-infected patients significantly suppressed T20’s antiviral activity, these antibodies neither recognizedmore » AP3, nor attenuated its anti-HIV-1 activity. Structurally different from T20, AP3 could fold into single-helix and interact with gp41 NHR. The two residues, Met and Thr, at the N-terminus of AP3 form a hook-like structure to stabilize interaction between AP3 and NHR helices. Therefore, AP3 has potential for further development as a new HIV fusion inhibitor with improved antiviral efficacy, resistance profile and pharmacological properties over enfuvirtide. Meanwhile, this study highlighted the advantages of artificially designed peptides, and confirmed that this strategy could be used in developing artificial peptide-based viral fusion inhibitors against HIV and other enveloped viruses.« less

  13. The temperate Burkholderia phage AP3 of the Peduovirinae shows efficient antimicrobial activity against B. cenocepacia of the IIIA lineage.

    PubMed

    Roszniowski, Bartosz; Latka, Agnieszka; Maciejewska, Barbara; Vandenheuvel, Dieter; Olszak, Tomasz; Briers, Yves; Holt, Giles S; Valvano, Miguel A; Lavigne, Rob; Smith, Darren L; Drulis-Kawa, Zuzanna

    2017-02-01

    Burkholderia phage AP3 (vB_BceM_AP3) is a temperate virus of the Myoviridae and the Peduovirinae subfamily (P2likevirus genus). This phage specifically infects multidrug-resistant clinical Burkholderia cenocepacia lineage IIIA strains commonly isolated from cystic fibrosis patients. AP3 exhibits high pairwise nucleotide identity (61.7 %) to Burkholderia phage KS5, specific to the same B. cenocepacia host, and has 46.7-49.5 % identity to phages infecting other species of Burkholderia. The lysis cassette of these related phages has a similar organization (putative antiholin, putative holin, endolysin, and spanins) and shows 29-98 % homology between specific lysis genes, in contrast to Enterobacteria phage P2, the hallmark phage of this genus. The AP3 and KS5 lysis genes have conserved locations and high amino acid sequence similarity. The AP3 bacteriophage particles remain infective up to 5 h at pH 4-10 and are stable at 60 °C for 30 min, but are sensitive to chloroform, with no remaining infective particles after 24 h of treatment. AP3 lysogeny can occur by stable genomic integration and by pseudo-lysogeny. The lysogenic bacterial mutants did not exhibit any significant changes in virulence compared to wild-type host strain when tested in the Galleria mellonella moth wax model. Moreover, AP3 treatment of larvae infected with B. cenocepacia revealed a significant increase (P < 0.0001) in larvae survival in comparison to AP3-untreated infected larvae. AP3 showed robust lytic activity, as evidenced by its broad host range, the absence of increased virulence in lysogenic isolates, the lack of bacterial gene disruption conditioned by bacterial tRNA downstream integration site, and the absence of detected toxin sequences. These data suggest that the AP3 phage is a promising potent agent against bacteria belonging to the most common B. cenocepacia IIIA lineage strains.

  14. Functions of Adaptor Protein (AP)-3 and AP-1 in Tyrosinase Sorting from Endosomes to MelanosomesD⃞

    PubMed Central

    Theos, Alexander C.; Tenza, Danièle; Martina, José A.; Hurbain, Ilse; Peden, Andrew A.; Sviderskaya, Elena V.; Stewart, Abigail; Robinson, Margaret S.; Bennett, Dorothy C.; Cutler, Daniel F.; Bonifacino, Juan S.; Marks, Michael S.; Raposo, Graça

    2005-01-01

    Specialized cells exploit adaptor protein complexes for unique post-Golgi sorting events, providing a unique model system to specify adaptor function. Here, we show that AP-3 and AP-1 function independently in sorting of the melanocyte-specific protein tyrosinase from endosomes to the melanosome, a specialized lysosome-related organelle distinguishable from lysosomes. AP-3 and AP-1 localize in melanocytes primarily to clathrin-coated buds on tubular early endosomes near melanosomes. Both adaptors recognize the tyrosinase dileucine-based melanosome sorting signal, and tyrosinase largely colocalizes with each adaptor on endosomes. In AP-3-deficient melanocytes, tyrosinase accumulates inappropriately in vacuolar and multivesicular endosomes. Nevertheless, a substantial fraction still accumulates on melanosomes, concomitant with increased association with endosomal AP-1. Our data indicate that AP-3 and AP-1 function in partially redundant pathways to transfer tyrosinase from distinct endosomal subdomains to melanosomes and that the AP-3 pathway ensures that tyrosinase averts entrapment on internal membranes of forming multivesicular bodies. PMID:16162817

  15. Validation of membrane vesicle-based breast cancer resistance protein and multidrug resistance protein 2 assays to assess drug transport and the potential for drug-drug interaction to support regulatory submissions.

    PubMed

    Elsby, Robert; Smith, Veronica; Fox, Lisa; Stresser, David; Butters, Caroline; Sharma, Pradeep; Surry, Dominic D

    2011-09-01

    Breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) can play a role in the absorption, distribution, metabolism, and excretion of drugs, impacting on the potential for drug-drug interactions. This study has characterized insect cell- and mammalian cell-derived ABC-transporter-expressing membrane vesicle test systems and validated methodologies for evaluation of candidate drugs as substrates or inhibitors of BCRP or MRP2. Concentration-dependent uptake of BCRP ([³H]oestrone 3-sulfate, [³H]methotrexate, [³H]rosuvastatin) and MRP2 ([³H]oestradiol 17β-glucuronide, [³H]pravastatin, carboxydichlorofluorescein) substrates, and inhibitory potencies (IC₅₀) of BCRP (sulfasalazine, novobiocin, fumitremorgin C) and MRP2 (benzbromarone, MK-571, terfenadine) inhibitors were determined. The apparent K(m) for probes [³H]oestrone 3-sulfate and [³H]oestradiol 17β-glucuronide was determined in insect cell vesicles to be 7.4 ± 1.7 and 105 ± 8.3 µM, respectively. All other substrates exhibited significant uptake ratios. Positive control inhibitors sulfasalazine and benzbromarone gave IC₅₀ values of 0.74 ± 0.18 and 36 ± 6.1 µM, respectively. All other inhibitors exhibited concentration-dependent inhibition. There was no significant difference in parameters generated between test systems. On the basis of the validation results, acceptance criteria to identify substrates/inhibitors of BCRP and MRP2 were determined for insect cell vesicles. The approach builds on earlier validations to support drug registration and extends from those cell-based systems to encompass assay formats using membrane vesicles.

  16. Hydrolysis of bound GTP by ARF protein triggers uncoating of Golgi- derived COP-coated vesicles

    PubMed Central

    1993-01-01

    The cycle of nucleotide exchange and hydrolysis by a small GTP-binding protein, ADP-ribosylation factor (ARF), helps to provide vectoriality to vesicle transport. Coat assembly is triggered when ARF binds GTP, initiating transport vesicle budding, and coat disassembly is triggered when ARF hydrolyzes GTP, allowing the uncoated vesicle to fuse. PMID:8253837

  17. Characterization of Alternaria infectoria extracellular vesicles

    PubMed Central

    Silva, Branca M.A.; Prados-Rosales, Rafael; Espadas-Moreno, Javier; Wolf, Julie M.; Luque-Garcia, Jose L.; Gonçalves, Teresa; Casadevall, Arturo

    2015-01-01

    Many fungi use membrane vesicles to transport complex molecules across their cell walls. Like mammalian exosomes, fungal vesicles contain lipids, proteins, and polysaccharides, many of which are associated with virulence. Here we identify and characterize extracellular vesicles (EVs) in Alternaria infectoria, a ubiquitous, environmental filamentous fungus that is also an opportunistic human pathogen. Examination of the A. infectoria EVs revealed a morphology similar to that of vesicles described in other fungal species. Of note, proteomic analysis detected a reduced number of vesicle-associated proteins. There were two prevalent categories among the 20 identified proteins, including the polysaccharide metabolism group, probably related to plant host invasion or biosynthesis/degradation of cell wall components, and the nuclear proteins, especially DNA repair enzymes. We also found enzymes related to pigment synthesis, adhesion to the host cell, and trafficking of vesicles/organelles/molecules. This is the first time EV secretions have been identified in a filamentous fungus. We believe that these vesicles might have a role in virulence. PMID:24576997

  18. Adhesion of Polymer Vesicles

    NASA Astrophysics Data System (ADS)

    Lin, John J.; Bates, Frank S.; Hammer, Daniel A.; Silas, James A.

    2005-07-01

    The adhesion and bending modulus of polybutadiene-poly(ethylene oxide) block copolymer vesicles made from a bidisperse mixture of polymers is measured using micropipette aspiration. The adhesion energy between biotinylated vesicles and avidin beads is modeled by incorporating the extension of the adhesive ligands above the surface brush of the vesicle according to the blob model of bidisperse polymer mixtures of Komura and Safran assuming the polymer brush at the surface of the vesicle is compact. The same model accurately reproduces the scaling of the bending modulus with polymer composition.

  19. Phospholipid Vesicles in Materials Science

    SciTech Connect

    Granick, Steve

    2016-05-11

    The objective of this research was to develop the science basis needed to deploy phospholipid vesicles as functional materials in energy contexts. Specifically, we sought to: (1) Develop an integrated molecular-level understanding of what determines their dynamical shape, spatial organization, and responsiveness to complex, time-varying environments; and (2) Develop understanding of their active transportation in crowded environments, which our preliminary measurements in cells suggest may hold design principles for targeting improved energy efficiency in new materials systems. The methods to do this largely involved fluorescence imaging and other spectroscopy involving single particles, vesicles, particles, DNA, and endosomes. An unexpected importance outcome was a new method to image light-emitting diodes during actual operation using super-resolution spectroscopy.

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

    PubMed Central

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

    1988-01-01

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

  1. Na(+)-I- symport activity is present in membrane vesicles from thyrotropin-deprived non-I(-)-transporting cultured thyroid cells.

    PubMed Central

    Kaminsky, S M; Levy, O; Salvador, C; Dai, G; Carrasco, N

    1994-01-01

    The active accumulation of I- in the thyroid gland is mediated by the Na(+)-I- symporter and driven by the Na+ gradient generated by the Na+/K(+)-ATPase. Thyrotropin (TSH) stimulates thyroidal I- accumulation. Rat thyroid-derived FRTL-5 cells require TSH to accumulate I-. TSH withdrawal for over 7 days results in complete loss of Na(+)-I-symport activity in these cells [Weiss, S. J., Philp, N. J. and Grollman, E. F. (1984) Endocrinology 114, 1090-1098]. Surprisingly, membrane vesicles prepared from FRTL-5 cells maintained in TSH-free medium [TSH(-)cells]accumulate I-, suggesting that the absence of Na(+)-I- symport activity in TSH(-) cells cannot be due solely to a decrease in the biosynthesis of either the symporter or a putative activating factor. This finding indicates that the Na(+)-I- symporter is present, probably in an inactive state, in TSH(-) cells despite their lack of Na(+)-I- symport activity. Na(+)-I- symport activity in thyroid membrane vesicles is enhanced when conditions for vesicle preparation favor proteolysis. Subcellular fractionation studies in both TSH(+) and TSH(-) cells show that Na(+)-I- symport activity is mostly associated with fractions enriched in plasma membrane rather than in intracellular membranes, suggesting that the Na(+)-I- symporter may constitutively reside in the plasma membrane and may be activated by TSH. Images PMID:8170988

  2. Permeability of human erythrocyte membrane vesicles to alkali cations.

    PubMed

    Sze, H; Solomon, A K

    1979-02-02

    The permeability of inside-out and right-side-out vesicles from erythrocyte membranes to inorganic cations was determined quantitatively. Using 86Rb as a K analog, we have measured the rate constant of 86Rb efflux from vesicles under equilibrium exchange conditions, using a dialysis procedure. The permeability coefficients of the vesicles to Rb are only about an order of magnitude greater than that of whole erythrocytes. Furthermore, we have measured many of the specialized transport systems known to exist in erythrocytes and have shown that glucose, sulfate, ATP-dependent Ca and ATP-dependent Na transport activities are retained by the vesicle membranes. These results suggest that inside-out and right-side-out vesicles can be used effectively to study transport properties of erythrocyte membranes.

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

    PubMed

    Frost, Adam

    2011-10-11

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

  4. Large vesicles record pathways of degassing at basalic volcanoes

    SciTech Connect

    Polacci, M.; Baker, D.R.; Bai, L.; Mancini, L.

    2008-10-08

    Volcanic degassing is directly linked to magma dynamics and controls the style of eruptive activity. To better understand how gas is transported within basaltic magma we perform a 3D investigation of vesicles preserved in scoria from the 2005 activity at Stromboli volcano (Italy). We find that clasts are characterized by the ubiquitous occurrence of one to a few large vesicles, exhibiting mostly irregular, tortuous, channel-like textures, orders of magnitude greater in volume than all the other vesicles in the sample. We compare observations on natural samples with results from numerical simulations and experimental investigations of vesicle size distributions and demonstrate that this type of vesicle invariably forms in magmas with vesicularities > 0.30 (and possibly > 0.10). We suggest that large vesicles represent pathways used by gas to flow non-explosively to the surface and that they indicate the development of an efficient system that sustains persistent degassing in basaltic systems.

  5. Vesicle extrusion in nanopores

    NASA Astrophysics Data System (ADS)

    Joóos, B. Éla; Bertrand, Martin; Ouellet, S. Ébastien

    2010-03-01

    Monodisperse vesicles of nearly circular shape or liposomes are used as drug delivery systems. Their fabrication involves repeated passage of large vesicles through small pores. At each passage the vesicle ruptures and the fragments reform into smaller vesicles. We report on the last stages of the process where small liposomes are pushed by pressure differences into nano-sized pores, and we study the stress distribution along the lipid bilayer to determine the rupture lines. This is done by performing coarse grained Molecular Dynamics simulations. We have developed a technique to measure the stress in the membrane based on a tessellation of the surface which allows us to monitor the local area per lipid fluctuations. The results show subtle and complex flow phenomena. We can predict the final size distribution after many passages. Comparisons will be made with existing experimental data.

  6. Human Immunodeficiency Virus Type 2 (HIV-2) Gag Is Trafficked in an AP-3 and AP-5 Dependent Manner

    PubMed Central

    Alford, Justine E.; Marongiu, Michela; Watkins, Gemma L.

    2016-01-01

    Although human immunodeficiency virus (HIV) types 1 and 2 are closely related lentiviruses with similar replication cycles, HIV-2 infection is associated with slower progression to AIDS, a higher proportion of long term non-progressors, and lower rates of transmission than HIV-1, likely as a consequence of a lower viral load during HIV-2 infection. A mechanistic explanation for the differential viral load remains unclear but knowledge of differences in particle production between HIV-1 and HIV-2 may help to shed light on this issue. In contrast to HIV-1, little is known about the assembly of HIV-2 particles, and the trafficking of HIV-2 Gag, the structural component of the virus, within cells. We have established that HIV-2 Gag accumulates in intracellular CD63 positive compartments, from which it may be delivered or recycled to the cell surface, or degraded. HIV-2 particle release was dependent on the adaptor protein complex AP-3 and the newly identified AP-5 complex, but much less so on AP-1. In contrast, HIV-1 particle release required AP-1 and AP-3, but not AP-5. AP-2, an essential component of clathrin-mediated endocytosis, which was previously shown to be inhibitory to HIV-1 particle release, had no effect on HIV-2. The differential requirement for adaptor protein complexes confirmed that HIV-1 and HIV-2 Gag have distinct cellular trafficking pathways, and that HIV-2 particles may be more susceptible to degradation prior to release. PMID:27392064

  7. Human Immunodeficiency Virus Type 2 (HIV-2) Gag Is Trafficked in an AP-3 and AP-5 Dependent Manner.

    PubMed

    Alford, Justine E; Marongiu, Michela; Watkins, Gemma L; Anderson, Emma C

    2016-01-01

    Although human immunodeficiency virus (HIV) types 1 and 2 are closely related lentiviruses with similar replication cycles, HIV-2 infection is associated with slower progression to AIDS, a higher proportion of long term non-progressors, and lower rates of transmission than HIV-1, likely as a consequence of a lower viral load during HIV-2 infection. A mechanistic explanation for the differential viral load remains unclear but knowledge of differences in particle production between HIV-1 and HIV-2 may help to shed light on this issue. In contrast to HIV-1, little is known about the assembly of HIV-2 particles, and the trafficking of HIV-2 Gag, the structural component of the virus, within cells. We have established that HIV-2 Gag accumulates in intracellular CD63 positive compartments, from which it may be delivered or recycled to the cell surface, or degraded. HIV-2 particle release was dependent on the adaptor protein complex AP-3 and the newly identified AP-5 complex, but much less so on AP-1. In contrast, HIV-1 particle release required AP-1 and AP-3, but not AP-5. AP-2, an essential component of clathrin-mediated endocytosis, which was previously shown to be inhibitory to HIV-1 particle release, had no effect on HIV-2. The differential requirement for adaptor protein complexes confirmed that HIV-1 and HIV-2 Gag have distinct cellular trafficking pathways, and that HIV-2 particles may be more susceptible to degradation prior to release.

  8. Rho-GTPase-regulated vesicle trafficking in plant cell polarity.

    PubMed

    Chen, Xu; Friml, Jiří

    2014-02-01

    ROPs (Rho of plants) belong to a large family of plant-specific Rho-like small GTPases that function as essential molecular switches to control diverse cellular processes including cytoskeleton organization, cell polarization, cytokinesis, cell differentiation and vesicle trafficking. Although the machineries of vesicle trafficking and cell polarity in plants have been individually well addressed, how ROPs co-ordinate those processes is still largely unclear. Recent progress has been made towards an understanding of the co-ordination of ROP signalling and trafficking of PIN (PINFORMED) transporters for the plant hormone auxin in both root and leaf pavement cells. PIN transporters constantly shuttle between the endosomal compartments and the polar plasma membrane domains, therefore the modulation of PIN-dependent auxin transport between cells is a main developmental output of ROP-regulated vesicle trafficking. The present review focuses on these cellular mechanisms, especially the integration of ROP-based vesicle trafficking and plant cell polarity.

  9. Vesicles in Poiseuille flow.

    PubMed

    Danker, Gerrit; Vlahovska, Petia M; Misbah, Chaouqi

    2009-04-10

    Blood microcirculation critically depends on the migration of red cells towards the flow centerline. We identify theoretically the ratio of the inner over the outer fluid viscosities lambda as a key parameter. At low lambda, the vesicle deforms into a tank-treading ellipsoid shape far away from the flow centerline. The migration is always towards the flow centerline, unlike drops. Above a critical lambda, the vesicle tumbles or breaths and migration is suppressed. A surprising coexistence of two types of shapes at the centerline, a bulletlike and a parachutelike shape, is predicted.

  10. Brefeldin A sensitive mechanisms contribute to endocytotic membrane retrieval and vesicle recycling in cerebellar granule cells.

    PubMed

    Rampérez, Alberto; Sánchez-Prieto, José; Torres, Magdalena

    2017-03-11

    The recycling of synaptic vesicle (SV) proteins and transmitter release both occur at multiple sites along the axon. These processes are sensitive to inhibition of the small GTP binding protein ARF1, which regulates the AP-1/AP-3 complex. As the axon matures, SV recycling becomes restricted to the presynaptic bouton, and its machinery undergoes a complex process of maturation. We used the styryl dye FM1-43 to highlight differences in the efficiency of membrane recycling at different sites in cerebellar granule cells cultured for 7 days in vitro. We used Brefeldin A (BFA) to inhibit AP-1/AP-3-mediated recycling and to test the contribution of this pathway to the heterogeneity of the responses when these cells are strongly stimulated. Combining imaging techniques and ultrastructural analyses, we found a significant decrease in the density of functional boutons and an increase in the presence of endosome-like structures within the boutons of cells incubated with BFA prior to FM1-43 loading. Such effects were not observed when BFA was added 5 minutes after the end of the loading step, when endocytosis was almost fully completed. In this situation, vesicles were found closer to the active zone (AZ) in boutons exposed to BFA. Together, these data suggest that the AP-1/AP-3 pathway contributes to SV recycling, affecting different steps in all boutons but not equally, and thus being partly responsible for the heterogeneity of the different recycling efficiencies. This article is protected by copyright. All rights reserved.

  11. Dielectrophoresis of Functional Phospholipid Vesicles

    NASA Astrophysics Data System (ADS)

    Froude, Victoria; Zhu, Yingxi Elaine

    2008-03-01

    Recently, there has been an emerging interest in using AC-dielectrophoresis (DEP) to transport and assemble phospholipid vesicles (liposomes) and nanoparticles to form functional bio-assemblies where the underlying charge polarization mechanism of colloids in AC fields strongly depends on nano-scaled surface charge. In this work, we study liposomes segregation and aggregation in the presence of nanocolloids and salts in which the biological functionality of liposomes is augmented by the physical functionality of inorganic coating and particles. Liposomes, synthesized by sonication with 1,2-Dioleoyl-sn-Glycero-3-Phosphate (DOPA), are manipulated at varied AC-field frequencies across fabricated micro-electrodes in a quadrapole configuration on glass. We observe the co-assembly of liposome and opposite-charged nanocolloids by confocal microscopy and SEM, where the smaller nanocolloids are captured in between liposome junctions to form stabilized composite vesicles at several distinct frequencies. We observe a strong dependence of the liposome DEP mobility on the number of nanoparticles present in suspension and propose a new mechanism based on charge segregation and charged nanocolloid entrainment in the double layer.

  12. Interaction between silicon dioxide and dipalmitoylphosphatidylcholine (DPPC) vesicles

    SciTech Connect

    Mohd, Hur Munawar Kabir; Ahmad, Ainee Fatimah; Radiman, Shahidan; Mohamed, Faizal; Rosli, Nur Ratasha Alia Md; Ayob, Muhammad Taqiyuddin Mawardi; Rahman, Irman Abdul

    2014-09-03

    Many of the cellular process depend on the ability of the membrane to separate areas while allowing exchange and tightly regulated transport of material within and across the membrane to occur, which is the driving principle behind cell communication. The complexity of biological membranes has motivated the development of a wide variety of simpler model systems whose size, geometry and composition can be tailored with precision. This study was conducted to investigate the interactions between silica nanoparticles and Dipalmitoylphosphatidylcholine (DPPC) vesicles. The size range of DPPC vesicles formed was from 50 to 150 nm. Concentration of silica added to the vesicles was varied from 0.25 to 1.5 mg/ml. The change in vesicle size distribution, localization and positioning of silica nanoparticles in vesicles was studied via transmission electron microscopy (TEM) and differential scanning calorimetry (DSC)

  13. Vesicle coats: structure, function, and general principles of assembly.

    PubMed

    Faini, Marco; Beck, Rainer; Wieland, Felix T; Briggs, John A G

    2013-06-01

    The transport of proteins and lipids between distinct cellular compartments is conducted by coated vesicles. These vesicles are formed by the self-assembly of coat proteins on a membrane, leading to collection of the vesicle cargo and membrane bending to form a bud. Scission at the bud neck releases the vesicle. X-ray crystallography and electron microscopy (EM) have recently generated models of isolated coat components and assembled coats. Here, we review these data to present a structural overview of the three main coats: clathrin, COPII, and COPI. The three coats have similar function, common ancestry, and structural similarities, but exhibit fundamental differences in structure and assembly. We describe the implications of structural similarities and differences for understanding the function, assembly principles, and evolution of vesicle coats.

  14. Complexity of vesicle microcirculation

    NASA Astrophysics Data System (ADS)

    Kaoui, B.; Tahiri, N.; Biben, T.; Ez-Zahraouy, H.; Benyoussef, A.; Biros, G.; Misbah, C.

    2011-10-01

    This study focuses numerically on dynamics in two dimensions of vesicles in microcirculation. The method used is based on boundary integral formulation. This study is inspired by the behavior of red blood cells (RBCs) in the microvasculature. Red RBCs carry oxygen from the lungs and deliver it through the microvasculature. The shape adopted by RBCs can affect blood flow and influence oxygen delivery. Our simulation using vesicles (a simple model for RBC) reveals unexpected complexity as compared to the case where a purely unbounded Poiseuille flow is considered [Kaoui, Biros, and Misbah, Phys. Rev. Lett.10.1103/PhysRevLett.103.188101 103, 188101 (2009)]. In sufficiently large channels (in the range of 100μm; the vesicle size and its reduced volume are taken in the range of those of a human RBC), such as arterioles, a slipperlike (asymmetric) shape prevails. A parachutelike (symmetric) shape is adopted in smaller channels (in the range of 20μm, as in venules), but this shape loses stability and again changes to a pronounced slipperlike morphology in channels having a size typical of capillaries (5-10 μm). Stiff membranes, mimicking malaria infection, for example, adopt a centered or off-centered snakelike locomotion instead (the denomination snaking is used for this regime). A general scenario of how and why vesicles adopt their morphologies and dynamics among several distinct possibilities is provided. This finding potentially points to nontrivial RBCs dynamics in the microvasculature.

  15. Direct Imaging of RAB27B-Enriched Secretory Vesicle Biogenesis in Lacrimal Acinar Cells Reveals Origins on a Nascent Vesicle Budding Site

    PubMed Central

    Chiang, Lilian; Karvar, Serhan; Hamm-Alvarez, Sarah F.

    2012-01-01

    This study uses YFP-tagged Rab27b expression in rabbit lacrimal gland acinar cells, which are polarized secretory epithelial cells, to characterize early stages of secretory vesicle trafficking. Here we demonstrate the utility of YFP-Rab27b to delineate new perspectives on the mechanisms of early vesicle biogenesis in lacrimal gland acinar cells, where information is significantly limited. Protocols were developed to deplete the mature YFP-Rab27b-enriched secretory vesicle pool in the subapical region of the cell, and confocal fluorescence microscopy was used to track vesicle replenishment. This analysis revealed a basally-localized organelle, which we termed the “nascent vesicle site,” from which nascent vesicles appeared to emerge. Subapical vesicular YFP-Rab27b was co-localized with p150Glued, a component of the dynactin cofactor of cytoplasmic dynein. Treatment with the microtubule-targeted agent, nocodazole, did not affect release of mature secretory vesicles, although during vesicle repletion it significantly altered nascent YFP-Rab27b-enriched secretory vesicle localization. Instead of moving to the subapical region, these vesicles were trapped at the nascent vesicle site which was adjacent to, if not a sub-compartment of, the trans-Golgi network. Finally, YFP-Rab27b-enriched secretory vesicles which reached the subapical cytoplasm appeared to acquire the actin-based motor protein, Myosin 5C. Our findings show that Rab27b enrichment occurs early in secretory vesicle formation, that secretory vesicles bud from a visually discernable nascent vesicle site, and that transport from the nascent vesicle site to the subapical region requires intact microtubules. PMID:22363735

  16. Cellular COPII Proteins Are Involved in Production of the Vesicles That Form the Poliovirus Replication Complex

    PubMed Central

    Rust, René C.; Landmann, Lukas; Gosert, Rainer; Tang, Bor Luen; Hong, Wanjin; Hauri, Hans-Peter; Egger, Denise; Bienz, Kurt

    2001-01-01

    Poliovirus (PV) replicates its genome in association with membranous vesicles in the cytoplasm of infected cells. To elucidate the origin and mode of formation of PV vesicles, immunofluorescence labeling with antibodies against the viral vesicle marker proteins 2B and 2BC, as well as cellular markers of the endoplasmic reticulum (ER), anterograde transport vesicles, and the Golgi complex, was performed in BT7-H cells. Optical sections obtained by confocal laser scanning microscopy were subjected to a deconvolution process to enhance resolution and signal-to-noise ratio and to allow for a three-dimensional representation of labeled membrane structures. The mode of formation of the PV vesicles was, on morphological grounds, similar to the formation of anterograde membrane traffic vesicles in uninfected cells. ER-resident membrane markers were excluded from both types of vesicles, and the COPII components Sec13 and Sec31 were both found to be colocalized on the vesicular surface, indicating the presence of a functional COPII coat. PV vesicle formation during early time points of infection did not involve the Golgi complex. The expression of PV protein 2BC or the entire P2 and P3 genomic region led to the production of vesicles carrying a COPII coat and showing the same mode of formation as vesicles produced after PV infection. These results indicate that PV vesicles are formed at the ER by the cellular COPII budding mechanism and thus are homologous to the vesicles of the anterograde membrane transport pathway. PMID:11559814

  17. Regulation of synaptic activity by snapin-mediated endolysosomal transport and sorting

    PubMed Central

    Di Giovanni, Jerome; Sheng, Zu-Hang

    2015-01-01

    Recycling synaptic vesicles (SVs) transit through early endosomal sorting stations, which raises a fundamental question: are SVs sorted toward endolysosomal pathways? Here, we used snapin mutants as tools to assess how endolysosomal sorting and trafficking impact presynaptic activity in wild-type and snapin−/− neurons. Snapin acts as a dynein adaptor that mediates the retrograde transport of late endosomes (LEs) and interacts with dysbindin, a subunit of the endosomal sorting complex BLOC-1. Expressing dynein-binding defective snapin mutants induced SV accumulation at presynaptic terminals, mimicking the snapin−/− phenotype. Conversely, over-expressing snapin reduced SV pool size by enhancing SV trafficking to the endolysosomal pathway. Using a SV-targeted Ca2+ sensor, we demonstrate that snapin–dysbindin interaction regulates SV positional priming through BLOC-1/AP-3-dependent sorting. Our study reveals a bipartite regulation of presynaptic activity by endolysosomal trafficking and sorting: LE transport regulates SV pool size, and BLOC-1/AP-3-dependent sorting fine-tunes the Ca2+ sensitivity of SV release. Therefore, our study provides new mechanistic insights into the maintenance and regulation of SV pool size and synchronized SV fusion through snapin-mediated LE trafficking and endosomal sorting. PMID:26108535

  18. Regulation of synaptic activity by snapin-mediated endolysosomal transport and sorting.

    PubMed

    Di Giovanni, Jerome; Sheng, Zu-Hang

    2015-08-04

    Recycling synaptic vesicles (SVs) transit through early endosomal sorting stations, which raises a fundamental question: are SVs sorted toward endolysosomal pathways? Here, we used snapin mutants as tools to assess how endolysosomal sorting and trafficking impact presynaptic activity in wild-type and snapin(-/-) neurons. Snapin acts as a dynein adaptor that mediates the retrograde transport of late endosomes (LEs) and interacts with dysbindin, a subunit of the endosomal sorting complex BLOC-1. Expressing dynein-binding defective snapin mutants induced SV accumulation at presynaptic terminals, mimicking the snapin(-/-) phenotype. Conversely, over-expressing snapin reduced SV pool size by enhancing SV trafficking to the endolysosomal pathway. Using a SV-targeted Ca(2+) sensor, we demonstrate that snapin-dysbindin interaction regulates SV positional priming through BLOC-1/AP-3-dependent sorting. Our study reveals a bipartite regulation of presynaptic activity by endolysosomal trafficking and sorting: LE transport regulates SV pool size, and BLOC-1/AP-3-dependent sorting fine-tunes the Ca(2+) sensitivity of SV release. Therefore, our study provides new mechanistic insights into the maintenance and regulation of SV pool size and synchronized SV fusion through snapin-mediated LE trafficking and endosomal sorting.

  19. Identification of Atg2 and ArfGAP1 as Candidate Genetic Modifiers of the Eye Pigmentation Phenotype of Adaptor Protein-3 (AP-3) Mutants in Drosophila melanogaster.

    PubMed

    Rodriguez-Fernandez, Imilce A; Dell'Angelica, Esteban C

    2015-01-01

    The Adaptor Protein (AP)-3 complex is an evolutionary conserved, molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes and related organelles. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs) such as mammalian melanosomes and insect eye pigment granules. In this work, we have performed a forward screening for genetic modifiers of AP-3 function in the fruit fly, Drosophila melanogaster. Specifically, we have tested collections of large multi-gene deletions--which together covered most of the autosomal chromosomes-to identify chromosomal regions that, when deleted in single copy, enhanced or ameliorated the eye pigmentation phenotype of two independent AP-3 subunit mutants. Fine-mapping led us to define two non-overlapping, relatively small critical regions within fly chromosome 3. The first critical region included the Atg2 gene, which encodes a conserved protein involved in autophagy. Loss of one functional copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, namely Blos1 and lightoid, and even increased the eye pigment content of wild-type flies. The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family. Loss of a single functional copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants but did not to modify the eye pigmentation of wild-type flies or mutants in Blos1 or lightoid. Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively. These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with molecules implicated in

  20. Transfection of mammalian cells using block copolypeptide vesicles.

    PubMed

    Sun, Victor Z; Choe, Uh-Joo; Rodriguez, April R; Dai, Howard; Deming, Timothy J; Kamei, Daniel T

    2013-05-01

    An arginine-leucine block copolypeptide (R60 L20 ) is synthesized, which is capable of forming vesicles with controllable sizes, able to transport hydrophilic cargo across the cell membrane, and exhibit relatively low cytotoxicity. The R60 L20 vesicles also possess the ability to deliver DNA into mammalian cells for transfection. Although the transfection efficiency is lower than that of the commercially available transfection agent Lipofectamine 2000, the R60 L20 vesicles are able to achieve transfection with significantly lower cytotoxicity and immunogenicity. This behavior is potentially due to its stronger interaction with DNA which subsequently provides better protection against anionic heparin.

  1. Equilibrium of nematic vesicles

    NASA Astrophysics Data System (ADS)

    Napoli, Gaetano; Vergori, Luigi

    2010-11-01

    A variational scheme is proposed which allows the derivation of a concise and elegant formulation of the equilibrium equations for closed fluid membranes, endowed with a nematic microstructure. The nematic order is described by an in-plane nematic director and a degree of orientation, as customary in the theory of uniaxial nematics. The only constitutive ingredient in this scheme is a free-energy density which depends on the vesicle geometry and order parameters. The stress and the couple stress tensors related to this free-energy density are provided. As an application of the proposed scheme, a certain number of special theories are deduced: soap bubbles, lipid vesicles, chiral and achiral nematic membranes, and nematics on curved substrates.

  2. Limited intermixing of synaptic vesicle components upon vesicle recycling.

    PubMed

    Opazo, Felipe; Punge, Annedore; Bückers, Johanna; Hoopmann, Peer; Kastrup, Lars; Hell, Stefan W; Rizzoli, Silvio O

    2010-06-01

    Synaptic vesicles recycle repeatedly in order to maintain synaptic transmission. We have previously proposed that upon exocytosis the vesicle components persist as clusters, which would be endocytosed as whole units. It has also been proposed that the vesicle components diffuse into the plasma membrane and are then randomly gathered into new vesicles. We found here that while strong stimulation (releasing the entire recycling pool) causes the diffusion of the vesicle marker synaptotagmin out of synaptic boutons, moderate stimulation (releasing approximately 19% of all vesicles) is followed by no measurable diffusion. In agreement with this observation, synaptotagmin molecules labeled with different fluorescently tagged antibodies did not appear to mix upon vesicle recycling, when investigated by subdiffraction resolution stimulated emission depletion (STED) microscopy. Finally, as protein diffusion from vesicles has been mainly observed using molecules tagged with pH-sensitive green fluorescent protein (pHluorin), we have also investigated the membrane patterning of several native and pHluorin-tagged proteins. While the native proteins had a clustered distribution, the GFP-tagged ones were diffused in the plasma membrane. We conclude that synaptic vesicle components intermix little, at least under moderate stimulation, possibly because of the formation of clusters in the plasma membrane. We suggest that several pHluorin-tagged vesicle proteins are less well integrated in clusters.

  3. Studying calcium triggered vesicle fusion in a single vesicle-vesicle content/lipid mixing system

    PubMed Central

    Kyoung, Minjoung; Zhang, Yunxiang; Diao, Jiajie; Chu, Steven; Brunger, Axel T.

    2013-01-01

    This Protocol describes a single vesicle-vesicle microscopy system to study Ca2+-triggered vesicle fusion. Donor vesicles contain reconstituted synaptobrevin and synaptotagmin-1. Acceptor vesicles contain reconstituted syntaxin and SNAP-25, and are tethered to a PEG-coated glass surface. Donor vesicles are mixed with the tethered acceptor vesicles and incubated for several minutes at zero Ca2+-concentration, resulting in a collection of single interacting vesicle pairs. The donor vesicles also contain two spectrally distinct fluorophores that allow simultaneous monitoring of temporal changes of the content and membrane. Upon Ca2+-injection into the sample chamber, our system therefore differentiates between hemifusion and complete fusion of interacting vesicle pairs and determines the temporal sequence of these events on a sub-hundred millisecond timescale. Other factors, such as complexin, can be easily added. Our system is unique by monitoring both content and lipid mixing, and by starting from a metastable state of interacting vesicle pairs prior to Ca2+-injection. PMID:23222454

  4. 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.

  5. Effects of deuterium oxide on cell growth and vesicle speed in RBL-2H3 cells

    PubMed Central

    Triplett, Ashley R.

    2014-01-01

    For the first time we show the effects of deuterium oxide on cell growth and vesicle transport in rat basophilic leukemia (RBL-2H3) cells. RBL-2H3 cells cultured with 15 moles/L deuterium showed decreased cell growth which was attributed to cells not doubling their DNA content. Experimental observations also showed an increase in vesicle speed for cells cultured in deuterium oxide. This increase in vesicle speed was not observed in deuterium oxide cultures treated with a microtubule-destabilizing drug, suggesting that deuterium oxide affects microtubule-dependent vesicle transport. PMID:25237603

  6. Determination of floral organ identity by Arabidopsis MADS domain homeotic proteins AP1, AP3, PI, and AG is independent of their DNA-binding specificity.

    PubMed Central

    Riechmann, J L; Meyerowitz, E M

    1997-01-01

    The MADS domain homeotic proteins APETALA1 (AP1), APETALA3 (AP3), PISTILLATA (PI), and AGAMOUS (AG) combinatorially specify the identity of Arabidopsis floral organs. AP1/AP1, AG/AG, and AP3/PI dimers bind to similar CArG box sequences; thus, differences in DNA-binding specificity among these proteins do not seem to be the origin of their distinct organ identity properties. To assess the overall contribution that specific DNA binding could make to their biological specificity, we have generated chimeric genes in which the amino-terminal half of the MADS domain of AP1, AP3, PI, and AG was substituted by the corresponding sequences of human SRF and MEF2A proteins. In vitro DNA-binding assays reveal that the chimeric proteins acquired the respective, and distinct, DNA-binding specificity of SRF or MEF2A. However, ectopic expression of the chimeric genes reproduces the dominant gain-of-function phenotypes exhibited by plants ectopically expressing the corresponding Arabidopsis wild-type genes. In addition, both the SRF and MEF2 chimeric genes can complement the pertinent ap1-1, ap3-3, pi-1, or ag-3 mutations to a degree similar to that of AP1, AP3, PI, and AG when expressed under the control of the same promoter. These results indicate that determination of floral organ identity by the MADS domain homeotic proteins AP1, AP3, PI, and AG is independent of their DNA-binding specificity. In addition, the DNA-binding experiments show that either one of the two MADS domains of a dimer can be sufficient to confer a particular DNA-binding specificity to the complex and that sequences outside the amino-terminal basic region of the MADS domain can, in some cases, contribute to the DNA-binding specificity of the proteins. Images PMID:9243505

  7. Alpha 1-antitrypsin activates lung cancer cell survival by acting on cap-dependent protein translation, vesicle-mediated transport, and metastasis.

    PubMed

    Chang, Seung-Hee; Cho, Kyung-Cho; Yu, Kyeong-Nam; Hong, Seong-Ho; Park, Sungjin; Lee, Ah Young; Kim, Sanghwa; Lee, Somin; Kang, Jeong Won; Chae, Chanhee; Park, Jongsun; Kim, Kwang Pyo; Cho, Myung-Haing

    2016-07-19

    Lung cancer remains the leading cause of cancer-related deaths worldwide. Although elevated expression levels of alpha 1-antitrypsin (AAT) have been reported in lung cancer patients, the precise role of AAT in lung cancer progression and prevention has not yet been fully elucidated. We have explored the mechanisms by which AAT stimulates in lung cancer progression. Here, we used proteomic analyses to compare protein levels following AAT overexpression in normal lung L132 cells containing fundamentally low level of AAT. Overexpression of AAT increased levels of proteins involved in transcription and translation, such as signal transducer and activator of transcription 5B (STAT5B) and eukaryotic translation elongation factor 1-alpha 2 (EEF1A2). Furthermore, dual luciferase activity for cap-dependent protein translation increased a 53% at 24 h and 45% at 48 h in AAT-overexpressing cells compared with control. Overexpression of AAT also increased levels of the vesicular transport protein, GOPC, which inhibited the expression of the autophagy protein, BECN1, thereby possibly increasing cell survival. In addition, overexpression of AAT promoted angiogenesis and cell adhesion through increasing expression of the metastatic protein, thrombospondin 1 (THBS1). In contrast, down-regulation of AAT by short hairpin RNA (shRNA) suppressed cell proliferation, metastasis, and adhesion in human lung adenocarcinoma A549 cells and in the lung tissue of K-rasLA1 lung cancer model mice. These findings strongly suggest that AAT regulation shows promise as an alternative avenue for lung cancer treatment and prevention.

  8. Shapes of Mixed Phospholipid Vesicles

    PubMed Central

    Aranda-Espinoza, Helim; Maldonado, Amir

    2006-01-01

    We studied the shape of phospholipid vesicles prepared by hydration of a mixture of phosphatidylcholine (SOPC) and phosphatidylserine (SOPS) in different proportions. The aim of the work is to obtain some insight into the influence of the chemical composition of a biomembrane on its shape. The optical microscopy results show that the shape of the vesicles depend on the SOPC:SOPS composition. For low SOPS contents, coiled cylindrical vesicles are observed. The results suggest that specific compositions of the SOPC:SOPS vesicles produce some spontaneous curvature on the membrane and then a coiling instability. PMID:19669461

  9. Imaging Synaptic Vesicle Exocytosis-Endocytosis with pH-Sensitive Fluorescent Proteins.

    PubMed

    Afuwape, Olusoji A T; Kavalali, Ege T

    2016-01-01

    The introduction of pHluorin, a pH-sensitive GFP, by Miesenbock and colleagues provided a versatile tool to studies of vesicle trafficking, in particular synaptic vesicle exocytosis and endocytosis. By tagging pHluorin to the luminal region of the synaptic vesicular protein synaptobrevin (also called VAMP, vesicle-associated membrane protein) or other synaptic vesicle-specific proteins such as the vesicular glutamate transporter-1, we are able to directly track synaptic vesicle endocytosis in response to stimuli in a molecularly specific manner. Here, we describe the process of imaging synaptic vesicle endocytosis in response to extracellular stimulation in dissociated neuronal cultures of hippocampal neurons obtained from rats-also applicable to mice-using pHluorin-tagged vesicular glutamate transporter-1 as a reporter.

  10. Mapping organelle motion reveals a vesicular conveyor belt spatially replenishing secretory vesicles in stimulated chromaffin cells.

    PubMed

    Maucort, Guillaume; Kasula, Ravikiran; Papadopulos, Andreas; Nieminen, Timo A; Rubinsztein-Dunlop, Halina; Meunier, Frederic A

    2014-01-01

    How neurosecretory cells spatially adjust their secretory vesicle pools to replenish those that have fused and released their hormonal content is currently unknown. Here we designed a novel set of image analyses to map the probability of tracked organelles undergoing a specific type of movement (free, caged or directed). We then applied our analysis to time-lapse z-stack confocal imaging of secretory vesicles from bovine Chromaffin cells to map the global changes in vesicle motion and directionality occurring upon secretagogue stimulation. We report a defined region abutting the cortical actin network that actively transports secretory vesicles and is dissipated by actin and microtubule depolymerizing drugs. The directionality of this "conveyor belt" towards the cell surface is activated by stimulation. Actin and microtubule networks therefore cooperatively probe the microenvironment to transport secretory vesicles to the periphery, providing a mechanism whereby cells globally adjust their vesicle pools in response to secretagogue stimulation.

  11. 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

  12. The native structure of cytoplasmic dynein at work translocating vesicles in Paramecium.

    PubMed

    Ishida, Masaki; Aihara, Marilynn S; Allen, Richard D; Fok, Agnes K

    2011-01-01

    In Paramecium multimicronucleatum, the discoidal vesicles, the acidosomes and the 100-nm carrier vesicles are involved in phagosome formation, phagosome acidification and endosomal processing, respectively. Numerous cross bridges link these vesicles to the kinetic side of the microtubules of a cytopharyngeal microtubular ribbon. Vesicles are translocated along these ribbons in a minus-end direction towards the cytopharynx. A monoclonal antibody specific for the light vanadate-photocleaved fragment of the heavy chain of cytoplasmic dynein was used to show that this dynein is located between the discoidal vesicles and the ribbons as well as on the cytosolic surface of the acidosomes and the 100-nm carrier vesicles. This antibody inhibited the docking of the vesicles to the microtubular ribbons so that the transport of discoidal vesicles and acidosomes were reduced by 60% and 70%, respectively. It had little effect on the dynein's velocity of translocation. These results show that cytoplasmic dynein is the motor for vesicle translocation and its location, between the vesicles and the ribbons, indicates that the cross bridges seen at this location in thin sections and in quick-frozen, deep-etched replicas are apparently the working dyneins. Such a working dynein cross bridge, as preserved by ultra-rapid freezing, is 54 nm long and has two legs arising from a globular head that appears to be firmly bound to its cargo vesicle and each leg consists of ≥3 beaded subunits with the last subunit making contact with the microtubular ribbon.

  13. 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.

  14. The COPII cage: unifying principles of vesicle coat assembly.

    PubMed

    Gürkan, Cemal; Stagg, Scott M; Lapointe, Paul; Balch, William E

    2006-10-01

    Communication between compartments of the exocytic and endocytic pathways in eukaryotic cells involves transport carriers - vesicles and tubules - that mediate the vectorial movement of cargo. Recent studies of transport-carrier formation in the early secretory pathway have provided new insights into the mechanisms of cargo selection by coat protein complex-II (COPII) adaptor proteins, the construction of cage-protein scaffolds and fission. These studies are beginning to produce a unifying molecular and structural model of coat function in the formation and fission of vesicles and tubules in endomembrane traffic.

  15. Vesicles with a double bilayer.

    PubMed

    Zawada, Zygmunt H

    2004-01-01

    A modified reverse phase evaporation method was used to prepare intermediate unilamellar vesicles coated with an additional membrane, or large vesicles in which several vesicles were coated with a common membrane. In both kinds of vesicle, the outer and inner membranes are usually of different phospholipid composition. The preparation involves the formation of a double emulsion: vesicles in a buffer are emerged in a low-boiling point organic solution of phospholipids. Then the organic solvent is evaporated during the heating and mixing process. As result large unilamellar vesicles (LUVs), about 100 nm in diameter, were coated with an additional membrane from egg lecithin or dipalmitoylphosphatidylcholine and cholesterol. The highest yield of the coating was about 50%. When DPPC was used for coating above the phase transition temperature Tm, the data suggested the formation of vesicles that were slightly larger than the starting LUVs. It might be concluded that many of these had a double bilayer. If the coating was done below Tm, the micrographs suggested the formation of structures resembling multi-vesicular vesicles. They looked like LUV clusters coated with a common membrane.

  16. Electrical Properties of Phospholipid Vesicles

    PubMed Central

    Schwan, H. P.; Takashima, S.; Miyamoto, V. K.; Stoeckenius, W.

    1970-01-01

    The capacitance of the membrane of phospholipid vesicles and the electrical properties of the vesicle interior have been determined. To this end the electrical properties of phospholipid vesicles have been investigated over a frequency range extending from 1 kHz to 100 MHz. The dielectric behavior is characterized by two dispersions, one placed between 1 kHz and 1 MHz and the other between 1 and 100 MHz. The relaxational behavior at low frequencies is explained by counterion movement tangential to the vesicle surface and a reasonable value for the fixed charge of the vesicles is calculated from the dispersion magnitude. The relaxation at high frequencies is of the Maxwell-Wagner type and appears caused by the phospholipid bilayer bounding the interior phase of the vesicles. It is consistent with the existence of a closed bilayer with a capacitance of about 2 μF/cm2 and an internal phase similar to the vesicle suspending medium. There is no indication of other than normally structured water inside the small vesicles. PMID:5471701

  17. Identification of Atg2 and ArfGAP1 as Candidate Genetic Modifiers of the Eye Pigmentation Phenotype of Adaptor Protein-3 (AP-3) Mutants in Drosophila melanogaster

    PubMed Central

    Rodriguez-Fernandez, Imilce A.; Dell’Angelica, Esteban C.

    2015-01-01

    The Adaptor Protein (AP)-3 complex is an evolutionary conserved, molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes and related organelles. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs) such as mammalian melanosomes and insect eye pigment granules. In this work, we have performed a forward screening for genetic modifiers of AP-3 function in the fruit fly, Drosophila melanogaster. Specifically, we have tested collections of large multi-gene deletions–which together covered most of the autosomal chromosomes–to identify chromosomal regions that, when deleted in single copy, enhanced or ameliorated the eye pigmentation phenotype of two independent AP-3 subunit mutants. Fine-mapping led us to define two non-overlapping, relatively small critical regions within fly chromosome 3. The first critical region included the Atg2 gene, which encodes a conserved protein involved in autophagy. Loss of one functional copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, namely Blos1 and lightoid, and even increased the eye pigment content of wild-type flies. The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family. Loss of a single functional copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants but did not to modify the eye pigmentation of wild-type flies or mutants in Blos1 or lightoid. Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively. These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with molecules implicated

  18. Proton pump of clathrin-coated vesicles

    SciTech Connect

    Xie, X.

    1985-01-01

    Clathrin-coated vesicles were prepared from bovine brain catalyze ATP-driven proton translocation and a /sup 32/Pi-ATP exchange reaction. N-ethylmaleimide (NEM) at 1 mM and dicyclohexylcarbodiimide (DCCD) at 0.5 mM inhibit the pump completely, whereas neither vanadate, efrapeptin, NaN/sub 3/, nor mitochondrial ATPase inhibitor has an effect. The coated vesicle proton pump is characterized by ATP specificity. dATP, but no other nucleotide, can replace ATP as a substrate. The pump is electrogenic and the electrogenicity is neutralized by chloride or bromide serving as co-ions. ATP-driven proton translocation can be observed in the absence of chloride, provided that the membrane potential is collapsed by K/sup +/ moving out in the presence of valinomycin. Chloride transport can be observed independent of proton movements in the absence of ATP, provided that an inside positive membrane potential is generated by K/sup +/ influx in the presence of valinomycin. The proton-translocating ATPase of coated vesicles was solubilized with a nonionic detergent polyoxyethylene 9 lauryl ether, and purified about 700 fold to near homogeneity. During purification the enzymatic activity was lost. A purified brain phospholipid fraction restored the activity and was subsequently identified as phosphatidylserine.

  19. Transport of Ca2+ across Phosphatidylcholine Vesicles.

    DTIC Science & Technology

    1983-01-01

    ws close to one with an error limit of about 10%. Therefore, in contradiction ith previous findings ( Kafka and Dolz, 1976) our results Indicate a...lariat Pether series. L •9 tETERENCES , yono, A. , Hendriks. T., Daement, F.J.H. and Bonting, S.L. 11975) Biochim. Biophys. Act& M 34-46. Kafka , M.S

  20. Intracellular fates of cell-penetrating block copolypeptide vesicles.

    PubMed

    Sun, Victor Z; Li, Zhibo; Deming, Timothy J; Kamei, Daniel T

    2011-01-10

    The block copolypeptide poly(l-homoarginine)(60)-b-poly(l-leucine)(20) (R(60)L(20)) was previously found to self-assemble into versatile vesicles with controllable size and encapsulate hydrophilic cargo. These R(60)L(20) vesicles also demonstrated the ability to cross the cell membrane and transport encapsulated cargo into different cell lines. To assess the potential for using the R(60)L(20) vesicles as drug delivery vehicles further, we have investigated their endocytosis and intracellular trafficking behavior. Using drugs that inhibit different endocytosis pathways, we identified macropinocytosis to be a major process by which the R(60)L(20) vesicles enter HeLa cells. Subsequent immunostaining experiments demonstrated that the vesicles entered the early endosomes but not the lysosomes, suggesting that they recycle back to the cell surface. Overall, our studies indicate that the R(60)L(20) vesicles are able to enter cells intact with their cargos, and although some manage to escape from early endosomes, most are trapped within these intracellular compartments.

  1. Isolation and Characterization of Calcifying Matrix Vesicles from Epiphyseal Cartilage*

    PubMed Central

    Ali, S. Y.; Sajdera, S. W.; Anderson, H. C.

    1970-01-01

    Matrix vesicles, associated with initial calcification in cartilage, have been isolated from bovine fetal epiphyseal cartilage. Cartilage was digested with collagenase, then partitioned into seven fractions by differential centrifugation. The cellular fractions contained over 80% of the DNA in the digest. The extracellular fraction that contained matrix vesicles, in which apatite crystals were often seen on electron microscopy, also displayed the highest specific activity for alkaline phosphatase, pyrophosphatase, ATPase, and 5′-AMPase (EC 3.1.3.1., 3.6.1.1, 3.6.1.3, and 3.1.3.5, respectively). Most of the acid phosphatase (EC 3.1.3.2) activity, on the other hand, was found in the cellular fractions, indicating that matrix vesicles are quite distinct from lysosomes. This appears to be the first instance of isolation of membrane-bounded extracellular particles from any normal tissue. The matrix vesicles possess enzymes that can increase the local concentration of orthophosphate and thus could lead to the formation of hydroxyapatite. The membrane-bounded matrix vesicles may also provide a mechanism for ATP-dependent transport of calcium or phosphate into the lumen of the vesicles with resultant mineralization. Images PMID:5274475

  2. Na sup + uptake into colonic enterocyte membrane vesicles

    SciTech Connect

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

    1988-04-01

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

  3. Effective Mechanism for Synthesis of Neurotransmitter Glutamate and its Loading into Synaptic Vesicles.

    PubMed

    Takeda, Kouji; Ueda, Tetsufumi

    2017-01-01

    Glutamate accumulation into synaptic vesicles is a pivotal step in glutamate transmission. This process is achieved by a vesicular glutamate transporter (VGLUT) coupled to v-type proton ATPase. Normal synaptic transmission, in particular during intensive neuronal firing, would demand rapid transmitter re-filling of emptied synaptic vesicles. We have previously shown that isolated synaptic vesicles are capable of synthesizing glutamate from α-ketoglutarate (not from glutamine) by vesicle-bound aspartate aminotransferase for immediate uptake, in addition to ATP required for uptake by vesicle-bound glycolytic enzymes. This suggests that local synthesis of these substances, essential for glutamate transmission, could occur at the synaptic vesicle. Here we provide evidence that synaptosomes (pinched-off nerve terminals) also accumulate α-ketoglutarate-derived glutamate into synaptic vesicles within, at the expense of ATP generated through glycolysis. Glutamine-derived glutamate is also accumulated into synaptic vesicles in synaptosomes. The underlying mechanism is discussed. It is suggested that local synthesis of both glutamate and ATP at the presynaptic synaptic vesicle would represent an efficient mechanism for swift glutamate loading into synaptic vesicles, supporting maintenance of normal synaptic transmission.

  4. Synaptic Vesicle Exocytosis

    PubMed Central

    Südhof, Thomas C.; Rizo, Josep

    2011-01-01

    Presynaptic nerve terminals release neurotransmitters by synaptic vesicle exocytosis. Membrane fusion mediating synaptic exocytosis and other intracellular membrane traffic is affected by a universal machinery that includes SNARE (for “soluble NSF-attachment protein receptor”) and SM (for “Sec1/Munc18-like”) proteins. During fusion, vesicular and target SNARE proteins assemble into an α-helical trans-SNARE complex that forces the two membranes tightly together, and SM proteins likely wrap around assembling trans-SNARE complexes to catalyze membrane fusion. After fusion, SNARE complexes are dissociated by the ATPase NSF (for “N-ethylmaleimide sensitive factor”). Fusion-competent conformations of SNARE proteins are maintained by chaperone complexes composed of CSPα, Hsc70, and SGT, and by nonenzymatically acting synuclein chaperones; dysfunction of these chaperones results in neurodegeneration. The synaptic membrane-fusion machinery is controlled by synaptotagmin, and additionally regulated by a presynaptic protein matrix (the “active zone”) that includes Munc13 and RIM proteins as central components. PMID:22026965

  5. A role for Yip1p in COPII vesicle biogenesis

    PubMed Central

    Heidtman, Matthew; Chen, Catherine Z.; Collins, Ruth N.; Barlowe, Charles

    2003-01-01

    Yeast Ypt1p-interacting protein (Yip1p) belongs to a conserved family of transmembrane proteins that interact with Rab GTPases. We encountered Yip1p as a constituent of ER-derived transport vesicles, leading us to hypothesize a direct role for this protein in transport through the early secretory pathway. Using a cell-free assay that recapitulates protein transport from the ER to the Golgi complex, we find that affinity-purified antibodies directed against the hydrophilic amino terminus of Yip1p potently inhibit transport. Surprisingly, inhibition is specific to the COPII-dependent budding stage. In support of this in vitro observation, strains bearing the temperature-sensitive yip1-4 allele accumulate ER membranes at a nonpermissive temperature, with no apparent accumulation of vesicle intermediates. Genetic interaction analyses of the yip1-4 mutation corroborate a function in ER budding. Finally, ordering experiments show that preincubation of ER membranes with COPII proteins decreases sensitivity to anti-Yip1p antibodies, indicating an early requirement for Yip1p in vesicle formation. We propose that Yip1p has a previously unappreciated role in COPII vesicle biogenesis. PMID:14557247

  6. A role for Yip1p in COPII vesicle biogenesis.

    PubMed

    Heidtman, Matthew; Chen, Catherine Z; Collins, Ruth N; Barlowe, Charles

    2003-10-13

    Yeast Ypt1p-interacting protein (Yip1p) belongs to a conserved family of transmembrane proteins that interact with Rab GTPases. We encountered Yip1p as a constituent of ER-derived transport vesicles, leading us to hypothesize a direct role for this protein in transport through the early secretory pathway. Using a cell-free assay that recapitulates protein transport from the ER to the Golgi complex, we find that affinity-purified antibodies directed against the hydrophilic amino terminus of Yip1p potently inhibit transport. Surprisingly, inhibition is specific to the COPII-dependent budding stage. In support of this in vitro observation, strains bearing the temperature-sensitive yip1-4 allele accumulate ER membranes at a nonpermissive temperature, with no apparent accumulation of vesicle intermediates. Genetic interaction analyses of the yip1-4 mutation corroborate a function in ER budding. Finally, ordering experiments show that preincubation of ER membranes with COPII proteins decreases sensitivity to anti-Yip1p antibodies, indicating an early requirement for Yip1p in vesicle formation. We propose that Yip1p has a previously unappreciated role in COPII vesicle biogenesis.

  7. Open Syntaxin Docks Synaptic Vesicles

    PubMed Central

    Olsen, Shawn; Jorgensen, Erik M

    2007-01-01

    Synaptic vesicles dock to the plasma membrane at synapses to facilitate rapid exocytosis. Docking was originally proposed to require the soluble N-ethylmaleimide–sensitive fusion attachment protein receptor (SNARE) proteins; however, perturbation studies suggested that docking was independent of the SNARE proteins. We now find that the SNARE protein syntaxin is required for docking of all vesicles at synapses in the nematode Caenorhabditis elegans. The active zone protein UNC-13, which interacts with syntaxin, is also required for docking in the active zone. The docking defects in unc-13 mutants can be fully rescued by overexpressing a constitutively open form of syntaxin, but not by wild-type syntaxin. These experiments support a model for docking in which UNC-13 converts syntaxin from the closed to the open state, and open syntaxin acts directly in docking vesicles to the plasma membrane. These data provide a molecular basis for synaptic vesicle docking. PMID:17645391

  8. Identification of a QTL in Mus musculus for Alcohol Preference, Withdrawal, and Ap3m2 Expression Using Integrative Functional Genomics and Precision Genetics

    PubMed Central

    Bubier, Jason A.; Jay, Jeremy J.; Baker, Christopher L.; Bergeson, Susan E.; Ohno, Hiroshi; Metten, Pamela; Crabbe, John C.; Chesler, Elissa J.

    2014-01-01

    Extensive genetic and genomic studies of the relationship between alcohol drinking preference and withdrawal severity have been performed using animal models. Data from multiple such publications and public data resources have been incorporated in the GeneWeaver database with >60,000 gene sets including 285 alcohol withdrawal and preference-related gene sets. Among these are evidence for positional candidates regulating these behaviors in overlapping quantitative trait loci (QTL) mapped in distinct mouse populations. Combinatorial integration of functional genomics experimental results revealed a single QTL positional candidate gene in one of the loci common to both preference and withdrawal. Functional validation studies in Ap3m2 knockout mice confirmed these relationships. Genetic validation involves confirming the existence of segregating polymorphisms that could account for the phenotypic effect. By exploiting recent advances in mouse genotyping, sequence, epigenetics, and phylogeny resources, we confirmed that Ap3m2 resides in an appropriately segregating genomic region. We have demonstrated genetic and alcohol-induced regulation of Ap3m2 expression. Although sequence analysis revealed no polymorphisms in the Ap3m2-coding region that could account for all phenotypic differences, there are several upstream SNPs that could. We have identified one of these to be an H3K4me3 site that exhibits strain differences in methylation. Thus, by making cross-species functional genomics readily computable we identified a common QTL candidate for two related bio-behavioral processes via functional evidence and demonstrate sufficiency of the genetic locus as a source of variation underlying two traits. PMID:24923803

  9. Identification of a QTL in Mus musculus for alcohol preference, withdrawal, and Ap3m2 expression using integrative functional genomics and precision genetics.

    PubMed

    Bubier, Jason A; Jay, Jeremy J; Baker, Christopher L; Bergeson, Susan E; Ohno, Hiroshi; Metten, Pamela; Crabbe, John C; Chesler, Elissa J

    2014-08-01

    Extensive genetic and genomic studies of the relationship between alcohol drinking preference and withdrawal severity have been performed using animal models. Data from multiple such publications and public data resources have been incorporated in the GeneWeaver database with >60,000 gene sets including 285 alcohol withdrawal and preference-related gene sets. Among these are evidence for positional candidates regulating these behaviors in overlapping quantitative trait loci (QTL) mapped in distinct mouse populations. Combinatorial integration of functional genomics experimental results revealed a single QTL positional candidate gene in one of the loci common to both preference and withdrawal. Functional validation studies in Ap3m2 knockout mice confirmed these relationships. Genetic validation involves confirming the existence of segregating polymorphisms that could account for the phenotypic effect. By exploiting recent advances in mouse genotyping, sequence, epigenetics, and phylogeny resources, we confirmed that Ap3m2 resides in an appropriately segregating genomic region. We have demonstrated genetic and alcohol-induced regulation of Ap3m2 expression. Although sequence analysis revealed no polymorphisms in the Ap3m2-coding region that could account for all phenotypic differences, there are several upstream SNPs that could. We have identified one of these to be an H3K4me3 site that exhibits strain differences in methylation. Thus, by making cross-species functional genomics readily computable we identified a common QTL candidate for two related bio-behavioral processes via functional evidence and demonstrate sufficiency of the genetic locus as a source of variation underlying two traits.

  10. Variation in PTCHD2, CRISP3, NAP1L4, FSCB, and AP3B2 associated with spherical equivalent

    PubMed Central

    Chen, Fei; Duggal, Priya; Klein, Barbara E.K.; Lee, Kristine E.; Truitt, Barbara; Klein, Ronald; Iyengar, Sudha K.

    2016-01-01

    Purpose Ocular refraction is measured in spherical equivalent as the power of the external lens required to focus images on the retina. Myopia (nearsightedness) and hyperopia (farsightedness) are the most common refractive errors, and the leading causes of visual impairment and blindness in the world. The goal of this study is to identify rare and low-frequency variants that influence spherical equivalent. Methods We conducted variant-level and gene-level quantitative trait association analyses for mean spherical equivalent, using data from 1,560 individuals in the Beaver Dam Eye Study. Genotyping was conducted using the Illumina exome array. We analyzed 34,976 single nucleotide variants and 11,571 autosomal genes across the genome, using single-variant tests as well as gene-based tests. Results Spherical equivalent was significantly associated with five genes in gene-based analysis: PTCHD2 at 1p36.22 (p = 3.6 × 10−7), CRISP3 at 6p12.3 (p = 4.3 × 10−6), NAP1L4 at 11p15.5 (p = 3.6 × 10−6), FSCB at 14q21.2 (p = 1.5 × 10−7), and AP3B2 at 15q25.2 (p = 1.6 × 10−7). The variant-based tests identified evidence suggestive of association with two novel variants in linkage disequilibrium (pairwise r2 = 0.80) in the TCTE1 gene region at 6p21.1 (rs2297336, minor allele frequency (MAF) = 14.1%, β = –0.62 p = 3.7 × 10−6; rs324146, MAF = 16.9%, β = –0.55, p = 1.4 × 10−5). In addition to these novel findings, we successfully replicated a previously reported association with rs634990 near GJD2 at 15q14 (MAF = 47%, β = –0.29, p=1.8 × 10−3). We also found evidence of association with spherical equivalent on 2q37.1 in PRSS56 at rs1550094 (MAF = 31%, β = –0.33, p = 1.7 × 10−3), a region previously associated with myopia. Conclusions We identified several novel candidate genes that may play a role in the control of spherical equivalent. However, further studies are needed to replicate these findings. In addition, our results contribute to the

  11. Two-stage comprehensive evaluation of genetic susceptibility of common variants in FBXO38, AP3B2 and WHAMM to severe chronic periodontitis.

    PubMed

    Shang, Dong; Dong, Li; Zeng, Lingfang; Yang, Rui; Xu, Jing; Wu, Yue; Xu, Ran; Tao, Hong; Zhang, Nan

    2015-12-08

    Chronic periodontitis is an oral disorder characterized with gingival inflammation and bone destruction. As the sixth-most prevalent condition affecting more than 743 million people around the world, it is classified as one of the seven destructive oral disorders. Early genetic epidemiological evidence indicated a major role for genetics in periodontal disease development. In this study, we conducted a two-stage comprehensive evaluation of the genetic susceptibility of FBXO38, AP3B2 and WHAMM with the diagnosis of severe chronic periodontitis. A total of 5,065 study subjects from the Han Chinese population consisting of 1,264 cases and 3,801 healthy controls were recruited, and 65 single nucleotide markers related to the three candidate genes were genotyped to investigate the susceptibility of patients with these polymorphisms to severe chronic periodontitis. To increase the coverage of genetic markers, we implemented imputation techniques to extend the number of tested makers to 416. Single marker and haplotype-based analyses were performed, and significant results were obtained for FBXO38 (rs10043775, P = 0.0009) and AP3B2 (rs11631963-rs11637433, CA, P = 9.98 × 10(-5); rs1864699-rs2099259-rs2278355, ATC, P = 3.84 × 10(-8)). Our findings provide direct evidence for the association of FBXO38 and AP3B2 with severe chronic periodontitis in the Han Chinese population.

  12. The protein machinery of vesicle budding and fusion.

    PubMed Central

    Rothman, J. E.

    1996-01-01

    A general protein machinery that buds and fuses transport vesicles is harnessed to generate the complex web of intracellular transport pathways critical for such diverse processes as cell growth, endocytosis, hormone release, and neurotransmission. With this appreciation, the challenge of understanding the precise molecular mechanisms of these many facets of cell biology has been reduced to a series of problems in protein structure and chemistry. PMID:8745395

  13. Functional Advantages Conferred by Extracellular Prokaryotic Membrane Vesicles

    PubMed Central

    Manning, Andrew J.; Kuehn, Meta J.

    2015-01-01

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

  14. Functional advantages conferred by extracellular prokaryotic membrane vesicles.

    PubMed

    Manning, Andrew J; Kuehn, Meta J

    2013-01-01

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

  15. Durable vesicles for reconstitution of membrane proteins in biotechnology

    PubMed Central

    Khan, Sanobar; Muench, Stephen P.; Jeuken, Lars J.C.

    2017-01-01

    The application of membrane proteins in biotechnology requires robust, durable reconstitution systems that enhance their stability and support their functionality in a range of working environments. Vesicular architectures are highly desirable to provide the compartmentalisation to utilise the functional transmembrane transport and signalling properties of membrane proteins. Proteoliposomes provide a native-like membrane environment to support membrane protein function, but can lack the required chemical and physical stability. Amphiphilic block copolymers can also self-assemble into polymersomes: tough vesicles with improved stability compared with liposomes. This review discusses the reconstitution of membrane proteins into polymersomes and the more recent development of hybrid vesicles, which blend the robust nature of block copolymers with the biofunctionality of lipids. These novel synthetic vesicles hold great promise for enabling membrane proteins within biotechnologies by supporting their enhanced in vitro performance and could also contribute to fundamental biochemical and biophysical research by improving the stability of membrane proteins that are challenging to work with. PMID:28202656

  16. Botulinum neurotoxin type-A enters a non-recycling pool of synaptic vesicles.

    PubMed

    Harper, Callista B; Papadopulos, Andreas; Martin, Sally; Matthews, Daniel R; Morgan, Garry P; Nguyen, Tam H; Wang, Tong; Nair, Deepak; Choquet, Daniel; Meunier, Frederic A

    2016-01-25

    Neuronal communication relies on synaptic vesicles undergoing regulated exocytosis and recycling for multiple rounds of fusion. Whether all synaptic vesicles have identical protein content has been challenged, suggesting that their recycling ability may differ greatly. Botulinum neurotoxin type-A (BoNT/A) is a highly potent neurotoxin that is internalized in synaptic vesicles at motor nerve terminals and induces flaccid paralysis. Recently, BoNT/A was also shown to undergo retrograde transport, suggesting it might enter a specific pool of synaptic vesicles with a retrograde trafficking fate. Using high-resolution microscopy techniques including electron microscopy and single molecule imaging, we found that the BoNT/A binding domain is internalized within a subset of vesicles that only partially co-localize with cholera toxin B-subunit and have markedly reduced VAMP2 immunoreactivity. Synaptic vesicles loaded with pHrodo-BoNT/A-Hc exhibited a significantly reduced ability to fuse with the plasma membrane in mouse hippocampal nerve terminals when compared with pHrodo-dextran-containing synaptic vesicles and pHrodo-labeled anti-GFP nanobodies bound to VAMP2-pHluorin or vGlut-pHluorin. Similar results were also obtained at the amphibian neuromuscular junction. These results reveal that BoNT/A is internalized in a subpopulation of synaptic vesicles that are not destined to recycle, highlighting the existence of significant molecular and functional heterogeneity between synaptic vesicles.

  17. Botulinum neurotoxin type-A enters a non-recycling pool of synaptic vesicles

    PubMed Central

    Harper, Callista B.; Papadopulos, Andreas; Martin, Sally; Matthews, Daniel R.; Morgan, Garry P.; Nguyen, Tam H.; Wang, Tong; Nair, Deepak; Choquet, Daniel; Meunier, Frederic A.

    2016-01-01

    Neuronal communication relies on synaptic vesicles undergoing regulated exocytosis and recycling for multiple rounds of fusion. Whether all synaptic vesicles have identical protein content has been challenged, suggesting that their recycling ability may differ greatly. Botulinum neurotoxin type-A (BoNT/A) is a highly potent neurotoxin that is internalized in synaptic vesicles at motor nerve terminals and induces flaccid paralysis. Recently, BoNT/A was also shown to undergo retrograde transport, suggesting it might enter a specific pool of synaptic vesicles with a retrograde trafficking fate. Using high-resolution microscopy techniques including electron microscopy and single molecule imaging, we found that the BoNT/A binding domain is internalized within a subset of vesicles that only partially co-localize with cholera toxin B-subunit and have markedly reduced VAMP2 immunoreactivity. Synaptic vesicles loaded with pHrodo-BoNT/A-Hc exhibited a significantly reduced ability to fuse with the plasma membrane in mouse hippocampal nerve terminals when compared with pHrodo-dextran-containing synaptic vesicles and pHrodo-labeled anti-GFP nanobodies bound to VAMP2-pHluorin or vGlut-pHluorin. Similar results were also obtained at the amphibian neuromuscular junction. These results reveal that BoNT/A is internalized in a subpopulation of synaptic vesicles that are not destined to recycle, highlighting the existence of significant molecular and functional heterogeneity between synaptic vesicles. PMID:26805017

  18. Souffle/Spastizin controls secretory vesicle maturation during zebrafish oogenesis.

    PubMed

    Kanagaraj, Palsamy; Gautier-Stein, Amandine; Riedel, Dietmar; Schomburg, Christoph; Cerdà, Joan; Vollack, Nadine; Dosch, Roland

    2014-06-01

    During oogenesis, the egg prepares for fertilization and early embryogenesis. As a consequence, vesicle transport is very active during vitellogenesis, and oocytes are an outstanding system to study regulators of membrane trafficking. Here, we combine zebrafish genetics and the oocyte model to identify the molecular lesion underlying the zebrafish souffle (suf) mutation. We demonstrate that suf encodes the homolog of the Hereditary Spastic Paraplegia (HSP) gene SPASTIZIN (SPG15). We show that in zebrafish oocytes suf mutants accumulate Rab11b-positive vesicles, but trafficking of recycling endosomes is not affected. Instead, we detect Suf/Spastizin on cortical granules, which undergo regulated secretion. We demonstrate genetically that Suf is essential for granule maturation into secretion competent dense-core vesicles describing a novel role for Suf in vesicle maturation. Interestingly, in suf mutants immature, secretory precursors accumulate, because they fail to pinch-off Clathrin-coated buds. Moreover, pharmacological inhibition of the abscission regulator Dynamin leads to an accumulation of immature secretory granules and mimics the suf phenotype. Our results identify a novel regulator of secretory vesicle formation in the zebrafish oocyte. In addition, we describe an uncharacterized cellular mechanism for Suf/Spastizin activity during secretion, which raises the possibility of novel therapeutic avenues for HSP research.

  19. Souffle/Spastizin Controls Secretory Vesicle Maturation during Zebrafish Oogenesis

    PubMed Central

    Riedel, Dietmar; Schomburg, Christoph; Cerdà, Joan; Vollack, Nadine; Dosch, Roland

    2014-01-01

    During oogenesis, the egg prepares for fertilization and early embryogenesis. As a consequence, vesicle transport is very active during vitellogenesis, and oocytes are an outstanding system to study regulators of membrane trafficking. Here, we combine zebrafish genetics and the oocyte model to identify the molecular lesion underlying the zebrafish souffle (suf) mutation. We demonstrate that suf encodes the homolog of the Hereditary Spastic Paraplegia (HSP) gene SPASTIZIN (SPG15). We show that in zebrafish oocytes suf mutants accumulate Rab11b-positive vesicles, but trafficking of recycling endosomes is not affected. Instead, we detect Suf/Spastizin on cortical granules, which undergo regulated secretion. We demonstrate genetically that Suf is essential for granule maturation into secretion competent dense-core vesicles describing a novel role for Suf in vesicle maturation. Interestingly, in suf mutants immature, secretory precursors accumulate, because they fail to pinch-off Clathrin-coated buds. Moreover, pharmacological inhibition of the abscission regulator Dynamin leads to an accumulation of immature secretory granules and mimics the suf phenotype. Our results identify a novel regulator of secretory vesicle formation in the zebrafish oocyte. In addition, we describe an uncharacterized cellular mechanism for Suf/Spastizin activity during secretion, which raises the possibility of novel therapeutic avenues for HSP research. PMID:24967841

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

    SciTech Connect

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

    1987-05-01

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

  1. Ellipsoidal Relaxation of Deformed Vesicles

    NASA Astrophysics Data System (ADS)

    Yu, Miao; Lira, Rafael B.; Riske, Karin A.; Dimova, Rumiana; Lin, Hao

    2015-09-01

    Theoretical analysis and experimental quantification on the ellipsoidal relaxation of vesicles are presented. The current work reveals the simplicity and universal aspects of this process. The Helfrich formula is shown to apply to the dynamic relaxation of moderate-to-high tension membranes, and a closed-form solution is derived which predicts the vesicle aspect ratio as a function of time. Scattered data are unified by a time scale, which leads to a similarity behavior, governed by a distinctive solution for each vesicle type. Two separate regimes in the relaxation are identified, namely, the "entropic" and the "constant-tension" regimes. The bending rigidity and the initial membrane tension can be simultaneously extracted from the data analysis, posing the current approach as an effective means for the mechanical analysis of biomembranes.

  2. Morphological docking of secretory vesicles

    PubMed Central

    2010-01-01

    Calcium-dependent secretion of neurotransmitters and hormones is essential for brain function and neuroendocrine-signaling. Prior to exocytosis, neurotransmitter-containing vesicles dock to the target membrane. In electron micrographs of neurons and neuroendocrine cells, like chromaffin cells many synaptic vesicles (SVs) and large dense-core vesicles (LDCVs) are docked. For many years the molecular identity of the morphologically docked state was unknown. Recently, we resolved the minimal docking machinery in adrenal medullary chromaffin cells using embryonic mouse model systems together with electron-microscopic analyses and also found that docking is controlled by the sub-membrane filamentous (F-)actin. Currently it is unclear if the same docking machinery operates in synapses. Here, I will review our docking assay that led to the identification of the LDCV docking machinery in chromaffin cells and also discuss whether identical docking proteins are required for SV docking in synapses. PMID:20577884

  3. Structural Basis for the Recognition of Tyrosine-based Sorting Signals by the μ3A Subunit of the AP-3 Adaptor Complex*

    PubMed Central

    Mardones, Gonzalo A.; Burgos, Patricia V.; Lin, Yimo; Kloer, Daniel P.; Magadán, Javier G.; Hurley, James H.; Bonifacino, Juan S.

    2013-01-01

    Tyrosine-based signals fitting the YXXØ motif mediate sorting of transmembrane proteins to endosomes, lysosomes, the basolateral plasma membrane of polarized epithelial cells, and the somatodendritic domain of neurons through interactions with the homologous μ1, μ2, μ3, and μ4 subunits of the corresponding AP-1, AP-2, AP-3, and AP-4 complexes. Previous x-ray crystallographic analyses identified distinct binding sites for YXXØ signals on μ2 and μ4, which were located on opposite faces of the proteins. To elucidate the mode of recognition of YXXØ signals by other members of the μ family, we solved the crystal structure at 1.85 Å resolution of the C-terminal domain of the μ3 subunit of AP-3 (isoform A) in complex with a peptide encoding a YXXØ signal (SDYQRL) from the trans-Golgi network protein TGN38. The μ3A C-terminal domain consists of an immunoglobulin-like β-sandwich organized into two subdomains, A and B. The YXXØ signal binds in an extended conformation to a site on μ3A subdomain A, at a location similar to the YXXØ-binding site on μ2 but not μ4. The binding sites on μ3A and μ2 exhibit similarities and differences that account for the ability of both proteins to bind distinct sets of YXXØ signals. Biochemical analyses confirm the identification of the μ3A site and show that this protein binds YXXØ signals with 14–19 μm affinity. The surface electrostatic potential of μ3A is less basic than that of μ2, in part explaining the association of AP-3 with intracellular membranes having less acidic phosphoinositides. PMID:23404500

  4. Structural basis for the recognition of tyrosine-based sorting signals by the μ3A subunit of the AP-3 adaptor complex.

    PubMed

    Mardones, Gonzalo A; Burgos, Patricia V; Lin, Yimo; Kloer, Daniel P; Magadán, Javier G; Hurley, James H; Bonifacino, Juan S

    2013-03-29

    Tyrosine-based signals fitting the YXXØ motif mediate sorting of transmembrane proteins to endosomes, lysosomes, the basolateral plasma membrane of polarized epithelial cells, and the somatodendritic domain of neurons through interactions with the homologous μ1, μ2, μ3, and μ4 subunits of the corresponding AP-1, AP-2, AP-3, and AP-4 complexes. Previous x-ray crystallographic analyses identified distinct binding sites for YXXØ signals on μ2 and μ4, which were located on opposite faces of the proteins. To elucidate the mode of recognition of YXXØ signals by other members of the μ family, we solved the crystal structure at 1.85 Å resolution of the C-terminal domain of the μ3 subunit of AP-3 (isoform A) in complex with a peptide encoding a YXXØ signal (SDYQRL) from the trans-Golgi network protein TGN38. The μ3A C-terminal domain consists of an immunoglobulin-like β-sandwich organized into two subdomains, A and B. The YXXØ signal binds in an extended conformation to a site on μ3A subdomain A, at a location similar to the YXXØ-binding site on μ2 but not μ4. The binding sites on μ3A and μ2 exhibit similarities and differences that account for the ability of both proteins to bind distinct sets of YXXØ signals. Biochemical analyses confirm the identification of the μ3A site and show that this protein binds YXXØ signals with 14-19 μm affinity. The surface electrostatic potential of μ3A is less basic than that of μ2, in part explaining the association of AP-3 with intracellular membranes having less acidic phosphoinositides.

  5. Plasmadesmatal frequency, apoplast-symplast ratio, and photosynthetic transfer in grapefruit juice vesicles. [Citrus paradisi Macf

    SciTech Connect

    Koch, K.E.; Lowell, C.A.; Avigne, W.T.

    1986-04-01

    Structure and function were examined in phloem-free vesicles and vesicle stalks of grapefruit (Citrus paradisi Macf.) by light and electron microscopy and /sup 14/C-photosynthate transport in intact and dissected tissues. Plasmodesmatal frequencies were approximately 0.3 to 0.5 ..mu..m/sup -1/ cell wall interface (3 to 5 ..mu..m/sup -2/), less than that of known secretory structures but similar to root parenchyma. Cell wall or apoplast comprised 18 to 24% of the total cross-sectional area of the vesicle stalk. The mass of total photosynthate transfer through individual vesicle stalks was ca. 0.5 ..mu..g C h/sup -1/ and rate of /sup 14/C-movement 0.1 to 0.4 mm h/sup -1/. Transport continued in rows of vesicles dissected in association with a vascular bundle. If isolated from fully-expanded fruit, translocation was similar for systems with frozen vs. non-frozen vesicle stalks. Similar freezing treatment decreased transport in vesicles from younger fruit. Symplastic and apoplastic pathways may therefore both operate in this system.

  6. 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.

  7. ATP-Dependent Formation of Phosphatidylserine-Rich Vesicles from the Endoplasmic Reticulum of Leek Cells

    PubMed Central

    Sturbois-Balcerzak, Bénédicte; Vincent, Patrick; Maneta-Peyret, Lilly; Duvert, Michel; Satiat-Jeunemaitre, Béatrice; Cassagne, Claude; Moreau, Patrick

    1999-01-01

    Leek (Allium porrum) plasma membrane is enriched in phosphatidylserine (PS) by the vesicular pathway, in a way similar to that already observed in animal cells (B. Sturbois-Balcerzak, D.J. Morré, O. Loreau, J.P. Noel, P. Moreau, C. Cassagne [1995] Plant Physiol Biochem 33: 625–637). In this paper we document the formation of PS-rich small vesicles from leek endoplasmic reticulum (ER) membranes upon addition of ATP and other factors. The omission of ATP or its replacement by ATPγ-S prevents vesicle formation. These vesicles correspond to small structures (70–80 nm) and their phospholipid composition, characterized by a PS enrichment, is compatible with a role in PS transport. Moreover, the PS enrichment over phosphatidylinositol in the ER-derived vesicles is the first example, to our knowledge, of phospholipid sorting from the ER to ER-derived vesicles in plant cells. PMID:10318702

  8. Morphology of nematic and smectic vesicles

    PubMed Central

    Xing, Xiangjun; Shin, Homin; Bowick, Mark J.; Yao, Zhenwei; Jia, Lin; Li, Min-Hui

    2012-01-01

    Recent experiments on vesicles formed from block copolymers with liquid-crystalline side chains reveal a rich variety of vesicle morphologies. The additional internal order (“structure”) developed by these self-assembled block copolymer vesicles can lead to significantly deformed vesicles as a result of the delicate interplay between two-dimensional ordering and vesicle shape. The inevitable topological defects in structured vesicles of spherical topology also play an essential role in controlling the final vesicle morphology. Here we develop a minimal theoretical model for the morphology of the membrane structure with internal nematic/smectic order. Using both analytic and numerical approaches, we show that the possible low free energy morphologies include nano-size cylindrical micelles (nano-fibers), faceted tetrahedral vesicles, and ellipsoidal vesicles, as well as cylindrical vesicles. The tetrahedral vesicle is a particularly fascinating example of a faceted liquid-crystalline membrane. Faceted liquid vesicles may lead to the design of supramolecular structures with tetrahedral symmetry and new classes of nano-carriers. PMID:22431595

  9. 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.

  10. Biomimetic proteolipid vesicles for targeting inflamed tissues

    PubMed Central

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

    2016-01-01

    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 the 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. PMID:27213956

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

    PubMed

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

    2015-11-01

    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.

  12. TRANSPORT

    EPA Science Inventory

    Presentation outline: transport principles, effective solubility; gasoline composition; and field examples (plume diving).
    Presentation conclusions: MTBE transport follows from - phyiscal and chemical properties and hydrology. Field examples show: MTBE plumes > benzene plu...

  13. Harnessing fluid-driven vesicles to pick up and drop off Janus particles.

    PubMed

    Salib, Isaac; Yong, Xin; Crabb, Emily J; Moellers, Nicholas M; McFarlin, Gerald T; Kuksenok, Olga; Balazs, Anna C

    2013-02-26

    Using dissipative particle dynamics (DPD) simulations, we model the interaction between nanoscopic lipid vesicles and Janus nanoparticles in the presence of an imposed flow. Both the vesicle and Janus nanoparticles are localized on a hydrophilic substrate and immersed in a hydrophilic solution. The fluid-driven vesicle successfully picks up Janus particles on the substrate and transports these particles as cargo along the surface. The vesicle can carry up to four particles as its payload. Hence, the vesicles can act as nanoscopic "vacuum cleaners", collecting nanoscopic debris localized on the floors of the fluidic devices. Importantly, these studies reveal how an imposed flow can facilitate the incorporation of nanoparticles into nanoscale vesicles. With the introduction of a "sticky" domain on the substrate, the vesicles can also robustly drop off and deposit the particles on the surface. The controlled pickup and delivery of nanoparticles via lipid vesicles can play an important step in the bottom-up assembly of these nanoparticles within small-scale fluidic devices.

  14. Harnessing Fluid-Driven Vesicles to Pick Up and Drop Off Janus Particles

    NASA Astrophysics Data System (ADS)

    Yong, Xin; Salib, Isaac; Crabb, Emily; Moellers, Nicholas; McFarlin, Gerald; Kuksenok, Olga; Balazs, Anna

    2013-03-01

    Using dissipative particle dynamics (DPD) simulations, we model the interaction between nanoscopic lipid vesicles and Janus nanoparticles in the presence of an imposed flow. Both the vesicle and Janus nanoparticles are localized on a hydrophilic substrate and immersed in a hydrophilic solution. The fluid-driven vesicle successfully picks up Janus particles on the substrate and transports these particles as cargo along the surface. The vesicle can carry up to four particles as its payload. Hence, the vesicles can acts as nanoscopic ``vacuum cleaners'', collecting nanoscopic debris localized on the floors of the fluidic devices. Importantly, these studies reveal how an imposed flow can facilitate the incorporation of nanoparticles into nanoscale vesicles. With the introduction of a hydrophobic domain on the substrate, the vesicles can also robustly drop off and deposit the particles on the surface. The controlled pickup and delivery of nanoparticles via lipid vesicles can play an important step in the bottom-up assembly of these nanoparticles within small-scale fluidic devices.

  15. Protein binding onto surfactant-based synthetic vesicles.

    PubMed

    Letizia, Caterina; Andreozzi, Patrizia; Scipioni, Anita; La Mesa, Camillo; Bonincontro, Adalberto; Spigone, Elisabetta

    2007-02-01

    Synthetic vesicles were prepared by mixing anionic and cationic surfactants, aqueous sodium dodecylsulfate with didodecyltrimethylammonium or cetyltrimethylammonium bromide. The overall surfactant content and the (anionic/cationic) mole ratios allow one to obtain negatively charged vesicles. In the phase diagram, the vesicular region is located between a solution phase, a lamellar liquid crystalline dispersion, and a precipitate area. Characterization of the vesicles was performed by electrophoretic mobility, NMR, TEM, and DLS and we determined their uni-lamellar character, size, stability, and charge density. Negatively charged vesicular dispersions, made of sodium dodecylsulfate/didodecyltrimethylammonium bromide or sodium dodecylsulfate/cetyltrimethylammonium bromide, were mixed with lysozyme, to form lipoplexes. Depending on the protein/vesicle charge ratio, binding, surface saturation, and lipoplexes flocculation, or precipitation, occurs. The free protein in excess remains in solution, after binding saturation. The systems were investigated by thermodynamic (surface tension and solution calorimetry), DLS, CD, TEM, 1H NMR, transport properties, electrophoretic mobility, and dielectric relaxation. The latter two methods give information on the vesicle charge neutralization by adsorbed protein. Binding is concomitant to modifications in the double layer thickness of vesicles and in the surface charge density of the resulting lipoplexes. This is also confirmed by developing the electrophoretic mobility results in terms of a Langmuir-like adsorption isotherm. Charges in excess with respect to the amount required to neutralize the vesicle surface promote lipoplexes clustering and/or flocculation. Protein-vesicle interactions were observed by DLS, indicating changes in particle size (and in their distribution functions) upon addition of LYSO. According to CD, the bound protein retains its native conformation, at least in the SDS/CTAB vesicular system. In fact

  16. Hemifusion in Synaptic Vesicle Cycle

    PubMed Central

    Kweon, Dae-Hyuk; Kong, Byoungjae; Shin, Yeon-Kyun

    2017-01-01

    In the neuron, early neurotransmitters are released through the fusion pore prior to the complete vesicle fusion. It has been thought that the fusion pore is a gap junction-like structure made of transmembrane domains (TMDs) of soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins. However, evidence has accumulated that lipid mixing occurs prior to the neurotransmitter release through the fusion pore lined predominantly with lipids. To explain these observations, the hemifusion, a membrane structure in which two bilayers are partially merged, has emerged as a key step preceding the formation of the fusion pore. Furthermore, the hemifusion appears to be the bona fide intermediate step not only for the synaptic vesicle cycle, but for a wide range of membrane remodeling processes, such as viral membrane fusion and endocytotic membrane fission. PMID:28360835

  17. Axisymmetric Boundary Element Method for vesicles in a capillary

    NASA Astrophysics Data System (ADS)

    Trozzo, R.; Boedec, G.; Leonetti, M.; Jaeger, M.

    2015-05-01

    The problem of a vesicle transported by a fluid flow can present a large range of length scales. One example is the case of a vesicle producing a tether, and eventually pearls, in an elongational flow. Another case occurs when a lubrication film is formed, such as during the short range interaction between two vesicles. Such problems are still challenging for 3D simulations. On the other hand, a good understanding could be obtained by first considering the axisymmetric regime when such a regime exists. An axisymmetric model could then be used, without the criticisms that can be made of a 2D approach. We propose such a model, primarily interested in flows through narrow cylindrical capillaries. Two options are compared, with and without explicit representation of the capillary boundaries by a mesh. The numerical effort is characterized as a function of the vesicle's initial shape, the flow magnitude and the confinement. The model is able to treat typical configurations of red blood cells flowing through very narrow pores with extremely thin lubrication films.

  18. Ornithine decarboxylase antizyme inhibitor 2 regulates intracellular vesicle trafficking

    SciTech Connect

    Kanerva, Kristiina; Maekitie, Laura T.; Baeck, Nils; Andersson, Leif C.

    2010-07-01

    Antizyme inhibitor 1 (AZIN1) and 2 (AZIN2) are proteins that activate ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis. Both AZINs release ODC from its inactive complex with antizyme (AZ), leading to formation of the catalytically active ODC. The ubiquitously expressed AZIN1 is involved in cell proliferation and transformation whereas the role of the recently found AZIN2 in cellular functions is unknown. Here we report the intracellular localization of AZIN2 and present novel evidence indicating that it acts as a regulator of vesicle trafficking. We used immunostaining to demonstrate that both endogenous and FLAG-tagged AZIN2 localize to post-Golgi vesicles of the secretory pathway. Immuno-electron microscopy revealed that the vesicles associate mainly with the trans-Golgi network (TGN). RNAi-mediated knockdown of AZIN2 or depletion of cellular polyamines caused selective fragmentation of the TGN and retarded the exocytotic release of vesicular stomatitis virus glycoprotein. Exogenous addition of polyamines normalized the morphological changes and reversed the inhibition of protein secretion. Our findings demonstrate that AZIN2 regulates the transport of secretory vesicles by locally activating ODC and polyamine biosynthesis.

  19. Mechanics and stability of vesicles and droplets in confined spaces

    NASA Astrophysics Data System (ADS)

    Benet, Eduard; Vernerey, Franck J.

    2016-12-01

    The permeation and trapping of soft colloidal particles in the confined space of porous media are of critical importance in cell migration studies, design of drug delivery vehicles, and colloid separation devices. Our current understanding of these processes is however limited by the lack of quantitative models that can relate how the elasticity, size, and adhesion properties of the vesicle-pore complex affect colloid transport. We address this shortcoming by introducing a semianalytical model that predicts the equilibrium shapes of a soft vesicle driven by pressure in a narrow pore. Using this approach, the problem is recast in terms of pressure and energy diagrams that characterize the vesicle stability and permeation pressures in different conditions. We particularly show that the critical permeation pressure for a vesicle arises from a compromise between the critical entry pressure and exit pressure, both of which are sensitive to geometrical features, mechanics, and adhesion. We further find that these results can be leveraged to rationally design microfluidic devices and diodes that can help characterize, select, and separate colloids based on physical properties.

  20. Occurrence and Characteristics of a Rapid Exchange of Phosphate Oxygens Catalyzed by Sarcoplasmic Reticulum Vesicles

    DOE R&D Accomplishments Database

    Kanazawa, T.; Boyer, P. D.

    1972-01-01

    Sarcoplasmic reticulum vesicles isolated from skeletal muscle actively take up Ca{sup ++} from the medium in the presence of Mg{sup ++} and ATP. This transport is coupled to ATP hydrolysis catalyzed by membrane-bound Ca{sup++}, Mg{sup ++}-ATPase which is activated by concurrent presence of Ca{sup ++} and Mg{sup ++}. Considerable informations have accumulated that give insight into the ATPase and its coupling to the calcium transport. The hydrolysis of ATP by this enzyme occurs through a phosphorylated intermediate. Formation and decomposition of the intermediate show vectorial requirements for Ca{sup ++} and Mg{sup ++}, suggesting an intimate involvement of the intermediate in the transport process. ATP synthesis from P{sub i} and ADP coupled to outflow of Ca{sup ++} from sarcoplasmic reticulum vesicles has recently been demonstrated. This indicates the reversibility of the entire process of calcium transport in sarcoplasmic reticulum vesicles.

  1. Quantifying mixing in vesicle suspensions using numerical simulations in two dimensions

    NASA Astrophysics Data System (ADS)

    Kabacaoglu, Gokberk; Biros, George; Quaife, Bryan

    2015-11-01

    Vesicles, which resist bending and are locally inextensible, serve as an experimental and numerical proxy for red blood cells. In this work, we study the effect of the presence of vesicles to mixing. The motivating application is the study of transport phenomena in microcirculation. We investigate transport specifically in a Couette apparatus, which is governed by an advection-diffusion equation, and we consider mixing in the absence and presence of vesicles using numerical simulations in two dimensions. The advection-diffusion equation is discretized spectrally in space, and with a second-order L-stable Strang splitting in time. To our knowledge, there are no universally accepted measures of mixing. Here, we study two measures: the ``mix-norm'' defined by a Sobolev norm of negative index and a standard moment fluctuation of the transported species. We define mixing efficiency in terms of mixing measure in the absence of vesicles relative to the measure in the presence of vesicles. We then study the correlation of mixing efficiency with the Peclet number, the volume fraction of the vesicle suspension, and the type of initial conditions.

  2. Vesicles and actin are targeted to the cleavage furrow via furrow microtubules and the central spindle

    PubMed Central

    Albertson, Roger; Cao, Jian; Hsieh, Tao-shih; Sullivan, William

    2008-01-01

    During cytokinesis, cleavage furrow invagination requires an actomyosin-based contractile ring and addition of new membrane. Little is known about how this actin and membrane traffic to the cleavage furrow. We address this through live analysis of fluorescently tagged vesicles in postcellularized Drosophila melanogaster embryos. We find that during cytokinesis, F-actin and membrane are targeted as a unit to invaginating furrows through formation of F-actin–associated vesicles. F-actin puncta strongly colocalize with endosomal, but not Golgi-derived, vesicles. These vesicles are recruited to the cleavage furrow along the central spindle and a distinct population of microtubules (MTs) in contact with the leading furrow edge (furrow MTs). We find that Rho-specific guanine nucleotide exchange factor mutants, pebble (pbl), severely disrupt this F-actin–associated vesicle transport. These transport defects are a consequence of the pbl mutants' inability to properly form furrow MTs and the central spindle. Transport of F-actin–associated vesicles on furrow MTs and the central spindle is thus an important mechanism by which actin and membrane are delivered to the cleavage furrow. PMID:18504302

  3. Structures of yeast vesicle trafficking proteins.

    PubMed Central

    Tishgarten, T.; Yin, F. F.; Faucher, K. M.; Dluhy, R. A.; Grant, T. R.; Fischer von Mollard, G.; Stevens, T. H.; Lipscomb, L. A.

    1999-01-01

    In protein transport between organelles, interactions of v- and t-SNARE proteins are required for fusion of protein-containing vesicles with appropriate target compartments. Mammalian SNARE proteins have been observed to interact with NSF and SNAP, and yeast SNAREs with yeast homologues of NSF and SNAP proteins. This observation led to the hypothesis that, despite low sequence homology, SNARE proteins are structurally similar among eukaryotes. SNARE proteins can be classified into two groups depending on whether they interact with SNARE binding partners via conserved glutamine (Q-SNAREs) or arginine (R-SNAREs). Much of the published structural data available is for SNAREs involved in exocytosis (either in yeast or synaptic vesicles). This paper describes circular dichroism, Fourier transform infrared spectroscopy, and dynamic light scattering data for a set of yeast v- and t-SNARE proteins, Vti1p and Pep12p, that are Q-SNAREs involved in intracellular trafficking. Our results suggest that the secondary structure of Vti1p is highly alpha-helical and that Vti1p forms multimers under a variety of solution conditions. In these respects, Vti1p appears to be distinct from R-SNARE proteins characterized previously. The alpha-helicity of Vti1p is similar to that of Q-SNARE proteins characterized previously. Pep12p, a Q-SNARE, is highly alpha-helical. It is distinct from other Q-SNAREs in that it forms dimers under many of the solution conditions tested in our experiments. The results presented in this paper are among the first to suggest heterogeneity in the functioning of SNARE complexes. PMID:10595551

  4. Synaptic Vesicle Proteins and Active Zone Plasticity

    PubMed Central

    Kittel, Robert J.; Heckmann, Manfred

    2016-01-01

    Neurotransmitter is released from synaptic vesicles at the highly specialized presynaptic active zone (AZ). The complex molecular architecture of AZs mediates the speed, precision and plasticity of synaptic transmission. Importantly, structural and functional properties of AZs vary significantly, even for a given connection. Thus, there appear to be distinct AZ states, which fundamentally influence neuronal communication by controlling the positioning and release of synaptic vesicles. Vice versa, recent evidence has revealed that synaptic vesicle components also modulate organizational states of the AZ. The protein-rich cytomatrix at the active zone (CAZ) provides a structural platform for molecular interactions guiding vesicle exocytosis. Studies in Drosophila have now demonstrated that the vesicle proteins Synaptotagmin-1 (Syt1) and Rab3 also regulate glutamate release by shaping differentiation of the CAZ ultrastructure. We review these unexpected findings and discuss mechanistic interpretations of the reciprocal relationship between synaptic vesicles and AZ states, which has heretofore received little attention. PMID:27148040

  5. Extrasynaptic vesicle recycling in mature hippocampal neurons.

    PubMed

    Ratnayaka, Arjuna; Marra, Vincenzo; Branco, Tiago; Staras, Kevin

    2011-11-08

    Fast neuronal signalling relies on highly regulated vesicle fusion and recycling at specialized presynaptic terminals. Recently, examples of non-classical neurotransmission have also been reported, where fusion of vesicles can occur at sites remote from conventional synapses. This has potentially broad biological implications, but the underlying mechanisms are not well established. Here we show that a complete vesicle recycling pathway can occur at discrete axonal sites in mature hippocampal neurons and that extrasynaptic fusion is a robust feature of native tissue. We demonstrate that laterally mobile vesicle clusters trafficking between synaptic terminals become transiently stabilized by evoked action potentials and undergo complete but delayed Ca(2+)-dependent fusion along axons. This fusion is associated with dynamic actin accumulation and, subsequently, vesicles can be locally recycled, re-acidified and re-used. Immunofluorescence and ultrastructural work demonstrates that extrasynaptic fusion sites can have apposed postsynaptic specializations, suggesting that mobile vesicle recycling may underlie highly dynamic neuron-neuron communication.

  6. Ultrasound-responsive ultrathin multiblock copolyamide vesicles

    NASA Astrophysics Data System (ADS)

    Huang, Lei; Yu, Chunyang; Huang, Tong; Xu, Shuting; Bai, Yongping; Zhou, Yongfeng

    2016-02-01

    This study reports the self-assembly of novel polymer vesicles from an amphiphilic multiblock copolyamide, and the vesicles show a special structure with an ultrathin wall thickness of about 4.5 nm and a combined bilayer and monolayer packing model. Most interestingly, the vesicles are ultrasound-responsive and can release the encapsulated model drugs in response to ultrasonic irradiation.This study reports the self-assembly of novel polymer vesicles from an amphiphilic multiblock copolyamide, and the vesicles show a special structure with an ultrathin wall thickness of about 4.5 nm and a combined bilayer and monolayer packing model. Most interestingly, the vesicles are ultrasound-responsive and can release the encapsulated model drugs in response to ultrasonic irradiation. Electronic supplementary information (ESI) available: Details of experiments and characterization, and FT-IR, TEM, DPD, FL and micro-DSC results. See DOI: 10.1039/c5nr08596a

  7. 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

  8. Lipidomic Analysis of Extracellular Vesicles from the Pathogenic Phase of Paracoccidioides brasiliensis

    PubMed Central

    Longo, Larissa V. G.; Ganiko, Luciane; Lopes, Felipe G.; Matsuo, Alisson L.; Almeida, Igor C.; Puccia, Rosana

    2012-01-01

    Background Fungal extracellular vesicles are able to cross the cell wall and transport molecules that help in nutrient acquisition, cell defense, and modulation of the host defense machinery. Methodology/Principal Findings Here we present a detailed lipidomic analysis of extracellular vesicles released by Paracoccidioides brasiliensis at the yeast pathogenic phase. We compared data of two representative isolates, Pb3 and Pb18, which have distinct virulence profiles and phylogenetic background. Vesicle lipids were fractionated into different classes and analyzed by either electrospray ionization- or gas chromatography-mass spectrometry. We found two species of monohexosylceramide and 33 phospholipid species, including phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol. Among the phospholipid-bound fatty acids in extracellular vesicles, C181 predominated in Pb3, whereas C18:2 prevailed in Pb18. The prevalent sterol in Pb3 and Pb18 vesicles was brassicasterol, followed by ergosterol and lanosterol. Inter-isolate differences in sterol composition were observed, and also between extracellular vesicles and whole cells. Conclusions/Significance The extensive lipidomic analysis of extracellular vesicles from two P. brasiliensis isolates will help to understand the composition of these fungal components/organelles and will hopefully be useful to study their biogenesis and role in host-pathogen interactions. PMID:22745761

  9. Vesicles

    MedlinePlus

    ... Contact dermatitis (may be caused by poison ivy) Herpes simplex (cold sores, genital herpes ) Herpes zoster (shingles) Impetigo ... Hand, foot, and mouth disease on the soles Herpes simplex - close-up Herpes zoster (shingles) - close-up of ...

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

    PubMed

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

    2015-12-17

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

  11. Extracellular vesicles: small bricks for tissue repair/regeneration

    PubMed Central

    Taverna, Simona; Pucci, Marzia

    2017-01-01

    Extracellular vesicles (EVs) are nano-sized membrane vesicles involved in intercellular communication. EVs have pleiotropic actions in physiological and pathological conditions. The ability of EVs to transports proteins, drugs and nucleic acid, to target specific cells and to increase the stability of therapeutic cargo, make EVs interesting as new devices for the treatment of human disease. In a recently published issue of European journal of pharmaceutical sciences, Silva and colleagues reviewed the ability of EVs to modulate tissue repair and regeneration, focusing on their roles and therapeutic potential as immunomodulatory messengers. In this perspective, we discussed the open questions regarding the dual role of EVs in immune system, as well as the technical limitation of the procedure for EVs isolation and administration in clinical practices. EV-based therapies require further studies to consider EVs as promising candidate for a novel cell-free therapy in the context of regeneration medicine. PMID:28275628

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

    PubMed Central

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

    2015-01-01

    Summary Vesicular nucleo-cytoplasmic transport is becoming recognized as a general cellular mechanism for translocation of large cargoes across the nuclear envelope. Cargo is recruited, enveloped at the inner nuclear membrane (INM), and delivered by membrane fusion at the outer nuclear membrane. To understand the structural underpinning for this trafficking, we investigated nuclear egress of progeny herpesvirus capsids where capsid envelopment is mediated by two viral proteins, forming the nuclear egress complex (NEC). Using a multi-modal imaging approach, we visualized the NEC in situ forming coated vesicles of defined size. Cellular electron cryo-tomography revealed a protein layer showing two distinct hexagonal lattices at its membrane-proximal and membrane-distant faces, respectively. NEC coat architecture was determined by combining this information with integrative modeling using small-angle X-ray scattering data. The molecular arrangement of the NEC establishes the basic mechanism for budding and scission of tailored vesicles at the INM. PMID:26687357

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

    PubMed Central

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

    2013-01-01

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

  14. Construction of macroscopic cytomimetic vesicle aggregates based on click chemistry: controllable vesicle fusion and phase separation.

    PubMed

    Jin, Haibao; Huang, Wei; Zheng, Yongli; Zhou, Yongfeng; Yan, Deyue

    2012-07-09

    Vesicle-vesicle aggregation to mimic cell-cell aggregation has attracted much attention. Here, hyperbranched polymer vesicles (branched-polymersomes, BPs) with a cell-like size were selected as model membranes, and the vesicle aggregation process, triggered by click chemistry of the copper-catalysed azide-alkyne cycloaddition reaction, was systematically studied. For this purpose, azide and alkynyl groups were loaded on the membranes of BPs through the co-assembly method to obtain N(3)-BPs and Alk-BPs, respectively. Subsequently, macroscopic vesicle aggregates were obtained when these two kinds of functional BPs were mixed together with the ratio of azide to alkynyl groups of about 1:1. Both the vesicle fusion events and lateral phase separation on the vesicle membrane occurred during such a vesicle aggregation process, and the fusion rate and phase-separation degree could be controlled by adjusting the clickable group content. The vesicle aggregation process with N(3) -micelles as desmosome mimics to connect with Alk-BPs through click-chemistry reaction was also studied, and large-scale vesicle aggregates without vesicle fusion were obtained in this process. The present work has extended the controllable cytomimetic vesicle aggregation process with the use of covalent bonds, instead of noncovalent bonds, as the driving force.

  15. Vesicles protect activated acetic acid.

    PubMed

    Todd, Zoe R; House, Christopher H

    2014-10-01

    Abstract Methyl thioacetate, or activated acetic acid, has been proposed to be central to the origin of life and an important energy currency molecule in early cellular evolution. We have investigated the hydrolysis of methyl thioacetate under various conditions. Its uncatalyzed rate of hydrolysis is about 3 orders of magnitude faster (K=0.00663 s(-1); 100°C, pH 7.5, concentration=0.33 mM) than published rates for its catalyzed production, making it unlikely to accumulate under prebiotic conditions. However, our experiments showed that methyl thioacetate was protected from hydrolysis when inside its own hydrophobic droplets. Further, we found that methyl thioacetate protection from hydrolysis was also possible in droplets of hexane and in the membranes of nonanoic acid vesicles. Thus, the hydrophobic regions of prebiotic vesicles and early cell membranes could have offered a refuge for this energetic molecule, increasing its lifetime in close proximity to the reactions for which it would be needed. This model of early energy storage evokes an additional critical function for the earliest cell membranes.

  16. Proteomics of extracellular vesicles: Exosomes and ectosomes.

    PubMed

    Choi, Dong-Sic; Kim, Dae-Kyum; Kim, Yoon-Keun; Gho, Yong Song

    2015-01-01

    Almost all bacteria, archaea, and eukaryotic cells shed extracellular vesicles either constitutively or in a regulated manner. These nanosized membrane vesicles are spherical, bilayered proteolipids that harbor specific subsets of proteins, DNAs, RNAs, and lipids. Recent research has facilitated conceptual advancements in this emerging field that indicate that extracellular vesicles act as intercellular communicasomes by transferring signals to their target cell via surface ligands and delivering receptors and functional molecules. Recent progress in mass spectrometry-based proteomic analyses of mammalian extracellular vesicles derived from diverse cell types and body fluids has resulted in the identification of several thousand vesicular proteins that provide us with essential clues to the molecular mechanisms involved in vesicle cargo sorting and biogenesis. Furthermore, cell-type- or disease-specific vesicular proteins help us to understand the pathophysiological functions of extracellular vesicles and contribute to the discovery of diagnostic and therapeutic target proteins. This review focuses on the high-throughput mass spectrometry-based proteomic analyses of mammalian extracellular vesicles (i.e., exosomes and ectosomes), EVpedia (a free web-based integrated database of high-throughput data for systematic analyses of extracellular vesicles; http://evpedia.info), and the intravesicular protein-protein interaction network analyses of mammalian extracellular vesicles. The goal of this article is to encourage further studies to construct a comprehensive proteome database for extracellular vesicles that will help us to not only decode the biogenesis and cargo-sorting mechanisms during vesicle formation but also elucidate the pathophysiological roles of these complex extracellular organelles.

  17. Small Angle Neutron-Scattering Studies of the Core Structure of Intact Neurosecretory Vesicles.

    NASA Astrophysics Data System (ADS)

    Krueger, Susan Takacs

    Small angle neutron scattering (SANS) was used to study the state of the dense cores within intact neurosecretory vesicles. These vesicles transport the neurophysin proteins, along with their associated hormones, oxytocin or vasopressin, from the posterior pituitary gland to the bloodstream, where the entire vesicle contents are released. Knowledge of the vesicle core structure is important in developing an understanding of this release mechanism. Since the core constituents exist in a dense state at concentrations which cannot be reproduced (in solution) in the laboratory, a new method was developed to determine the core structure from SANS experiments performed on intact neurosecretory vesicles. These studies were complemented by biochemical assays performed to determine the role, if any, played by phospholipids in the interactions between the core constituents. H_2O/D_2 O ratio in the solvent can be adjusted, using the method of contrast variation, such that the scattering due to the vesicle membranes is minimized, thus emphasizing the scattering originating from the cores. The applicability of this method for examining the interior of biological vesicles was tested by performing an initial study on human red blood cells, which are similar in structure to other biological vesicles. Changes in intermolecular hemoglobin interactions, occurring when the ionic strength of the solvent was varied or when the cells were deoxygenated, were examined. The results agreed with those expected for dense protein solutions, indicating that the method developed was suitable for the study of hemoglobin within the cells. Similar SANS studies were then performed on intact neurosecretory vesicles. The experimental results were inconsistent with model calculations which assumed that the cores consisted of small, densely-packed particles or large, globular aggregates. Although a unique model could not be determined, the data suggest that the core constituents form long aggregates of

  18. Dynamical simulations of vesicle growth and division

    NASA Astrophysics Data System (ADS)

    Ruiz-Herrero, Teresa; Mahadevan, L.

    2015-03-01

    Prebiotic cells constitute a beautiful and intriguing example of self-replicating vesicles. How these cells managed to grow and divide without sophisticated machinery is still an open question. The properties of these primitive vesicles can shed light on the ways modern cells have evolved by exploiting those characteristics to develop their replication mechanisms. The equilibrium configurations of elastic shells are well understood, however the dynamical behavior during growth still lacks of a deep theoretical understanding. To study vesicle growth from a general perspective, we have developed a minimal generic model where vesicles are represented by a 2D spring network and characterized by a minimum set of magnitudes: growth rate, permeability, bending stiffness, viscosity and temperature. We have performed hybrid molecuar dynamic simulations as a function of a reduced set of dimensionless parameters. Three main outcomes were observed: vesicles that grow without division, vesicles that divide symmetrically, and vesicles that act as generators of daughter vesicles. The type of outcome depends on the system parameters and specifically on its dynamics via two timescales. Furthermore, we found sets of parameters where the system shows size homeostasis. TRH was supported by Ramon Areces Foundation.

  19. Functional Advantages of Porphyromonas gingivalis Vesicles

    PubMed Central

    Ho, Meng-Hsuan; Chen, Chin-Ho; Goodwin, J. Shawn; Wang, Bing-Yan; Xie, Hua

    2015-01-01

    Porphyromonas gingivalis is a keystone pathogen of periodontitis. Outer membrane vesicles (OMVs) have been considered as both offense and defense components of this bacterium. Previous studies indicated that like their originating cells, P. gingivalis vesicles, are able to invade oral epithelial cells and gingival fibroblasts, in order to promote aggregation of some specific oral bacteria and to induce host immune responses. In the present study, we investigated the invasive efficiency of P. gingivalis OMVs and compared results with that of the originating cells. Results revealed that 70–90% of human primary oral epithelial cells, gingival fibroblasts, and human umbilical vein endothelial cells carried vesicles from P. gingivalis 33277 after being exposed to the vesicles for 1 h, while 20–50% of the host cells had internalized P. gingivalis cells. We also detected vesicle-associated DNA and RNA and a vesicle-mediated horizontal gene transfer in P. gingivalis strains, which represents a novel mechanism for gene transfer between P. gingivalis strains. Moreover, purified vesicles of P. gingivalis appear to have a negative impact on biofilm formation and the maintenance of Streptococcus gordonii. Our results suggest that vesicles are likely the best offence weapon of P. gingivalis for bacterial survival in the oral cavity and for induction of periodontitis. PMID:25897780

  20. 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

  1. Extracellular vesicles in coronary artery disease.

    PubMed

    Boulanger, Chantal M; Loyer, Xavier; Rautou, Pierre-Emmanuel; Amabile, Nicolas

    2017-02-02

    Membrane vesicles released in the extracellular space are composed of a lipid bilayer enclosing soluble cytosolic material and nuclear components. Extracellular vesicles include apoptotic bodies, exosomes, and microvesicles (also known previously as microparticles). Originating from different subcellular compartments, the role of extracellular vesicles as regulators of transfer of biological information, acting locally and remotely, is now acknowledged. Circulating vesicles released from platelets, erythrocytes, leukocytes, and endothelial cells contain potential valuable biological information for biomarker discovery in primary and secondary prevention of coronary artery disease. Extracellular vesicles also accumulate in human atherosclerotic plaques, where they affect major biological pathways, including inflammation, proliferation, thrombosis, calcification, and vasoactive responses. Extracellular vesicles also recapitulate the beneficial effect of stem cells to treat cardiac consequences of acute myocardial infarction, and now emerge as an attractive alternative to cell therapy, opening new avenues to vectorize biological information to target tissues. Although interest in microvesicles in the cardiovascular field emerged about 2 decades ago, that for extracellular vesicles, in particular exosomes, started to unfold a decade ago, opening new research and therapeutic avenues. This Review summarizes current knowledge on the role of extracellular vesicles in coronary artery disease, and their emerging potential as biomarkers and therapeutic agents.

  2. 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.

  3. Analysis of Yeast Extracellular Vesicles.

    PubMed

    Rodrigues, Marcio L; Oliveira, Debora L; Vargas, Gabriele; Girard-Dias, Wendell; Franzen, Anderson J; Frasés, Susana; Miranda, Kildare; Nimrichter, Leonardo

    2016-01-01

    Extracellular vesicles (EV) are important carriers of biologically active components in a number of organisms, including fungal cells. Experimental characterization of fungal EVs suggested that these membranous compartments are likely involved in the regulation of several biological events. In fungal pathogens, these events include mechanisms of disease progression and/or control, suggesting potential targets for therapeutic intervention or disease prophylaxis. In this manuscript we describe methods that have been used in the last 10 years for the characterization of EVs produced by yeast forms of several fungal species. Experimental approaches detailed in this chapter include ultracentrifugation methods for EV fractionation, chromatographic approaches for analysis of EV lipids, microscopy techniques for analysis of both intracellular and extracellular vesicular compartments, interaction of EVs with host cells, and physical chemical analysis of EVs by dynamic light scattering.

  4. 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

  5. ATP-dependent transport of glutathione conjugate of 7beta, 8alpha-dihydroxy-9alpha,10alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene in murine hepatic canalicular plasma membrane vesicles.

    PubMed Central

    Srivastava, S K; Hu, X; Xia, H; Bleicher, R J; Zaren, H A; Orchard, J L; Awasthi, S; Singh, S V

    1998-01-01

    Glutathione (GSH) S-transferases (GSTs) have an important role in the detoxification of (+)-anti-7,8-dihydroxy-9,10-oxy-7,8,9, 10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE], which is the ultimate carcinogen of benzo[a]pyrene. However, the fate and/or biological activity of the GSH conjugate of (+)-anti-BPDE [(-)-anti-BPD-SG] is not known. We now report that (-)-anti-BPD-SG is a competitive inhibitor (Ki 19 microM) of Pi-class isoenzyme mGSTP1-1, which among murine hepatic GSTs is most efficient in the GSH conjugation of (+)-anti-BPDE. Thus the inhibition of mGSTP1-1 activity by (-)-anti-BPD-SG might interfere with the GST-catalysed GSH conjugation of (+)-anti-BPDE unless one or more mechanisms exist for the removal of the conjugate. The results of the present study indicate that (-)-anti-BPD-SG is transported across canalicular liver plasma membrane (cLPM) in an ATP-dependent manner. The ATP-dependent transport of (-)-anti-[3H]BPD-SG followed Michaelis-Menten kinetics (Km 46 microM). The ATP dependence of the (-)-anti-BPD-SG transport was confirmed by measuring the stimulation of ATP hydrolysis (ATPase activity) by the conjugate in the presence of cLPM protein, which also followed Michaelis-Menten kinetics. In contrast, a kinetic analysis of ATP-dependent uptake of the model conjugate S-[3H](2,4-dinitrophenyl)-glutathione ([3H]DNP-SG) revealed the presence of a high-affinity and a low-affinity transport system in mouse cLPM, with apparent Km values of 18 and 500 microM respectively. The ATP-dependent transport of (-)-anti-BPD-SG was inhibited competitively by DNP-SG (Ki 1.65 microM). Likewise, (-)-anti-BPD-SG was found to be a potent competitive inhibitor of the high-affinity component of DNP-SG transport (Ki 6.3 microM). Our results suggest that GST-catalysed conjugation of (+)-anti-BPDE with GSH, coupled with ATP-dependent transport of the resultant conjugate across cLPM, might be the ultimate detoxification pathway for this carcinogen. PMID:9620885

  6. 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

  7. On the Computing Potential of Intracellular Vesicles.

    PubMed

    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.

  8. Extracellular Vesicles and a Novel Form of Communication in the Brain.

    PubMed

    Basso, Manuela; Bonetto, Valentina

    2016-01-01

    In numerous neurodegenerative diseases, the interplay between neurons and glia modulates the outcome and progression of pathology. One particularly intriguing mode of interaction between neurons, astrocytes, microglia, and oligodendrocytes is characterized by the release of extracellular vesicles that transport proteins, lipids, and nucleotides from one cell to another. Notably, several proteins that cause disease, including the prion protein and mutant SOD1, have been detected in glia-derived extracellular vesicles and observed to fuse with neurons and trigger pathology in vitro. Here we review the structural and functional characterization of such extracellular vesicles in neuron-glia interactions. Furthermore, we discuss possible mechanisms of extracellular vesicle biogenesis and release from activated glia and microglia, and their effects on neurons. Given that exosomes, the smallest type of extracellular vesicles, have been reported to recognize specific cellular populations and act as carriers of very specialized cargo, a thorough analysis of these vesicles may aid in their engineering in vitro and targeted delivery in vivo, opening opportunities for therapeutics.

  9. Extracellular vesicle sorting of α-Synuclein is regulated by sumoylation.

    PubMed

    Kunadt, Marcel; Eckermann, Katrin; Stuendl, Anne; Gong, Jing; Russo, Belisa; Strauss, Katrin; Rai, Surya; Kügler, Sebastian; Falomir Lockhart, Lisandro; Schwalbe, Martin; Krumova, Petranka; Oliveira, Luis M A; Bähr, Mathias; Möbius, Wiebke; Levin, Johannes; Giese, Armin; Kruse, Niels; Mollenhauer, Brit; Geiss-Friedlander, Ruth; Ludolph, Albert C; Freischmidt, Axel; Feiler, Marisa S; Danzer, Karin M; Zweckstetter, Markus; Jovin, Thomas M; Simons, Mikael; Weishaupt, Jochen H; Schneider, Anja

    2015-05-01

    Extracellular α-Synuclein has been implicated in interneuronal propagation of disease pathology in Parkinson's Disease. How α-Synuclein is released into the extracellular space is still unclear. Here, we show that α-Synuclein is present in extracellular vesicles in the central nervous system. We find that sorting of α-Synuclein in extracellular vesicles is regulated by sumoylation and that sumoylation acts as a sorting factor for targeting of both, cytosolic and transmembrane proteins, to extracellular vesicles. We provide evidence that the SUMO-dependent sorting utilizes the endosomal sorting complex required for transport (ESCRT) by interaction with phosphoinositols. Ubiquitination of cargo proteins is so far the only known determinant for ESCRT-dependent sorting into the extracellular vesicle pathway. Our study reveals a function of SUMO protein modification as a Ubiquitin-independent ESCRT sorting signal, regulating the extracellular vesicle release of α-Synuclein. We deciphered in detail the molecular mechanism which directs α-Synuclein into extracellular vesicles which is of highest relevance for the understanding of Parkinson's disease pathogenesis and progression at the molecular level. We furthermore propose that sumo-dependent sorting constitutes a mechanism with more general implications for cell biology.

  10. An endosomal tether undergoes an entropic collapse to bring vesicles together.

    PubMed

    Murray, David H; Jahnel, Marcus; Lauer, Janelle; Avellaneda, Mario J; Brouilly, Nicolas; Cezanne, Alice; Morales-Navarrete, Hernán; Perini, Enrico D; Ferguson, Charles; Lupas, Andrei N; Kalaidzidis, Yannis; Parton, Robert G; Grill, Stephan W; Zerial, Marino

    2016-09-01

    An early step in intracellular transport is the selective recognition of a vesicle by its appropriate target membrane, a process regulated by Rab GTPases via the recruitment of tethering effectors. Membrane tethering confers higher selectivity and efficiency to membrane fusion than the pairing of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) alone. Here we address the mechanism whereby a tethered vesicle comes closer towards its target membrane for fusion by reconstituting an endosomal asymmetric tethering machinery consisting of the dimeric coiled-coil protein EEA1 (refs 6, 7) recruited to phosphatidylinositol 3-phosphate membranes and binding vesicles harbouring Rab5. Surprisingly, structural analysis reveals that Rab5:GTP induces an allosteric conformational change in EEA1, from extended to flexible and collapsed. Through dynamic analysis by optical tweezers, we confirm that EEA1 captures a vesicle at a distance corresponding to its extended conformation, and directly measure its flexibility and the forces induced during the tethering reaction. Expression of engineered EEA1 variants defective in the conformational change induce prominent clusters of tethered vesicles in vivo. Our results suggest a new mechanism in which Rab5 induces a change in flexibility of EEA1, generating an entropic collapse force that pulls the captured vesicle towards the target membrane to initiate docking and fusion.

  11. Seminal Tract Amyloidosis: Synchronous Amyloidosis of the Seminal Vesicles, Deferent Ducts and Ejaculatory Ducts.

    PubMed

    Rath-Wolfson, Lea; Bubis, Golan; Shtrasburg, Shmuel; Shvero, Asaf; Koren, Rumelia

    2017-01-17

    Senile Seminal Vesicle Amyloidosis (SSVA) increases with age. Involvement of the whole seminal tract, i.e. the seminal vesicles, ejaculatory and deferent ducts was first reported by us in the International Symposium on Amyloidosis 1998. Since then we encountered four more cases of SSVA. In all these cases the ejaculatory and deferent ducts were also involved by amyloid. The amyloid was located mostly sub-epithelially, stained positively with Congo red, gave green birefringence under polarized light and was permanganate sensitive, slightly positive for lactoferrin immunostaining and negative for all known amyloid types. In recent years the amyloid was found to be derived from Semenogelin I, a major constituent of the seminal fluid which is present in the epithelial cells of the seminal vesicle and vas deference. This would explain the deposition of amyloid not only in the seminal vesicles but also in the deferent an ejaculatory ducts which transport the seminal fluid. In a review of the literature we found three more articles on SSVA in which the amyloid was not limited to the seminal vesicles alone. We propose to designate this type of amyloid as "Senile seminal Tract Amyloidosis" (SSTA) instead of "Senile Seminal Vesicle Amyloidosis (SSVA)".

  12. An endosomal tether undergoes an entropic collapse to bring vesicles together

    PubMed Central

    Lauer, Janelle; Avellaneda, Mario J.; Brouilly, Nicolas; Cezanne, Alice; Morales-Navarrete, Hernán; Perini, Enrico D.; Ferguson, Charles; Lupas, Andrei N.; Kalaidzidis, Yannis; Parton, Robert G.; Grill, Stephan W.; Zerial, Marino

    2016-01-01

    An early step in intracellular transport is the selective recognition of a vesicle by its appropriate target membrane, a process regulated by Rab GTPases via the recruitment of tethering effectors1–4. Membrane tethering confers higher selectivity and efficiency to membrane fusion than the pairing of SNAREs alone5,6,7. Here, we addressed the mechanism whereby a tethered vesicle comes closer towards its target membrane for fusion by reconstituting an endosomal asymmetric tethering machinery consisting of the dimeric coiled-coil protein EEA16,7 recruited to phosphatidylinositol 3-phosphate membranes and binding vesicles harboring Rab5. Surprisingly, structural analysis revealed that Rab5:GTP induces an allosteric conformational change in EEA1, from extended to flexible and collapsed. Through dynamic analysis by optical tweezers we confirmed that EEA1 captures a vesicle at a distance corresponding to its extended conformation, and directly measured its flexibility and the forces induced during the tethering reaction. Expression of engineered EEA1 variants defective in the conformational change induced prominent clusters of tethered vesicles in vivo. Our results suggest a new mechanism in which Rab5 induces a change in flexibility of EEA1, generating an entropic collapse force that pulls the captured vesicle toward the target membrane to initiate docking and fusion. PMID:27556945

  13. Extracellular Vesicles and a Novel Form of Communication in the Brain

    PubMed Central

    Basso, Manuela; Bonetto, Valentina

    2016-01-01

    In numerous neurodegenerative diseases, the interplay between neurons and glia modulates the outcome and progression of pathology. One particularly intriguing mode of interaction between neurons, astrocytes, microglia, and oligodendrocytes is characterized by the release of extracellular vesicles that transport proteins, lipids, and nucleotides from one cell to another. Notably, several proteins that cause disease, including the prion protein and mutant SOD1, have been detected in glia-derived extracellular vesicles and observed to fuse with neurons and trigger pathology in vitro. Here we review the structural and functional characterization of such extracellular vesicles in neuron-glia interactions. Furthermore, we discuss possible mechanisms of extracellular vesicle biogenesis and release from activated glia and microglia, and their effects on neurons. Given that exosomes, the smallest type of extracellular vesicles, have been reported to recognize specific cellular populations and act as carriers of very specialized cargo, a thorough analysis of these vesicles may aid in their engineering in vitro and targeted delivery in vivo, opening opportunities for therapeutics. PMID:27065789

  14. Comparative expression analysis of senescence gene CsNAP and B-class floral development gene CsAP3 during different stages of flower development in Saffron (Crocus sativus L.).

    PubMed

    Wafai, Asrar H; Bukhari, Shoiab; Mokhdomi, Taseem A; Amin, Asif; Wani, Zubair; Hussaini, Amjad; Mir, Javid I; Qadri, Raies A

    2015-07-01

    Crocus sativus, a monocot triploid species belonging to the Iridaceae family, is cultivated for its red stigmatic lobes of the carpel that constitute saffron. Flower development has been extensively studied in different plants. Different floral developmental pathways have been deciphered in many plants. In Crocus sativus, flower is the most important part and understanding the pathway underlying the flower development can pave the way for new avenues to improve its productivity and quality. The combination of class A genes (including APETALA1; CsAP1 and APETALA2; CsAP2), class B genes (including APETALA3; CsAP3 and PISTILLATA; CsPI) and class C genes (including AGAMOUS; CsAG) that are active in each whorl, determines the identity of the organs that will later develop in that whorl. CsAP3 is a class B homeotic gene which promotes petal and stamen formation and has a very important role in flower development. It also activates other genes playing pivotal role in flower development. It has been earlier reported that CsAP3 gene has direct role in activation of CsNAP gene which promotes senescence in plants. Present work was focused on study of relative gene expression changes of CsAP3 and CsNAP gene during different stages of flower development. CsAP3 gene expression was found maximum during late-preanthesis stages of stigma development. Expression increases from stage 5 to stage 6 of flower development and then reduces again from stage 6 to stage 7. CsNAP gene had moderate expression during stage 3 to stage 4 transition and its expression increased abruptly from stage 6 to stage 7 of flower development. There is no direct concordance in the expression of CsAP3 and CsNAP gene expression in saffron. We may conclude that some other factor(s) may be responsible for initiation of CsNAP expression and CsAP3 gene may directly/indirectly be involved in regulating the factors responsible for CsNAP activation.

  15. Vesicle membrane fluctuations at nm resolution

    NASA Astrophysics Data System (ADS)

    Chen, Kejia; Bae, Sung Chul; Min, Chang-Ki; Granick, Steve; Granick Group Team

    2011-03-01

    We measure membrane thermal fluctuations with nanometer spatial resolution and microsecond time resolution, extending a scattering technique used at the Curie Institute to study red blood cell dynamics (Timo Betz et al., Proc. Nat. Acad. Sci. USA 106, 15320, 2009). A laser beam is focused at the leading edge of a phospholipid vesicle membrane and the forward scattered light is detected by a quadrant photodiode. The measurements over 4 orders of magnitude of frequency allow quantification of more complete fluctuation spectra than competing methods, and therefore fuller understanding of the vesicle membrane mechanics. As a proof of concept, we quantify how adsorbed nanoparticles stiffen giant unilamellar vesicles (GUVs).

  16. Extracellular vesicles: Exosomes, microvesicles, and friends

    PubMed Central

    Stoorvogel, Willem

    2013-01-01

    Cells release into the extracellular environment diverse types of membrane vesicles of endosomal and plasma membrane origin called exosomes and microvesicles, respectively. These extracellular vesicles (EVs) represent an important mode of intercellular communication by serving as vehicles for transfer between cells of membrane and cytosolic proteins, lipids, and RNA. Deficiencies in our knowledge of the molecular mechanisms for EV formation and lack of methods to interfere with the packaging of cargo or with vesicle release, however, still hamper identification of their physiological relevance in vivo. In this review, we focus on the characterization of EVs and on currently proposed mechanisms for their formation, targeting, and function. PMID:23420871

  17. Evidence for an UDP-glucuronic acid/phenol glucuronide antiport in rat liver microsomal vesicles.

    PubMed

    Bánhegyi, G; Braun, L; Marcolongo, P; Csala, M; Fulceri, R; Mandl, J; Benedetti, A

    1996-04-01

    The transport of glucuronides synthesized in the luminal compartment of the endoplasmic reticulum by UDP-glucuronosyltransferase isoenzymes was studied in rat liver microsomal vesicles. Microsomal vesicles were loaded with p-nitrophenol glucuronide (5 mM), phenolphthalein glucuronide or UDP-glucuronic acid, by a freeze-thawing method. In was shown that: (i) the loading procedure resulted in millimolar intravesicular concentrations of the different loading compounds; (ii) addition of UDP-glucuronic acid (5 mM) to the vesicles released both intravesicular glucuronides within 1 min; (iii) glucuronides stimulated the release of UDP-glucuronic acid from UDP acid-loaded microsomal vesicles; (iv) trans-stimulation of UDP-glucuronic acid entry by loading of microsomal vesicles with p-nitrophenol glucuronide, phenolphthalein glucuronide, UDP-glucuronic acid and UDP-N-acetyl-glucosamine almost completely abolished the latency of UDP-glucuronosyltransferase, although mannose 6-phosphatase latency remained unaltered; (v) the loading compounds by themselves did not stimulate UDP-glucuronosyltransferase activity. This study indicates that glucuronides synthesized in the lumen of endoplasmic reticulum can leave by an antiport, which concurrently transports USP-glucuronic acid into the lumen of the endoplasmic reticulum.

  18. 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

  19. Extracellular vesicles in parasitic diseases

    PubMed Central

    Marcilla, Antonio; Martin-Jaular, Lorena; Trelis, Maria; de Menezes-Neto, Armando; Osuna, Antonio; Bernal, Dolores; Fernandez-Becerra, Carmen; Almeida, Igor C.; del Portillo, Hernando A.

    2014-01-01

    Parasitic diseases affect billions of people and are considered a major public health issue. Close to 400 species are estimated to parasitize humans, of which around 90 are responsible for great clinical burden and mortality rates. Unfortunately, they are largely neglected as they are mainly endemic to poor regions. Of relevance to this review, there is accumulating evidence of the release of extracellular vesicles (EVs) in parasitic diseases, acting both in parasite–parasite inter-communication as well as in parasite–host interactions. EVs participate in the dissemination of the pathogen and play a role in the regulation of the host immune systems. Production of EVs from parasites or parasitized cells has been described for a number of parasitic infections. In this review, we provide the most relevant findings of the involvement of EVs in intercellular communication, modulation of immune responses, involvement in pathology, and their potential as new diagnostic tools and therapeutic agents in some of the major human parasitic pathogens. PMID:25536932

  20. Mechanisms of amphetamine action illuminated through optical monitoring of dopamine synaptic vesicles in Drosophila brain

    PubMed Central

    Freyberg, Zachary; Sonders, Mark S.; Aguilar, Jenny I.; Hiranita, Takato; Karam, Caline S.; Flores, Jorge; Pizzo, Andrea B.; Zhang, Yuchao; Farino, Zachary J.; Chen, Audrey; Martin, Ciara A.; Kopajtic, Theresa A.; Fei, Hao; Hu, Gang; Lin, Yi-Ying; Mosharov, Eugene V.; McCabe, Brian D.; Freyberg, Robin; Wimalasena, Kandatege; Hsin, Ling-Wei; Sames, Dalibor; Krantz, David E.; Katz, Jonathan L.; Sulzer, David; Javitch, Jonathan A.

    2016-01-01

    Amphetamines elevate extracellular dopamine, but the underlying mechanisms remain uncertain. Here we show in rodents that acute pharmacological inhibition of the vesicular monoamine transporter (VMAT) blocks amphetamine-induced locomotion and self-administration without impacting cocaine-induced behaviours. To study VMAT's role in mediating amphetamine action in dopamine neurons, we have used novel genetic, pharmacological and optical approaches in Drosophila melanogaster. In an ex vivo whole-brain preparation, fluorescent reporters of vesicular cargo and of vesicular pH reveal that amphetamine redistributes vesicle contents and diminishes the vesicle pH-gradient responsible for dopamine uptake and retention. This amphetamine-induced deacidification requires VMAT function and results from net H+ antiport by VMAT out of the vesicle lumen coupled to inward amphetamine transport. Amphetamine-induced vesicle deacidification also requires functional dopamine transporter (DAT) at the plasma membrane. Thus, we find that at pharmacologically relevant concentrations, amphetamines must be actively transported by DAT and VMAT in tandem to produce psychostimulant effects. PMID:26879809

  1. Vesicle trafficking and cell surface membrane patchiness.

    PubMed Central

    Tang, Q; Edidin, M

    2001-01-01

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

  2. Ceramidase Regulates Synaptic Vesicle Exocytosis and Trafficking

    PubMed Central

    Rohrbough, Jeffrey; Rushton, Emma; Palanker, Laura; Woodruff, Elvin; Matthies, Heinrich J. G.; Acharya, Usha; Acharya, Jairaj K.; Broadie, Kendal

    2009-01-01

    A screen for Drosophila synaptic dysfunction mutants identified slug-a-bed (slab). The slab gene encodes ceramidase, a central enzyme in sphingolipid metabolism and regulation. Sphingolipids are major constituents of lipid rafts, membrane domains with roles in vesicle trafficking, and signaling pathways. Null slab mutants arrest as fully developed embryos with severely reduced movement. The SLAB protein is widely expressed in different tissues but enriched in neurons at all stages of development. Targeted neuronal expression of slab rescues mutant lethality, demonstrating the essential neuronal function of the protein. C5-ceramide applied to living preparations is rapidly accumulated at neuromuscular junction (NMJ) synapses dependent on the SLAB expression level, indicating that synaptic sphingolipid trafficking and distribution is regulated by SLAB function. Evoked synaptic currents at slab mutant NMJs are reduced by 50–70%, whereas postsynaptic glutamate-gated currents are normal, demonstrating a specific presynaptic impairment. Hypertonic saline-evoked synaptic vesicle fusion is similarly impaired by 50–70%, demonstrating a loss of readily releasable vesicles. In addition, FM1-43 dye uptake is reduced in slab mutant presynaptic terminals, indicating a smaller cycling vesicle pool. Ultrastructural analyses of mutants reveal a normal vesicle distribution clustered and docked at active zones, but fewer vesicles in reserve regions, and a twofold to threefold increased incidence of vesicles linked together and tethered at the plasma membrane. These results indicate that SLAB ceramidase function controls presynaptic terminal sphingolipid composition to regulate vesicle fusion and trafficking, and thus the strength and reliability of synaptic transmission. PMID:15356190

  3. Concentration-Independent Spontaneously Forming Biomimetric Vesicles

    NASA Astrophysics Data System (ADS)

    Nieh, M.-P.; Harroun, T. A.; Raghunathan, V. A.; Glinka, C. J.; Katsaras, J.

    2003-10-01

    In this Letter we present small-angle neutron scattering data from a biomimetic system composed of the phospholipids dimyristoyl and dihexanoyl phosphorylcholine (DMPC and DHPC, respectively). Doping DMPC-DHPC multilamellar vesicles with either the negatively charged lipid dimyristoyl phosphorylglycerol (DMPG, net charge -1) or the divalent cation, calcium (Ca2+), leads to the spontaneous formation of energetically stabilized monodisperse unilamellar vesicles whose radii are concentration independent and in contrast with previous experimental observations.

  4. Oxidative and other posttranslational modifications in extracellular vesicle biology.

    PubMed

    Szabó-Taylor, Katalin; Ryan, Brent; Osteikoetxea, Xabier; Szabó, Tamás G; Sódar, Barbara; Holub, Marcsilla; Németh, Andrea; Pálóczi, Krisztina; Pállinger, Éva; Winyard, Paul; Buzás, Edit I

    2015-04-01

    Extracellular vesicles including exosomes, microvesicles and apoptotic vesicles, are phospholipid bilayer surrounded structures secreted by cells universally, in an evolutionarily conserved fashion. Posttranslational modifications such as oxidation, citrullination, phosphorylation and glycosylation play diverse roles in extracellular vesicle biology. Posttranslational modifications orchestrate the biogenesis of extracellular vesicles. The signals extracellular vesicles transmit between cells also often function via modulating posttranslational modifications of target molecules, given that extracellular vesicles are carriers of several active enzymes catalysing posttranslational modifications. Posttranslational modifications of extracellular vesicles can also contribute to disease pathology by e.g. amplifying inflammation, generating neoepitopes or carrying neoepitopes themselves.

  5. Differential detergent sensitivity of extracellular vesicle subpopulations.

    PubMed

    Osteikoetxea, Xabier; Sódar, Barbara; Németh, Andrea; Szabó-Taylor, Katalin; Pálóczi, Krisztina; Vukman, Krisztina V; Tamási, Viola; Balogh, Andrea; Kittel, Ágnes; Pállinger, Éva; Buzás, Edit Irén

    2015-10-14

    Extracellular vesicles (including exosomes, microvesicles and apoptotic bodies) are currently attracting rapidly increasing attention from various fields of biology due to their ability to carry complex information and act as autocrine, paracrine and even endocrine intercellular messengers. In the present study we investigated the sensitivity of size-based subpopulations of extracellular vesicles to different concentrations of detergents including sodium dodecyl sulphate, Triton X-100, Tween 20 and deoxycholate. We determined the required detergent concentration that lysed each of the vesicle subpopulations secreted by Jurkat, THP-1, MiaPaCa and U937 human cell lines. We characterized the vesicles by tunable resistive pulse sensing, flow cytometry and transmission electron microscopy. Microvesicles and apoptotic bodies were found to be more sensitive to detergent lysis than exosomes. Furthermore, we found evidence that sodium dodecyl sulphate and Triton X-100 were more effective in vesicle lysis at low concentrations than deoxycholate or Tween 20. Taken together, our data suggest that a combination of differential detergent lysis with tunable resistive pulse sensing or flow cytometry may prove useful for simple and fast differentiation between exosomes and other extracellular vesicle subpopulations as well as between vesicular and non-vesicular structures.

  6. Ciliary Extracellular Vesicles: Txt Msg Organelles.

    PubMed

    Wang, Juan; Barr, Maureen M

    2016-04-01

    Cilia are sensory organelles that protrude from cell surfaces to monitor the surrounding environment. In addition to its role as sensory receiver, the cilium also releases extracellular vesicles (EVs). The release of sub-micron sized EVs is a conserved form of intercellular communication used by all three kingdoms of life. These extracellular organelles play important roles in both short and long range signaling between donor and target cells and may coordinate systemic responses within an organism in normal and diseased states. EV shedding from ciliated cells and EV-cilia interactions are evolutionarily conserved phenomena, yet remarkably little is known about the relationship between the cilia and EVs and the fundamental biology of EVs. Studies in the model organisms Chlamydomonas and Caenorhabditis elegans have begun to shed light on ciliary EVs. Chlamydomonas EVs are shed from tips of flagella and are bioactive. Caenorhabditis elegans EVs are shed and released by ciliated sensory neurons in an intraflagellar transport-dependent manner. Caenorhabditis elegans EVs play a role in modulating animal-to-animal communication, and this EV bioactivity is dependent on EV cargo content. Some ciliary pathologies, or ciliopathies, are associated with abnormal EV shedding or with abnormal cilia-EV interactions. Until the 21st century, both cilia and EVs were ignored as vestigial or cellular junk. As research interest in these two organelles continues to gain momentum, we envision a new field of cell biology emerging. Here, we propose that the cilium is a dedicated organelle for EV biogenesis and EV reception. We will also discuss possible mechanisms by which EVs exert bioactivity and explain how what is learned in model organisms regarding EV biogenesis and function may provide insight to human ciliopathies.

  7. Ciliary extracellular vesicles: Txt msg orgnlls

    PubMed Central

    Wang, Juan; Barr, Maureen M.

    2016-01-01

    Cilia are sensory organelles that protrude from cell surfaces to monitor the surrounding environment. In addition to its role as sensory receiver, the cilium also releases extracellular vesicles (EVs). The release of sub-micron sized EVs is a conserved form of intercellular communication used by all three kingdoms of life. These extracellular organelles play important roles in both short and long range signaling between donor and target cells and may coordinate systemic responses within an organism in normal and diseased states. EV shedding from ciliated cells and EV-cilia interactions are evolutionarily conserved phenomena, yet remarkably little is known about the relationship between the cilia and EVs and the fundamental biology of EVs. Studies in the model organisms Chlamydomonas and C. elegans have begun to shed light on ciliary EVs. Chlamydomonas EVs are shed from tips of flagella and are bioactive. C. elegans EVs are shed and released by ciliated sensory neurons in an intraflagellar transport (IFT)-dependent manner. C. elegans EVs play a role in modulating animal-to-animal communication, and this EV bioactivity is dependent on EV cargo content. Some ciliary pathologies, or ciliopathies, are associated with abnormal EV shedding or with abnormal cilia-EV interactions, suggest the cilium may be an important organelle as an EV donor or as an EV target. Until the past few decades, both cilia and EVs were ignored as vestigial or cellular junk. As research interest in these two organelles continues to gain momentum, we envision a new field of cell biology emerging. Here, we propose that the cilium is a dedicated organelle for EV biogenesis and EV reception. We will also discuss possible mechanisms by which EVs exert bioactivity and explain how what is learned in model organisms regarding EV biogenesis and function may provide insight to human ciliopathies. PMID:26983828

  8. Phosphorylcholine substituted polyolefins: New syntheses, solution assemblies, and polymer vesicles

    NASA Astrophysics Data System (ADS)

    Kratz, Katrina A.

    This thesis describes the synthesis and applications of a new series of amphiphilic homopolymers and copolymers consisting of hydrophobic polyolefin backbone and hydrophilic phosphorylcholine (PC) pendant groups. These polymers are synthesized by ring opening metathesis polymerization (ROMP) of a novel PC- cyclooctene monomer, and copolymerization of various functionalized cyclooctene comonomers. Incorporation of different comonomers into the PC-polyolefin backbone affords copolymers with different functionalities, including crosslinkers, fluorophores, and other reactive groups, that tune the range of applications of these polymers, and their hydrophobic/hydrophilic balance. The amphiphilic nature of PC-polyolefins was exploited in oil-water interfacial assembly, providing robust polymer capsules to encapsulate and deliver nanoparticles to damaged regions of a substrate in a project termed `repair-and-go.' In repair-and-go, a flexible microcapsule filled with a solution of nanoparticles probes an imperfection-riddled substrate as it rolls over the surface. The thin capsule wall allows the nanoparticles to escape the capsules and enter into the cracks, driven in part by favorable interactions between the nanoparticle ligands and the cracked surface (i.e., hydrophobic-hydrophobic interactions). The capsules then continue their transport along the surface, filling more cracks and depositing particles into them. The amphiphilic nature of PC-polyolefins was also exploited in aqueous assembly, forming novel polymer vesicles in water. PC-polyolefin vesicles ranged in size from 50 nm to 30 µm. The mechanical properties of PC-polyolefin vesicles were measured by micropipette aspiration techniques, and found to be more robust than conventional liposomes or polymersomes prepared from block copolymers. PC-polyolefin vesicles have potential use in drug delivery; it was found that the cancer drug doxorubicin could be encapsulated efficiently in PC-polyolefin vesicles. In

  9. The C-Terminal Sequence and PI motif of the Orchid (Oncidium Gower Ramsey) PISTILLATA (PI) Ortholog Determine its Ability to Bind AP3 Orthologs and Enter the Nucleus to Regulate Downstream Genes Controlling Petal and Stamen Formation.

    PubMed

    Mao, Wan-Ting; Hsu, Hsing-Fun; Hsu, Wei-Han; Li, Jen-Ying; Lee, Yung-I; Yang, Chang-Hsien

    2015-11-01

    This study focused on the investigation of the effects of the PI motif and C-terminus of the Oncidium Gower Ramsey MADS box gene 8 (OMADS8), a PISTILLATA (PI) ortholog, on floral organ formation. 35S::OMADS8 completely rescued and 35S::OMADS8-PI (with the PI motif deleted) partially rescued petal/stamen formation, whereas these deficiencies were not rescued by 35S::OMADS8-C (C-terminal 29 amino acids deleted) in pi-1 mutants. OMADS8 could interact with Arabidopsis APETALA3 (AP3) and enter the nucleus. The nuclear entry efficiency was reduced for OMADS8-PI/AP3 and OMADS8-C/AP3. OMADS8 could also interact with OMADS5/OMADS9 (the Oncidium AP3 ortholog) and enter the nucleus with an efficiency only slightly affected by the deletion of the C-terminal sequence or PI motif. However, the stability of the OMADS8/OMADS5 and OMADS8/OMADS9 complexes was significantly reduced by deletion of the C-terminal sequence or PI motif. Further analysis indicated that the expression of genes downstream of AP3/PI (BNQ1/BNQ2/GNC/At4g30270) was compensated by 35S::OMADS8 and 35S::OMADS8-PI to a level similar to wild-type plants but was not affected by 35S::OMADS8-C in the pi-1 mutants. A similar FRET (fluorescence resonance energy transfer) efficiency was observed for Arabidopsis AGAMOUS (AG) and the Oncidium AG ortholog OMADS4 for OMADS8, OMADS8-PI and OMADS8-C. These results indicated that the OMADS8 PI motif and C-terminus were valuable for the interaction of OMADS8 with the AP3 orthologs to form higher order heterotetrameric complexes that regulated petal/stamen formation in both Oncidium orchids and transgenic Arabidopsis. However, the C-terminal sequence and PI motif were dispensable for the interaction of OMADS8 with the AG orthologs.

  10. Sodium dodecyl sulfate/β-cyclodextrin vesicles embedded in chitosan gel for insulin delivery with pH-selective release.

    PubMed

    Li, Zhuo; Li, Haiyan; Wang, Caifen; Xu, Jianghui; Singh, Vikramjeet; Chen, Dawei; Zhang, Jiwen

    2016-07-01

    In an answer to the challenge of enzymatic instability and low oral bioavailability of proteins/peptides, a new type of drug-delivery vesicle has been developed. The preparation, based on sodium dodecyl sulfate (SDS) and β-cyclodextrin (β-CD) embedded in chitosan gel, was used to successfully deliver the model drug-insulin. The self-assembled SDS/β-CD vesicles were prepared and characterized by particle size, zeta potential, appearance, microscopic morphology and entrapment efficiency. In addition, both the interaction of insulin with vesicles and the stability of insulin loaded in vesicles in the presence of pepsin were investigated. The vesicles were crosslinked into thermo-sensitive chitosan/β-glycerol phosphate solution for an in-situ gel to enhance the dilution stability. The in vitro release characteristics of insulin from gels in media at different pH values were investigated. The insulin loaded vesicles-chitosan hydrogel (IVG) improved the dilution stability of the vesicles and provided pH-selective sustained release compared with insulin solution-chitosan hydrogel (ISG). In vitro, IVG exhibited slow release in acidic solution and relatively quick release in neutral solutions to provide drug efficacy. In simulated digestive fluid, IVG showed better sustained release and insulin protection properties compared with ISG. Thus IVG might improve the stability of insulin during its transport in vivo and contribute to the bioavailability and therapeutic effect of insulin.

  11. A two phase field model for tracking vesicle-vesicle adhesion.

    PubMed

    Gu, Rui; Wang, Xiaoqiang; Gunzburger, Max

    2016-11-01

    A multi-phase-field model for simulating the adhesion between two vesicles is constructed. Two phase field functions are introduced to simulate each of the two vesicles. An energy model is defined which accounts for the elastic bending energy of each vesicle and the contact potential energy between the two vesicles; the vesicle volume and surface area constraints are imposed using a penalty method. Numerical results are provided to verify the efficacy of our model and to provide visual illustrations of the different types of contact. The method can be adjusted to solve endocytosis problems by modifying the bending rigidity coefficients of the two elastic bending energies. The method can also be extended to simulate multi-cell adhesions, one example of which is erythrocyte rouleaux. A comparison with laboratory observations demonstrates the effectiveness of the multi-phase field approach.

  12. Microfluidic approaches for isolation, detection, and characterization of extracellular vesicles: Current status and future directions.

    PubMed

    Gholizadeh, Shima; Shehata Draz, Mohamed; Zarghooni, Maryam; Sanati-Nezhad, Amir; Ghavami, Saeid; Shafiee, Hadi; Akbari, Mohsen

    2017-05-15

    Extracellular vesicles (EVs) are cell-derived vesicles present in body fluids that play an essential role in various cellular processes, such as intercellular communication, inflammation, cellular homeostasis, survival, transport, and regeneration. Their isolation and analysis from body fluids have a great clinical potential to provide information on a variety of disease states such as cancer, cardiovascular complications and inflammatory disorders. Despite increasing scientific and clinical interest in this field, there are still no standardized procedures available for the purification, detection, and characterization of EVs. Advances in microfluidics allow for chemical sampling with increasingly high spatial resolution and under precise manipulation down to single molecule level. In this review, our objective is to give a brief overview on the working principle and examples of the isolation and detection methods with the potential to be used for extracellular vesicles. This review will also highlight the integrated on-chip systems for isolation and characterization of EVs.

  13. ON THE POSSIBLE FUNCTION OF COATED VESICLES IN MELANOGENESIS OF THE REGENERATING FOWL FEATHER

    PubMed Central

    Maul, Gerd G.; Brumbaugh, J. A.

    1971-01-01

    How tyrosinase becomes associated with the premelanosomes was investigated by histochemical demonstration of tyrosinase activity by the use of dihydroxyphenylalanine (DOPA) in melanocytes of regenerating fowl feathers. The reaction product of DOPA was localized in the anastomosing membrane tubules associated with the concave side of some dictyosomes of the Golgi apparatus and in coated vesicles most of which were in connection with the dictyosomes. No reaction product was found in early premelanosomes. In premelanosomes, the reaction product of DOPA appears first in vesicles approximately 400 A in diameter which are surrounded by a matrix with a characteristic periodicity. These observations seem to allow the speculation that the coated vesicles function in the transport of tyrosinase, and that the premelanosomes are formed in a process which is not necessarily dependent on the Golgi apparatus as was assumed earlier. PMID:4993485

  14. Exosomes and other extracellular vesicles in neural cells and neurodegenerative diseases.

    PubMed

    Janas, Anna M; Sapoń, Karolina; Janas, Teresa; Stowell, Michael H B; Janas, Tadeusz

    2016-06-01

    The function of human nervous system is critically dependent on proper interneuronal communication. Exosomes and other extracellular vesicles are emerging as a novel form of information exchange within the nervous system. Intraluminal vesicles within multivesicular bodies (MVBs) can be transported in neural cells anterogradely or retrogradely in order to be released into the extracellular space as exosomes. RNA loading into exosomes can be either via an interaction between RNA and the raft-like region of the MVB limiting membrane, or via an interaction between an RNA-binding protein-RNA complex with this raft-like region. Outflow of exosomes from neural cells and inflow of exosomes into neural cells presumably take place on a continuous basis. Exosomes can play both neuro-protective and neuro-toxic roles. In this review, we characterize the role of exosomes and microvesicles in normal nervous system function, and summarize evidence for defective signaling of these vesicles in disease pathogenesis of some neurodegenerative diseases.

  15. Chibby promotes ciliary vesicle formation and basal body docking during airway cell differentiation.

    PubMed

    Burke, Michael C; Li, Feng-Qian; Cyge, Benjamin; Arashiro, Takeshi; Brechbuhl, Heather M; Chen, Xingwang; Siller, Saul S; Weiss, Matthew A; O'Connell, Christopher B; Love, Damon; Westlake, Christopher J; Reynolds, Susan D; Kuriyama, Ryoko; Takemaru, Ken-Ichi

    2014-10-13

    Airway multiciliated epithelial cells play crucial roles in the mucosal defense system, but their differentiation process remains poorly understood. Mice lacking the basal body component Chibby (Cby) exhibit impaired mucociliary transport caused by defective ciliogenesis, resulting in chronic airway infection. In this paper, using primary cultures of mouse tracheal epithelial cells, we show that Cby facilitates basal body docking to the apical cell membrane through proper formation of ciliary vesicles at the distal appendage during the early stages of ciliogenesis. Cby is recruited to the distal appendages of centrioles via physical interaction with the distal appendage protein CEP164. Cby then associates with the membrane trafficking machinery component Rabin8, a guanine nucleotide exchange factor for the small guanosine triphosphatase Rab8, to promote recruitment of Rab8 and efficient assembly of ciliary vesicles. Thus, our study identifies Cby as a key regulator of ciliary vesicle formation and basal body docking during the differentiation of airway ciliated cells.

  16. Role of vesicle tethering factors in the ER-Golgi membrane traffic

    PubMed Central

    Sztul, Elizabeth; Lupashin, Vladimir

    2009-01-01

    Tethers are a diverse group of loosely related proteins and protein complexes grouped into 3 families based on structural and functional similarities. A well-accepted role for tethering factors is the initial attachment of transport carriers to acceptor membranes prior to fusion. However, accumulating evidence indicates that tethers are more than static bridges. Tethers have been shown to interact with components of the fusion machinery and with components involved in vesicle formation. Tethers belonging to the 3 families act at the same stage of traffic, suggesting that they mediate distinct events during vesicle tethering. Thus, multiple tether-facilitated events are required to provide selectivity to vesicle fusion. In this review, we highlight findings that support this model. PMID:19887069

  17. Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network

    PubMed Central

    Klemm, Robin W.; Ejsing, Christer S.; Surma, Michal A.; Kaiser, Hermann-Josef; Gerl, Mathias J.; Sampaio, Julio L.; de Robillard, Quentin; Ferguson, Charles; Proszynski, Tomasz J.; Shevchenko, Andrej

    2009-01-01

    The trans-Golgi network (TGN) is the major sorting station in the secretory pathway of all eukaryotic cells. How the TGN sorts proteins and lipids to generate the enrichment of sphingolipids and sterols at the plasma membrane is poorly understood. To address this fundamental question in membrane trafficking, we devised an immunoisolation procedure for specific recovery of post-Golgi secretory vesicles transporting a transmembrane raft protein from the TGN to the cell surface in the yeast Saccharomyces cerevisiae. Using a novel quantitative shotgun lipidomics approach, we could demonstrate that TGN sorting selectively enriched ergosterol and sphingolipid species in the immunoisolated secretory vesicles. This finding, for the first time, indicates that the TGN exhibits the capacity to sort membrane lipids. Furthermore, the observation that the immunoisolated vesicles exhibited a higher membrane order than the late Golgi membrane, as measured by C-Laurdan spectrophotometry, strongly suggests that lipid rafts play a role in the TGN-sorting machinery. PMID:19433450

  18. Tube extrusion from permeabilized giant vesicles

    NASA Astrophysics Data System (ADS)

    Borghi, N.; Kremer, S.; Askovic, V.; Brochard-Wyart, F.

    2006-08-01

    This letter reports the permeabilization effects of chemical additives on mechanical properties of Giant Unilamellar Vesicles (GUVs). We use a surfactant, Tween 20, inducing transient pores and a protein, Streptolysin O, inducing permanent pores in the membrane. Lipid tubes are extracted from GUVs anchored onto the tip of a micro-needle and submitted to hydrodynamic flows. On bare vesicles, tube extrusion is governed by the entropic elasticity of the membrane. The vesicle tension increases until it balances the flow velocity U and the tube reaches a stationary length. In permeabilized vesicles, the membrane tension is maintained at a constant value σp by the permeation of inner solution through nanometric pores. This allows extrusion of "infinite" tubes at constant velocity that never reach a stationary length. Tween-20 preliminary results suggest that σp strongly depends on surfactant concentration. For Streptolysin O, we have measured σp vs. U and found two regimes: a "high-porosity" regime for U > Up0 and a "low-porosity" regime for U < Up0, where Up0 is related to the number of pores on the vesicle surface.

  19. Getting to know the extracellular vesicle glycome.

    PubMed

    Gerlach, Jared Q; Griffin, Matthew D

    2016-04-01

    Extracellular vesicles (EVs) are a diverse population of complex biological particles with diameters ranging from approximately 20 to 1000 nm. Tremendous interest in EVs has been generated following a number of recent, high-profile reports describing their potential utility in diagnostic, prognostic, drug delivery, and therapeutic roles. Subpopulations, such as exosomes, are now known to directly participate in cell-cell communication and direct material transfer. Glycomics, the 'omic' portion of the glycobiology field, has only begun to catalog the surface oligosaccharide and polysaccharide structures and also the carbohydrate-binding proteins found on and inside EVs. The EV glycome undoubtedly contains vital clues essential to better understanding the function, biogenesis, release and transfer of vesicles, however getting at this information is technically challenging and made even more so because of the small physical size of the vesicles and the typically minute yield from physiological-scale biological samples. Vesicle micro-heterogeneity which may be related to specific vesicle origins and functions presents a further challenge. A number of primary studies carried out over the past decade have turned up specific and valuable clues regarding the composition and roles of glycan structures and also glycan binding proteins involved EV biogenesis and transfer. This review explores some of the major EV glycobiological research carried out to date and discusses the potential implications of these findings across the life sciences.

  20. 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

  1. Lysozyme binding onto cat-anionic vesicles.

    PubMed

    Bonincontro, A; Spigone, E; Ruiz Peña, M; Letizia, C; La Mesa, C

    2006-12-15

    Mixing aqueous sodium dodecylsulfate with cetyltrimethylammonium bromide solutions in mole ratios close to (1.7/1.0) allows the formation of cat-anionic vesicles with an excess of negative charges on the outer surface. The vesicular dispersions are mixed with lysozyme, and interact electrostatically with the positive charges on the protein, forming lipo-plexes. Dielectric relaxation, zeta-potential, and light scattering indicate the occurrence of interactions between vesicles and the protein. According to CD, the vesicle-adsorbed protein retains its native conformation. Binding and surface saturation, inferred by dielectric relaxation and zeta-potential, fulfil a charge neutralisation stoichiometry. Adsorbed lysozyme promotes the vesicle clustering and is concomitant with the lipo-plexes flocculation. Above the charge neutralisation threshold, lysozyme in excess remains dispersed in molecular form. Attempts were made to determine in what conditions protein release from the vesicles occurs. Accordingly, the full neutralisation of sodium dodecylsulfate in excess by cetyltrimethylammonium bromide ensures the lipo-plexes break-up, the precipitation of the mixed surfactants and the protein release in native form.

  2. Activation of calcineurin by phosphotidylserine containing vesicles

    SciTech Connect

    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% PS 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.

  3. 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

  4. Specific Stimulated Uptake of Acetylcholine by Torpedo Electric Organ Synaptic Vesicles

    NASA Astrophysics Data System (ADS)

    Parsons, Stanley M.; Koenigsberger, Robert

    1980-10-01

    The specificity of acetylcholine uptake by synaptic vesicles isolated from the electric organ of Torpedo californica was studied. In the absence of cofactors, [3H]acetylcholine was taken up identically to [14C]choline in the same solution (passive uptake), and the equilibrium concentration achieved inside the vesicles was equal to the concentration outside. In the presence of MgATP, [3H]acetylcholine and [14C]choline in the same solution were taken up identically, except only about half as much of each was taken up (suppressed uptake). [3H]Acetylcholine uptake was stimulated by MgATP and HCO3 about 4-fold relative to suppressed uptake, for a net concentrative uptake of about 2:1 (stimulated uptake). Uptake of [14C]choline in the same solution remained at the suppressed level. [3H]Acetylcholine taken up under stimulated conditions migrated with vesicles containing [14C]mannitol on analytical glycerol density gradients during centrifugation. Vesicles were treated with nine protein modification reagents under mild conditions. Two reagents had no effect on, dithiothreitol potentiated, and six reagents strongly inhibited subsequent stimulated uptake of [3H]acetylcholine. The results indicate that uptake of acetylcholine is conditionally specific for the transported substrate, is carried out by the synaptic vesicles rather than a contaminant of the preparation, and requires a functional protein system containing a critical sulfhydryl group.

  5. Response of unilamellar DPPC and DPPC:SM vesicles to hypo and hyper osmotic shocks: A comparison.

    PubMed

    Ahumada, M; Calderon, C; Alvarez, C; Lanio, M E; Lissi, E A

    2015-05-01

    DPPC and DPPC:SM large unilamellar vesicles (LUVs), prepared by extrusion, readily respond to osmotic shocks (hypo- and hyper-osmotic) by water influx/efflux (evaluated by changes in turbidity) and by entrapped calcein liberation (measured by an increase in dye fluorescence intensity). On the other hand, small unilamellar vesicles (SUVs) prepared by sonication are almost osmotically insensitive. LUVs water transport, both in hypo- and hyper-osmotic conditions, takes place faster than calcein ejection towards the external solvent. Similarly, response to a hypotonic imbalance is faster than that associated to a hypertonic stress. This difference is particularly noticeable for the increase in calcein fluorescence intensity and can be related to the large reorganization of the bilayer needed to form pores and/or to adsorb the dye to the inner leaflet of the vesicle after water efflux. Conversely, addition of SM to the vesicles barely modify the rate of calcein permeation across the bilayer.

  6. Carbon Nanotubes Mediate Fusion of Lipid Vesicles.

    PubMed

    Bhaskara, Ramachandra M; Linker, Stephanie M; Vögele, Martin; Köfinger, Jürgen; Hummer, Gerhard

    2017-02-28

    The fusion of lipid membranes is opposed by high energetic barriers. In living organisms, complex protein machineries carry out this biologically essential process. Here we show that membrane-spanning carbon nanotubes (CNTs) can trigger spontaneous fusion of small lipid vesicles. In coarse-grained molecular dynamics simulations, we find that a CNT bridging between two vesicles locally perturbs their lipid structure. Their outer leaflets merge as the CNT pulls lipids out of the membranes, creating an hourglass-shaped fusion intermediate with still intact inner leaflets. As the CNT moves away from the symmetry axis connecting the vesicle centers, the inner leaflets merge, forming a pore that completes fusion. The distinct mechanism of CNT-mediated membrane fusion may be transferable, providing guidance in the development of fusion agents, e.g., for the targeted delivery of drugs or nucleic acids.

  7. Dynamics of fibers growing inside soft vesicles

    NASA Astrophysics Data System (ADS)

    Marenduzzo, D.; Orlandini, E.

    2007-11-01

    We present 3D stochastic dynamic simulations of the growth of a semiflexible polymer inside a soft vesicle. We find that very stiff fibers stall soon and lock the membrane into a strongly deformed prolate shape. Fibers of intermediate stiffness buckle and form a toroidal configuration which distorts the membrane into an oblate shape. Finally, more flexible polymers form massive spool-like condensates with ordered domains, while the vesicle inflates isotropically. We discuss our results with respect to observations on cell shape in sickle red blood cells, developing erythrocytes, and genome packing inside bacteriophages. We quantify how the force felt by the fiber tip, and the vesicle aspect ratio, change during growth, and we discuss possible "synthetic biology" experiments to validate our results.

  8. Variable priming of a docked synaptic vesicle

    PubMed Central

    Jung, Jae Hoon; Szule, Joseph A.; Marshall, Robert M.; McMahan, Uel J.

    2016-01-01

    The priming of a docked synaptic vesicle determines the probability of its membrane (VM) fusing with the presynaptic membrane (PM) when a nerve impulse arrives. To gain insight into the nature of priming, we searched by electron tomography for structural relationships correlated with fusion probability at active zones of axon terminals at frog neuromuscular junctions. For terminals fixed at rest, the contact area between the VM of docked vesicles and PM varied >10-fold with a normal distribution. There was no merging of the membranes. For terminals fixed during repetitive evoked synaptic transmission, the normal distribution of contact areas was shifted to the left, due in part to a decreased number of large contact areas, and there was a subpopulation of large contact areas where the membranes were hemifused, an intermediate preceding complete fusion. Thus, fusion probability of a docked vesicle is related to the extent of its VM–PM contact area. For terminals fixed 1 h after activity, the distribution of contact areas recovered to that at rest, indicating the extent of a VM–PM contact area is dynamic and in equilibrium. The extent of VM–PM contact areas in resting terminals correlated with eccentricity in vesicle shape caused by force toward the PM and with shortness of active zone material macromolecules linking vesicles to PM components, some thought to include Ca2+ channels. We propose that priming is a variable continuum of events imposing variable fusion probability on each vesicle and is regulated by force-generating shortening of active zone material macromolecules in dynamic equilibrium. PMID:26858418

  9. Elastic energy of polyhedral bilayer vesicles

    NASA Astrophysics Data System (ADS)

    Haselwandter, Christoph A.; Phillips, Rob

    2011-06-01

    In recent experiments [M. Dubois, B. Demé, T. Gulik-Krzywicki, J.-C. Dedieu, C. Vautrin, S. Désert, E. Perez, and T. Zemb, Nature (London)NATUAS0028-083610.1038/35079541 411, 672 (2001)] the spontaneous formation of hollow bilayer vesicles with polyhedral symmetry has been observed. On the basis of the experimental phenomenology it was suggested [M. Dubois, V. Lizunov, A. Meister, T. Gulik-Krzywicki, J. M. Verbavatz, E. Perez, J. Zimmerberg, and T. Zemb, Proc. Natl. Acad. Sci. USAPNASA60027-842410.1073/pnas.0400837101 101, 15082 (2004)] 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.

  10. Assets of the non-pathogenic microorganism Dictyostelium discoideum as a model for the study of eukaryotic extracellular vesicles.

    PubMed

    Tatischeff, Irène

    2013-01-01

    Dictyostelium discoideum microvesicles have recently been presented as a valuable model for eukaryotic extracellular vesicles. Here, the advantages of D. discoideum for unraveling important biological functions of extracellular vesicles in general are detailed. D. discoideum, a non-pathogenic eukaryotic microorganism, belongs to a billion-year-old Amoeboza lineage, which diverged from the animal-fungal lineage after the plant animal-split. During growth and early starvation-induced development, it presents analogies with lymphocytes and macrophages with regard to motility and phagocytosis capability, respectively. Its 6-chromosome genome codes for about 12,500 genes, some showing analogies with human genes. The presence of extracellular vesicles during cell growth has been evidenced as a detoxification mechanism of various structurally unrelated drugs. Controls led to the discovery of constitutive extracellular vesicle secretion in this microorganism, which was an important point. It means that the secretion of extracellular vesicles occurs, in the absence of any drug, during both cell growth and early development. This constitutive secretion of D. discoideum cells is very likely to play a role in intercellular communication. The detoxifying secreted vesicles, which can transport drugs outside the cells, can also act as "Trojan horses", capable of transferring these drugs not only into naïve D. discoideum cells, but into human cells as well. Therefore, these extracellular vesicles were proposed as a new biological drug delivery tool. Moreover, Dictyostelium, chosen by the NIH (USA) as a new model organism for biomedical research, has already been used for studying some human diseases. These cells, which are much easier to manipulate than human cells, can be easily designed in simple conditioned medium experiments. Owing to the increasing consensus that extracellular vesicles are probably important mediators of intercellular communication, D. discoideum is here

  11. Assets of the non-pathogenic microorganism Dictyostelium discoideum as a model for the study of eukaryotic extracellular vesicles.

    PubMed

    Tatischeff, Irène

    2013-03-04

    Dictyostelium discoideum microvesicles have recently been presented as a valuable model for eukaryotic extracellular vesicles. Here, the advantages of D. discoideum for unraveling important biological functions of extracellular vesicles in general are detailed. D. discoideum, a non-pathogenic eukaryotic microorganism, belongs to a billion-year-old Amoeboza lineage, which diverged from the animal-fungal lineage after the plant animal-split. During growth and early starvation-induced development, it presents analogies with lymphocytes and macrophages with regard to motility and phagocytosis capability, respectively. Its 6-chromosome genome codes for about 12,500 genes, some showing analogies with human genes. The presence of extracellular vesicles during cell growth has been evidenced as a detoxification mechanism of various structurally unrelated drugs. Controls led to the discovery of constitutive extracellular vesicle secretion in this microorganism, which was an important point. It means that the secretion of extracellular vesicles occurs, in the absence of any drug, during both cell growth and early development. This constitutive secretion of D. discoideum cells is very likely to play a role in intercellular communication. The detoxifying secreted vesicles, which can transport drugs outside the cells, can also act as "Trojan horses", capable of transferring these drugs not only into naïve D. discoideum cells, but into human cells as well. Therefore, these extracellular vesicles were proposed as a new biological drug delivery tool. Moreover, Dictyostelium, chosen by the NIH (USA) as a new model organism for biomedical research, has already been used for studying some human diseases. These cells, which are much easier to manipulate than human cells, can be easily designed in simple conditioned medium experiments. Owing to the increasing consensus that extracellular vesicles are probably important mediators of intercellular communication, D. discoideum is here

  12. Toroidal membrane vesicles in spherical confinement.

    PubMed

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

    2015-09-01

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

  13. Toroidal membrane vesicles in spherical confinement

    NASA Astrophysics Data System (ADS)

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

    2015-09-01

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

  14. Asymmetric Requirements for a Rab Gtpase and Snare Proteins in Fusion of Copii Vesicles with Acceptor Membranes

    PubMed Central

    Cao, Xiaochun; Barlowe, Charles

    2000-01-01

    Soluble NSF attachment protein receptor (SNARE) proteins are essential for membrane fusion in transport between the yeast ER and Golgi compartments. Subcellular fractionation experiments demonstrate that the ER/Golgi SNAREs Bos1p, Sec22p, Bet1p, Sed5p, and the Rab protein, Ypt1p, are distributed similarly but localize primarily with Golgi membranes. All of these SNARE proteins are efficiently packaged into COPII vesicles and suggest a dynamic cycling of SNARE machinery between ER and Golgi compartments. Ypt1p is not efficiently packaged into vesicles under these conditions. To determine in which membranes protein function is required, temperature-sensitive alleles of BOS1, BET1, SED5, SLY1, and YPT1 that prevent ER/Golgi transport in vitro at restrictive temperatures were used to selectively inactivate these gene products on vesicles or on Golgi membranes. Vesicles bearing mutations in Bet1p or Bos1p inhibit fusion with wild-type acceptor membranes, but acceptor membranes containing these mutations are fully functional. In contrast, vesicles bearing mutations in Sed5p, Sly1p, or Ypt1p are functional, whereas acceptor membranes containing these mutations block fusion. Thus, this set of SNARE proteins is symmetrically distributed between vesicle and acceptor compartments, but they function asymmetrically such that Bet1p and Bos1p are required on vesicles and Sed5p activity is required on acceptor membranes. We propose the asymmetry in SNARE protein function is maintained by an asymmetric distribution and requirement for the Ypt1p GTPase in this fusion event. When a transmembrane-anchored form of Ypt1p is used to restrict this GTPase to the acceptor compartment, vesicles depleted of Ypt1p remain competent for fusion. PMID:10747087

  15. A small pool of vesicles maintains synaptic activity in vivo

    PubMed Central

    Denker, Annette; Bethani, Ioanna; Kröhnert, Katharina; Körber, Christoph; Horstmann, Heinz; Wilhelm, Benjamin G.; Barysch, Sina V.; Kuner, Thomas; Neher, Erwin; Rizzoli, Silvio O.

    2011-01-01

    Chemical synapses contain substantial numbers of neurotransmitter-filled synaptic vesicles, ranging from approximately 100 to many thousands. The vesicles fuse with the plasma membrane to release neurotransmitter and are subsequently reformed and recycled. Stimulation of synapses in vitro generally causes the majority of the synaptic vesicles to release neurotransmitter, leading to the assumption that synapses contain numerous vesicles to sustain transmission during high activity. We tested this assumption by an approach we termed cellular ethology, monitoring vesicle function in behaving animals (10 animal models, nematodes to mammals). Using FM dye photooxidation, pHluorin imaging, and HRP uptake we found that only approximately 1–5% of the vesicles recycled over several hours, in both CNS synapses and neuromuscular junctions. These vesicles recycle repeatedly, intermixing slowly (over hours) with the reserve vesicles. The latter can eventually release when recycling is inhibited in vivo but do not seem to participate under normal activity. Vesicle recycling increased only to ≈5% in animals subjected to an extreme stress situation (frog predation on locusts). Synapsin, a molecule binding both vesicles and the cytoskeleton, may be a marker for the reserve vesicles: the proportion of vesicles recycling in vivo increased to 30% in synapsin-null Drosophila. We conclude that synapses do not require numerous reserve vesicles to sustain neurotransmitter release and thus may use them for other purposes, examined in the accompanying paper. PMID:21903928

  16. Functions of Cancer-Derived Extracellular Vesicles in Immunosuppression.

    PubMed

    Czernek, Liliana; Düchler, Markus

    2017-01-18

    Extracellular vesicles, including exosomes, constitute an important element of intercellular communication by carrying a variety of molecules from producer to target cells. The transport of mRNA and miRNA can directly modulate gene expression in the target cells. The miRNA content in exosomes is characteristic for the cell from which the vesicles were derived enabling the usage of exosomes as biomarkers for the diagnosis various diseases, including cancer. Cancer-derived exosomes support the survival and progression of tumors in many ways and also contribute to the neutralization of the anti-cancer immune response. Exosomes participate in all known mechanisms by which cancer evades the immune system. They influence the differentiation and activation of immune suppressor cells, they modulate antigen presentation, and are able to induce T-cell apoptosis. Although cancer-derived exosomes mainly suppress the immune system and facilitate tumor progression, they are also important sources of tumor antigens with potential clinical application in stimulating immune responses. This review summarizes how exosomes assist cancer to escape immune recognition and to acquire control over the immune system.

  17. Preparation of vesicles entrapped lycopene extract.

    PubMed

    Luxsuwong, Dhitaree; Indranupakorn, Ratana; Wongtrakul, Paveena

    2014-01-01

    Lycopene, a lipophilic carotenoid, has been known as an effective antioxidant in supporting the cutaneous defensive system. However, it is unstable when exposed to light and water. In this study, lycopene was isolated from tomatoes and a vesicular delivery system was developed to entrap and stabilize the lycopene in the aqueous system. A simple process, maceration in ethyl acetate, was used to extract lycopene from the tomatoes. The extract was then chromatographed on the Sephadex LH20 column using acetone as a solvent system to yield 995 μg of lycopene per gram of dried tomato weight. The vesicular delivery system was prepared from a combination of ascorbic acid-6-palmitate (AP), cholesterol and dicetyl phosphate using a thin film hydration method. The formulation was composed of AP, cholesterol and dicetyl phosphate at a 44:44:12 molar ratio and with 2.12 μmol/ml of the isolated lycopene. Both blank vesicles and lycopene loaded vesicles were kept for a period of 3 months at 4±2°C and at the room temperature (28±2°C) to evaluate the effect of the encapsulation on the characteristic of the vesicles and on the antioxidant activity of the encapsulated lycopene. The result implied that lycopene could be stabilized in the vesicles and its scavenging activity against DPPH free radicals was superior to that of the free lycopene solution.

  18. Matrix vesicles: Are they anchored exosomes?

    PubMed

    Shapiro, Irving M; Landis, William J; Risbud, Makarand V

    2015-10-01

    Numerous studies have documented that matrix vesicles are unique extracellular membrane-bound microparticles that serve as initial sites for mineral formation in the growth plate and most other vertebrate mineralizing tissues. Microparticle generation is not confined to hard tissues, as cells in soft tissues generate similar structures; numerous studies have shown that a common type of extracellular particle, termed an exosome, a product of the endosomal pathway, shares many characteristics of matrix vesicles. Indeed, analyses of size, morphology and lipid and protein content indicate that matrix vesicles and exosomes are homologous structures. Such a possibility impacts our understanding of the biogenesis, processing and function of matrix vesicles (exosomes) in vertebrate hard tissues and explains in part how cells control the earliest stages of mineral deposition. Moreover, since exosomes influence a spectrum of functions, including cell-cell communication, it is suggested that this type of microparticle may provide a mechanism for the transfer of signaling molecules between cells within the growth plate and thereby regulate endochondral bone development and formation.

  19. 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.

  20. Vesicle dynamics in shear and capillary flows

    NASA Astrophysics Data System (ADS)

    Noguchi, Hiroshi; Gompper, Gerhard

    2005-11-01

    The deformation of vesicles in flow is studied by a mesoscopic simulation technique, which combines multi-particle collision dynamics for the solvent with a dynamically triangulated surface model for the membrane. Shape transitions are investigated both in simple shear flows and in cylindrical capillary flows. We focus on reduced volumes, where the discocyte shape of fluid vesicles is stable, and the prolate shape is metastable. In simple shear flow at low membrane viscosity, the shear induces a transformation from discocyte to prolate with increasing shear rate, while at high membrane viscosity, the shear induces a transformation from prolate to discocyte, or tumbling motion accompanied by oscillations between these two morphologies. In capillary flow, at small flow velocities the symmetry axis of the discocyte is found not to be oriented perpendicular to the cylinder axis. With increasing flow velocity, a transition to a prolate shape occurs for fluid vesicles, while vesicles with shear-elastic membranes (like red blood cells) transform into a coaxial parachute-like shape.

  1. Dihydrolipoyl dioleoylglycerol antioxidant capacity in phospholipid vesicles.

    PubMed

    Laszlo, Joseph A; Evans, Kervin O; Compton, David L; Appell, Michael

    2012-02-01

    Antioxidants have critical roles in maintaining cellular homeostasis and disease-state prevention. The multi-functional agent α-lipoic acid offers numerous beneficial effects to oxidatively stressed tissues. α-Lipoic acid was enzymatically incorporated into a triglyceride in conjunction with oleic acid, creating lipoyl dioleoylglycerol, and chemically reduced to form dihydrolipoyl dioleoylglycerol. The triglyceride forms of lipoic acid stabilized dioleoylphosphatidylcholine unilamellar liposomal vesicles, as judged by calcein-cobalt leakage. Stabilization resulted from increased packing density of phospholipid acyl chains. Scavenging activity against the 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) radical was monitored by oxidation of 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C(11)-Bodipy). Dihydrolipoyl dioleoylglycerol in vesicles demonstrated strong antioxidant capacity in comparison to the conventional Trolox standard. Fluorescence quenching measurements indicated the lipoyl moiety of dihydrolipoyl dioleoylglycerol is positioned near the vesicle aqueous/lipid boundary. Treatment of intact vesicles with a nonpenetrating sulfhydryl reagent indicated that 80% of the dihydrolipoyl dioleoylglycerol was available for reaction. Molecular modeling of lipoyl dioleoylglycerol and dihydrolipoyl dioleoylglycerol in a phospholipid layer confirmed the existence of an extended configuration for the molecules that accounts for the interfacial location of the lipoyl moiety, which may allow the antioxidant to readily react with radical species approaching membranes from the aqueous phase.

  2. Vesicle Pools: Lessons from Adrenal Chromaffin Cells

    PubMed Central

    Stevens, David R.; Schirra, Claudia; Becherer, Ute; Rettig, Jens

    2011-01-01

    The adrenal chromaffin cell serves as a model system to study fast Ca2+-dependent exocytosis. Membrane capacitance measurements in combination with Ca2+ uncaging offers a temporal resolution in the millisecond range and reveals that catecholamine release occurs in three distinct phases. Release of a readily releasable (RRP) and a slowly releasable (SRP) pool are followed by sustained release, due to maturation, and release of vesicles which were not release-ready at the start of the stimulus. Trains of depolarizations, a more physiological stimulus, induce release from a small immediately releasable pool of vesicles residing adjacent to calcium channels, as well as from the RRP. The SRP is poorly activated by depolarization. A sequential model, in which non-releasable docked vesicles are primed to a slowly releasable state, and then further mature to the readily releasable state, has been proposed. The docked state, dependent on membrane proximity, requires SNAP-25, synaptotagmin, and syntaxin. The ablation or modification of SNAP-25 and syntaxin, components of the SNARE complex, as well as of synaptotagmin, the calcium sensor, and modulators such complexins and Snapin alter the properties and/or magnitudes of different phases of release, and in particular can ablate the RRP. These results indicate that the composition of the SNARE complex and its interaction with modulatory molecules drives priming and provides a molecular basis for different pools of releasable vesicles. PMID:21423410

  3. Vesicle deformation by microtubules: A phase diagram

    NASA Astrophysics Data System (ADS)

    Emsellem, Virginie; Cardoso, Olivier; Tabeling, Patrick

    1998-10-01

    The experimental investigation of vesicles deformed by the growth of encapsulated microtubules shows that the axisymmetric morphologies can be classified into ovals, lemons, φ, cherries, dumbbells, and pearls. A geometrical phase diagram is established. Numerical minimization of the elastic energy of the membrane reproduces satisfactorily well the observed morphologies and the corresponding phase diagram.

  4. Amino Acid Neurotransmitters; Mechanisms of Their Uptake into Synaptic Vesicles

    DTIC Science & Technology

    1991-08-01

    same neuron, at least from the cerebellar Golgi cell terminals. It should be kept in mind that the uptake of noradrenaline and dopamine in synaptic...vesicles prepared from rat brain is relatively non-specific. Noradrenaline containing vesicles can take up noradrenaline, dopamine and serotonin. In...also shown that the vesicles isolated from corpus striatum exhibited the same ratio of uptake of dopamine /noradrenaline as did vesicles from cerebral

  5. Coupling a mechanosensitive channel with a vesicle under shear flow

    NASA Astrophysics Data System (ADS)

    Pak, On Shun; Young, Yuan Nan; Veerapaneni, Shravan; Stone, Howard

    2014-11-01

    Mechanosensitive channels enable cells to respond to their local environment. Continuum mechanical models have been proposed to describe how bilayer deformation induced by the transmembrane protein and the membrane tension influence the free energy of channel gating under static conditions. The dynamics of mechanosensitive channels under flow conditions however remains largely unexplored. Cells under flow display interesting features not observed under static environments. Here we present a model coupling a mechanosensitive channel with the dynamics of a vesicle under shear flow to investigate how the channel gating responds to hydrodynamic stress. The model could be used to investigate the release of signaling molecules, transport of ions or drugs across cell membranes under flow in biological systems, as well as the design and control of channel gating in synthetic cells.

  6. Mechanisms, pools, and sites of spontaneous vesicle release at synapses of rod and cone photoreceptors.

    PubMed

    Cork, Karlene M; Van Hook, Matthew J; Thoreson, Wallace B

    2016-08-01

    Photoreceptors have depolarized resting potentials that stimulate calcium-dependent release continuously from a large vesicle pool but neurons can also release vesicles without stimulation. We characterized the Ca(2+) dependence, vesicle pools, and release sites involved in spontaneous release at photoreceptor ribbon synapses. In whole-cell recordings from light-adapted horizontal cells (HCs) of tiger salamander retina, we detected miniature excitatory post-synaptic currents (mEPSCs) when no stimulation was applied to promote exocytosis. Blocking Ca(2+) influx by lowering extracellular Ca(2+) , by application of Cd(2+) and other agents reduced the frequency of mEPSCs but did not eliminate them, indicating that mEPSCs can occur independently of Ca(2+) . We also measured release presynaptically from rods and cones by examining quantal glutamate transporter anion currents. Presynaptic quantal event frequency was reduced by Cd(2+) or by increased intracellular Ca(2+) buffering in rods, but not in cones, that were voltage clamped at -70 mV. By inhibiting the vesicle cycle with bafilomycin, we found the frequency of mEPSCs declined more rapidly than the amplitude of evoked excitatory post-synaptic currents (EPSCs) suggesting a possible separation between vesicle pools in evoked and spontaneous exocytosis. We mapped sites of Ca(2+) -independent release using total internal reflectance fluorescence (TIRF) microscopy to visualize fusion of individual vesicles loaded with dextran-conjugated pHrodo. Spontaneous release in rods occurred more frequently at non-ribbon sites than evoked release events. The function of Ca(2+) -independent spontaneous release at continuously active photoreceptor synapses remains unclear, but the low frequency of spontaneous quanta limits their impact on noise.

  7. Acidification of endocytic vesicles by an ATP-dependent proton pump

    PubMed Central

    1983-01-01

    One of the early events in the pathway of receptor-mediated endocytosis is the acidification of the newly formed endocytic vesicle. To examine the mechanism of acidification, we used fluorescein-labeled alpha 2- macroglobulin (F-alpha 2M) as a probe for endocytic vesicle pH. Changes in pH were determined from the change in fluorescein fluorescence at 490-nm excitation as measured with a microscope spectrofluorometer. After endocytosis of F-alpha 2M, mouse fibroblast cells were permeabilized by brief exposure to the detergent digitonin. Treatment with the ionophore monensin or the protonophore carbonyl cyanide p- trifluoromethoxyphenylhydrazone (FCCP) caused a rapid increase in the pH of the endocytic vesicle. Upon removal of the ionophore, the endocytic vesicle rapidly acidified only when MgATP or MgGTP was added. Neither ADP nor the nonhydrolyzable analog, adenosine 5'-(beta, gamma- imido)triphosphate (AMP-PNP) could support acidification. The ATP- dependent acidification did not require a specific cation or anion in the external media. Acidification was insensitive to vanadate and amiloride but was inhibited by Zn2+ and the anion transport inhibitor diisothiocyanostilbene disulfonic acid (DIDS). We also examined the acidification of lysosomes with the permeabilized cell system, using fluorescein isothiocyanate dextran as probe. DIDS inhibited the ATP- dependent reacidification of lysosomes, although at a lower concentration than that for inhibition of endocytic vesicle reacidification. These results demonstrate that endocytic vesicles contain an ATP-dependent acidification mechanism that shares similar characteristics with the previously described lysosomal proton pump. PMID:6224803

  8. In vitro fusion between Saccharomyces cerevisiae secretory vesicles and cytoplasmic-side-out plasma membrane vesicles.

    PubMed Central

    Arrastua, Lorena; San Sebastian, Eider; Quincoces, Ana F; Antony, Claude; Ugalde, Unai

    2003-01-01

    The final step in the secretory pathway, which is the fusion event between secretory vesicles and the plasma membrane, was reconstructed using highly purified secretory vesicles and cytoplasmic-side-out plasma membrane vesicles from the yeast Saccharomyces cerevisiae. Both organelle preparations were obtained from a sec 6-4 temperature-sensitive mutant. Fusion was monitored by means of a fluorescence assay based on the dequenching of the lipophilic fluorescent probe octadecylrhodamine B-chloride (R18). The probe was incorporated into the membrane of secretory vesicles, and it diluted in unlabelled cytoplasmic-side-out plasma membrane vesicles as the fusion process took place. The obtained experimental dequenching curves were found by mathematical analysis to consist of two independent but simultaneous processes. Whereas one of them reflected the fusion process between both vesicle populations as confirmed by its dependence on the assay conditions, the other represented a non-specific transfer of the probe. The fusion process may now be examined in detail using the preparation, validation and analytical methods developed in this study. PMID:12435271

  9. Physicochemical characterization of drug-loaded rigid and elastic vesicles.

    PubMed

    Uchino, Tomonobu; Lefeber, Fons; Gooris, Gert; Bouwstra, Joke

    2011-06-30

    Ketorolac loaded rigid and elastic vesicles were prepared by sonication and the physicochemical properties of the drug loaded-vesicle formulations were examined. Rigid and elastic vesicles were prepared from the double chain surfactant sucrose-ester laurate (L-595) and the single chain surfactant octaoxyethylene-laurate ester (PEG-8-L). Sulfosuccinate (TR-70) was used as a negative charge inducer. Evaluation of the prepared vesicle was performed by dynamic light scattering, extrusion and by (1)H NMR (T(2) relaxation studies). The vesicles mean size varied between 90 and 150 nm. The elasticity of the vesicles was enhanced with increasing PEG-8-L/L-595 ratio, while an increase in loading of ketorolac resulted in a reduction in vesicle elasticity. (1)H NMR measurements showed that the molecular mobility of ketorolac was restricted, which indicates that ketorolac molecules were entrapped within the vesicle bilayers. The T(2) values of the aromatic protons of ketorolac increased gradually at higher PEG-8-L levels, indicating that ketorolac mobility increased in the vesicle bilayer. The chemical stability of ketorolac was dramatically improved in the vesicle formulation compared to a buffer solution. The strong interactions of ketorolac with the bilayers of the vesicles might be the explanation for this increased stability of ketorolac.

  10. Surface degassing and modifications to vesicle size distributions in active basalt flows

    USGS Publications Warehouse

    Cashman, K.V.; Mangan, M.T.; Newman, S.

    1994-01-01

    The character of the vesicle population in lava flows includes several measurable parameters that may provide important constraints on lava flow dynamics and rheology. Interpretation of vesicle size distributions (VSDs), however, requires an understanding of vesiculation processes in feeder conduits, and of post-eruption modifications to VSDs during transport and emplacement. To this end we collected samples from active basalt flows at Kilauea Volcano: (1) near the effusive Kupaianaha vent; (2) through skylights in the approximately isothermal Wahaula and Kamoamoa tube systems transporting lava to the coast; (3) from surface breakouts at different locations along the lava tubes; and (4) from different locations in a single breakout from a lava tube 1 km from the 51 vent at Pu'u 'O'o. Near-vent samples are characterized by VSDs that show exponentially decreasing numbers of vesicles with increasing vesicle size. These size distributions suggest that nucleation and growth of bubbles were continuous during ascent in the conduit, with minor associated bubble coalescence resulting from differential bubble rise. The entire vesicle population can be attributed to shallow exsolution of H2O-dominated gases at rates consistent with those predicted by simple diffusion models. Measurements of H2O, CO2 and S in the matrix glass show that the melt equilibrated rapidly at atmospheric pressure. Down-tube samples maintain similar VSD forms but show a progressive decrease in both overall vesicularity and mean vesicle size. We attribute this change to open system, "passive" rise and escape of larger bubbles to the surface. Such gas loss from the tube system results in the output of 1.2 ?? 106 g/day SO2, an output representing an addition of approximately 1% to overall volatile budget calculations. A steady increase in bubble number density with downstream distance is best explained by continued bubble nucleation at rates of 7-8/cm3s. Rates are ???25% of those estimated from the vent

  11. Novel cationic vesicle platform derived from vernonia oil for efficient delivery of DNA through plant cuticle membranes.

    PubMed

    Wiesman, Zeev; Dom, Naomi Ben; Sharvit, Efrat; Grinberg, Sarina; Linder, Charles; Heldman, Eli; Zaccai, Michele

    2007-05-31

    Novel cationic amphiphilic compounds were prepared from vernonia oil, a natural epoxidized triglyceride, and studied with respect to vesicle formation, encapsulation of biomaterials such as DNA, and their physical stability and transport through isolated plant cuticle membranes. The amphiphiles studied were a single-headed compound III (a quaternary ammonium head group with two alkyl chains) and a triple-headed compound IV, which is essentially three molecules of compound III bound together through a glycerol moiety. Vesicles of the two amphiphiles, prepared by sonication in water and solutions of uranyl acetate or the herbicide 2,4-D (2,4-dichloropenoxy acetic acid), were examined by TEM, SEM, AFM, and confocal laser systems and had a spherical shape which encapsulated the solutes with diameters between 40 and 110 nm. Vesicles from amphiphile IV could be made large enough to encapsulate a condensed 5.2kb DNA plasmid (pJD328). Vesicles of amphiphile IV were also shown to pass intact across isolated plant cuticle membranes and the rate of delivery of encapsulated radio-labeled 2,4-D through isolated plant cuticle membranes obtained with these vesicles was clearly greater in comparison to liposomes prepared from dipalmitopyl phosphatidylcholine (DPPC) and the control, nonencapsulated 2,4-D. Vesicles from amphiphiles III and IV were found to be more stable than those of liposomes from DPPC. The data indicate the potential of vesicles prepared from the novel amphiphile IV to be a relatively efficient nano-scale delivery system to transport DNA and other bioactive agents through plant biological barriers. This scientific approach may open the way for further development of efficient in vivo plant transformation systems.

  12. Photoionization of alkylphenothiazines in vesicles: Effects of the alkyl chain length and the vesicle surface charge

    SciTech Connect

    Sakaguchi, Masato; Hu, Ming; Kevan, L. )

    1990-01-25

    The photoionization of alkylphenothiazine (AP = alkylphenothiazine) in vesicles were observed by electron spin resonance (ESR) and electron spin echo modulation (ESEM) methods. Alkylphenothiazine derivatives including sodium 10-methylphenothiazinesulfonate (C{sub 1}PSO{sub 3}Na), sodium 10-dodecylphenothiazinesulfonate (C{sub 12}PSO{sub 3}Na), sodium 3-(10{prime}-phenothiazinyl)propane-1-sulfonate (PC{sub 3}SO{sub 3}Na), sodium 6-(10{prime}-phenothiazinyl)hexane-1-sulfonate (PC{sub 6}SO{sub 3}Na), and sodium 12-(10{prime}-phenothiazinyl)dodecane-1-sulfonate (PC{sub 12}SO{sub 3} Na) were synthesized and used to study the effects of the alkyl chain length, the position of the sulfonate group, and the vesicle surface charge on the photoionization. A single ESR spectrum due to the alkylphenothiazine cation radicals (AP{sup +}) was observed from rapidly frozen AP in dioctadecyldimethylammonium chloride (DODAC) or dihexadecyl phosphate (DHP) vesicles photoirradiated for 10 min with {lambda} > 300 nm. In DODAC vesicles with a positive surface charge, the photoionization yield of PC{sub 12}SO{sub 3}Na with a sulfonate group at the dodecyl chain end is higher than that of C{sub 12}PSO{sub 3}Na with a sulfonate group on the phenothiazine ring. The photoionization yields of AP having the sulfonate group at the alkyl chain end in DODAC vesicles increase with decreasing alkyl chain length. The highest photoionization yield was obtained from PC{sub 3}SO{sub 3}Na, which has the shortest alkyl chain in this study and has the sulfonate group at the end of the propyl chain. The photoionization yield of AP in DHP vesicles with a negative surface charge was not changed by added alkyl chains or the position of the sulfonate group in AP. The results are discussed in terms of the alkyl chain length, the position of the sulfonate group, and the vesicle surface charge.

  13. 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

  14. Reactive block copolymer vesicles with an epoxy wall.

    PubMed

    Zhu, Hui; Liu, Qingchun; Chen, Yongming

    2007-01-16

    Recently, block copolymer vesicles have attracted considerable attention because of their properties in encapsulation and release. To explore their applications in biorelated fields, functionalization of the polymer vesicle is necessary. Herein, a reactive unilamellar vesicle is reported by self-assembly of poly(ethylene oxide)-block-poly(glycidyl methacrylate) copolymer (PEO-b-PGMA) in solution. When water was added into the PEO-b-PGMA solution in THF, unilamellar vesicles were produced. If hydrophobic primary amine additives, such as hexamethylenediamine (HDA) and dodecylamine (DA), were introduced during block copolymer assembling, the vesicular morphology remained unchanged; instead, the amines reacted with the epoxys and the vesicles were fixed by cross-linking. Furthermore, when 3-aminopropyl trimethoxysilane (APS) was applied, the organic/inorganic hybrid vesicles were obtained, which were stable against the solvent change. Therefore, this research not only supplies a new way to fix the vesicular morphology but also a reactive vesicle scaffold for introducing functional species.

  15. Docking of Secretory Vesicles Is Syntaxin Dependent

    PubMed Central

    de Wit, Heidi; Cornelisse, L. Niels; Toonen, Ruud F.G.; Verhage, Matthijs

    2006-01-01

    Secretory vesicles dock at the plasma membrane before they undergo fusion. Molecular docking mechanisms are poorly defined but believed to be independent of SNARE proteins. Here, we challenged this hypothesis by acute deletion of the target SNARE, syntaxin, in vertebrate neurons and neuroendocrine cells. Deletion resulted in fusion arrest in both systems. No docking defects were observed in synapses, in line with previous observations. However, a drastic reduction in morphologically docked secretory vesicles was observed in chromaffin cells. Syntaxin-deficient chromaffin cells showed a small reduction in total and plasma membrane staining for the docking factor Munc18-1, which appears insufficient to explain the drastic reduction in docking. The sub-membrane cortical actin network was unaffected by syntaxin deletion. These observations expose a docking role for syntaxin in the neuroendocrine system. Additional layers of regulation may have evolved to make syntaxin redundant for docking in highly specialized systems like synaptic active zones. PMID:17205130

  16. In vitro formation of gap junction vesicles.

    PubMed

    Goodenough, D A

    1976-02-01

    A method is described that uses trypsin digestion combined with collagenase-hyaluronidase which produces a population of gap junction vesicles. The hexagonal lattice of subunits ("connexons") comprising the gapjunctions appears unaltered by various structural criteria and by buoyant density measurements. The gap junction vesciles are closed by either a single or a double profile of nonjunctional "membrane," which presents a smooth, particle-free fracture face. Horseradish peroxidase and cytochrome c studies have revealed that about 20% of the gap junction vesicles are impermeable to proteins 12,000 daltons or larger. The increased purity of the trypsinized junction preparation suggests that one of the disulfide reduction products of the gap-junction principal protein may be a nonjunctional contaminating peptide. The gap junction appears to be composed of a single 18,000-dalton protein, connexin, which may be reduced to a single 9,000-dalton peak. The number of peptides in this reduced peak are still unknown.

  17. Soft vesicles in the synthesis of hard materials.

    PubMed

    Dong, Renhao; Liu, Weimin; Hao, Jingcheng

    2012-04-17

    Vesicles of surfactants in aqueous solution have received considerable attention because of their use as simple model systems for biological membranes and their applications in various fields including colloids, pharmaceuticals, and materials. Because of their architecture, vesicles could prove useful as "soft" templates for the synthesis of "hard materials". The vesicle phase, however, has been challenging and difficult to work with in the construction of hard materials. In the solution-phase synthesis of various inorganic or macromolecular materials, templating methods provide a powerful strategy to control the size, morphology, and composition of the resulting micro- and nanostructures. In comparison with hard templates, soft templates are generally constructed using amphiphilic molecules, especially surfactants and amphiphilic polymers. These types of compounds offer advantages including the wide variety of available templates, simple fabrication processes under mild conditions, and easy removal of the templates with less damage to the final structures. Researchers have used many ordered molecular aggregates such as vesicles, micelles, liquid crystals, emulsion droplets, and lipid nanotubes as templates or structure-directing agents to control the synthesis or assembly hard micro- and nanomaterials composed from inorganic compounds or polymers. In addition to their range of sizes and morphologies, vesicles present unique structures that can simultaneously supply different microenvironments for the growth and assembly of hard materials: the inner chamber of vesicles, the outer surface of the vesicles, and the space between bilayers. Two main approaches for applying vesicles in the field of hard materials have been explored: (i) in situ synthesis of micro- or nanomaterials within a specific microenvironment by vesicle templating and (ii) the assembly or incorporation of guest materials during the formation of vesicles. This Account provides an in-depth look at

  18. Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles.

    PubMed

    Théry, C; Boussac, M; Véron, P; Ricciardi-Castagnoli, P; Raposo, G; Garin, J; Amigorena, S

    2001-06-15

    Dendritic cells constitutively secrete a population of small (50-90 nm diameter) Ag-presenting vesicles called exosomes. When sensitized with tumor antigenic peptides, dendritic cells produce exosomes, which stimulate anti-tumor immune responses and the rejection of established tumors in mice. Using a systematic proteomic approach, we establish the first extensive protein map of a particular exosome population; 21 new exosomal proteins were thus identified. Most proteins present in exosomes are related to endocytic compartments. New exosomal residents include cytosolic proteins most likely involved in exosome biogenesis and function, mainly cytoskeleton-related (cofilin, profilin I, and elongation factor 1alpha) and intracellular membrane transport and signaling factors (such as several annexins, rab 7 and 11, rap1B, and syntenin). Importantly, we also identified a novel category of exosomal proteins related to apoptosis: thioredoxin peroxidase II, Alix, 14-3-3, and galectin-3. These findings led us to analyze possible structural relationships between exosomes and microvesicles released by apoptotic cells. We show that although they both represent secreted populations of membrane vesicles relevant to immune responses, exosomes and apoptotic vesicles are biochemically and morphologically distinct. Therefore, in addition to cytokines, dendritic cells produce a specific population of membrane vesicles, exosomes, with unique molecular composition and strong immunostimulating properties.

  19. Membrane-elasticity model of Coatless vesicle budding induced by ESCRT complexes.

    PubMed

    Różycki, Bartosz; Boura, Evzen; Hurley, James H; Hummer, Gerhard

    2012-01-01

    The formation of vesicles is essential for many biological processes, in particular for the trafficking of membrane proteins within cells. The Endosomal Sorting Complex Required for Transport (ESCRT) directs membrane budding away from the cytosol. Unlike other vesicle formation pathways, the ESCRT-mediated budding occurs without a protein coat. Here, we propose a minimal model of ESCRT-induced vesicle budding. Our model is based on recent experimental observations from direct fluorescence microscopy imaging that show ESCRT proteins colocalized only in the neck region of membrane buds. The model, cast in the framework of membrane elasticity theory, reproduces the experimentally observed vesicle morphologies with physically meaningful parameters. In this parameter range, the minimum energy configurations of the membrane are coatless buds with ESCRTs localized in the bud neck, consistent with experiment. The minimum energy configurations agree with those seen in the fluorescence images, with respect to both bud shapes and ESCRT protein localization. On the basis of our model, we identify distinct mechanistic pathways for the ESCRT-mediated budding process. The bud size is determined by membrane material parameters, explaining the narrow yet different bud size distributions in vitro and in vivo. Our membrane elasticity model thus sheds light on the energetics and possible mechanisms of ESCRT-induced membrane budding.

  20. The t(10;11)(p13;q14) in the U937 cell line results in the fusion of the AF10 gene and CALM, encoding a new member of the AP-3 clathrin assembly protein family.

    PubMed Central

    Dreyling, M H; Martinez-Climent, J A; Zheng, M; Mao, J; Rowley, J D; Bohlander, S K

    1996-01-01

    The translocation t(10;11)(p13;q14) is a recurring chromosomal abnormality that has been observed in patients with acute lymphoblastic leukemia as well as acute myeloid leukemia. We have recently reported that the monocytic cell line U937 has a t(10;11)(p13;q14) translocation. Using a combination of positional cloning and candidate gene approach, we cloned the breakpoint and were able to show that AF10 is fused to a novel gene that we named CALM (Clathrin Assembly Lymphoid Myeloid leukemia gene) located at 11q14. AF10, a putative transcription factor, had recently been cloned as one of the fusion partners of MLL. CALM has a very high homology in its N-terminal third to the murine ap-3 gene which is one of the clathrin assembly proteins. The N-terminal region of ap-3 has been shown to bind to clathrin and to have a high-affinity binding site for phosphoinositols. The identification of the CALM/AF10 fusion gene in the widely used U937 cell line will contribute to our understanding of the malignant phenotype of this line. Images Fig. 1 Fig. 3 PMID:8643484

  1. ATP: The crucial component of secretory vesicles.

    PubMed

    Estévez-Herrera, Judith; Domínguez, Natalia; Pardo, Marta R; González-Santana, Ayoze; Westhead, Edward W; Borges, Ricardo; Machado, José David

    2016-07-12

    The colligative properties of ATP and catecholamines demonstrated in vitro are thought to be responsible for the extraordinary accumulation of solutes inside chromaffin cell secretory vesicles, although this has yet to be demonstrated in living cells. Because functional cells cannot be deprived of ATP, we have knocked down the expression of the vesicular nucleotide carrier, the VNUT, to show that a reduction in vesicular ATP is accompanied by a drastic fall in the quantal release of catecholamines. This phenomenon is particularly evident in newly synthesized vesicles, which we show are the first to be released. Surprisingly, we find that inhibiting VNUT expression also reduces the frequency of exocytosis, whereas the overexpression of VNUT drastically increases the quantal size of exocytotic events. To our knowledge, our data provide the first demonstration that ATP, in addition to serving as an energy source and purinergic transmitter, is an essential element in the concentration of catecholamines in secretory vesicles. In this way, cells can use ATP to accumulate neurotransmitters and other secreted substances at high concentrations, supporting quantal transmission.

  2. ATP: The crucial component of secretory vesicles

    PubMed Central

    Estévez-Herrera, Judith; Domínguez, Natalia; Pardo, Marta R.; González-Santana, Ayoze; Westhead, Edward W.; Borges, Ricardo; Machado, José David

    2016-01-01

    The colligative properties of ATP and catecholamines demonstrated in vitro are thought to be responsible for the extraordinary accumulation of solutes inside chromaffin cell secretory vesicles, although this has yet to be demonstrated in living cells. Because functional cells cannot be deprived of ATP, we have knocked down the expression of the vesicular nucleotide carrier, the VNUT, to show that a reduction in vesicular ATP is accompanied by a drastic fall in the quantal release of catecholamines. This phenomenon is particularly evident in newly synthesized vesicles, which we show are the first to be released. Surprisingly, we find that inhibiting VNUT expression also reduces the frequency of exocytosis, whereas the overexpression of VNUT drastically increases the quantal size of exocytotic events. To our knowledge, our data provide the first demonstration that ATP, in addition to serving as an energy source and purinergic transmitter, is an essential element in the concentration of catecholamines in secretory vesicles. In this way, cells can use ATP to accumulate neurotransmitters and other secreted substances at high concentrations, supporting quantal transmission. PMID:27342860

  3. The reserve pool of synaptic vesicles acts as a buffer for proteins involved in synaptic vesicle recycling

    PubMed Central

    Denker, Annette; Kröhnert, Katharina; Bückers, Johanna; Neher, Erwin; Rizzoli, Silvio O.

    2011-01-01

    Presynaptic nerve terminals contain between several hundred vesicles (for example in small CNS synapses) and several tens of thousands (as in neuromuscular junctions). Although it has long been assumed that such high numbers of vesicles are required to sustain neurotransmission during conditions of high demand, we found that activity in vivo requires the recycling of only a few percent of the vesicles. However, the maintenance of large amounts of reserve vesicles in many evolutionarily distinct species suggests that they are relevant for synaptic function. We suggest here that these vesicles constitute buffers for soluble accessory proteins involved in vesicle recycling, preventing their loss into the axon. Supporting this hypothesis, we found that vesicle clusters contain a large variety of proteins needed for vesicle recycling, but without an obvious function within the clusters. Disrupting the clusters by application of black widow spider venom resulted in the diffusion of numerous soluble proteins into the axons. Prolonged stimulation and ionomycin application had a similar effect, suggesting that calcium influx causes the unbinding of soluble proteins from vesicles. Confirming this hypothesis, we found that isolated synaptic vesicles in vitro sequestered soluble proteins from the cytosol in a process that was inhibited by calcium addition. We conclude that the reserve vesicles support neurotransmission indirectly, ensuring that soluble recycling proteins are delivered upon demand during synaptic activity. PMID:21903923

  4. Release of canine parvovirus from endocytic vesicles.

    PubMed

    Suikkanen, Sanna; Antila, Mia; Jaatinen, Anne; Vihinen-Ranta, Maija; Vuento, Matti

    2003-11-25

    Canine parvovirus (CPV) is a small nonenveloped virus with a single-stranded DNA genome. CPV enters cells by clathrin-mediated endocytosis and requires an acidic endosomal step for productive infection. Virion contains a potential nuclear localization signal as well as a phospholipase A(2) like domain in N-terminus of VP1. In this study we characterized the role of PLA(2) activity on CPV entry process. PLA(2) activity of CPV capsids was triggered in vitro by heat or acidic pH. PLA(2) inhibitors inhibited the viral proliferation suggesting that PLA(2) activity is needed for productive infection. The N-terminus of VP1 was exposed during the entry, suggesting that PLA(2) activity might have a role during endocytic entry. The presence of drugs modifying endocytosis (amiloride, bafilomycin A(1), brefeldin A, and monensin) caused viral proteins to remain in endosomal/lysosomal vesicles, even though the drugs were not able to inhibit the exposure of VP1 N-terminal end. These results indicate that the exposure of N-terminus of VP1 alone is not sufficient to allow CPV to proliferate. Some other pH-dependent changes are needed for productive infection. In addition to blocking endocytic entry, amiloride was able to block some postendocytic steps. The ability of CPV to permeabilize endosomal membranes was demonstrated by feeding cells with differently sized rhodamine-conjugated dextrans together with the CPV in the presence or in the absence of amiloride, bafilomycin A(1), brefeldin A, or monensin. Dextran with a molecular weight of 3000 was released from vesicles after 8 h of infection, while dextran with a molecular weight of 10,000 was mainly retained in vesicles. The results suggest that CPV infection does not cause disruption of endosomal vesicles. However, the permeability of endosomal membranes apparently changes during CPV infection, probably due to the PLA(2) activity of the virus. These results suggest that parvoviral PLA(2) activity is essential for productive

  5. Ultradeformable lipid vesicles can penetrate the skin and other semi-permeable barriers unfragmented. Evidence from double label CLSM experiments and direct size measurements.

    PubMed

    Cevc, Gregor; Schätzlein, Andreas; Richardsen, Holger

    2002-08-19

    The stability of various aggregates in the form of lipid bilayer vesicles was tested by three different methods before and after crossing different semi-permeable barriers. First, polymer membranes with pores significantly smaller than the average aggregate diameter were used as the skin barrier model; dynamic light scattering was employed to monitor vesicle size changes after barrier passage for several lipid mixtures with different bilayer elasticities. This revealed that vesicles must adapt their size and/or shape, dependent on bilayer stability and elasto-mechanics, to overcome an otherwise confining pore. For the mixed lipid aggregates with highly flexible bilayers (Transfersomes), the change is transient and only involves vesicle shape and volume adaptation. The constancy of ultradeformable vesicle size before and after pores penetration proves this. This is remarkable in light of the very strong aggregate deformation during an enforced barrier passage. Simple phosphatidylcholine vesicles, with less flexible bilayers, lack such capability and stability. Conventional liposomes are therefore fractured during transport through a semi-permeable barrier; as reported by other researchers, liposomes are fragmented to the size of a narrow pore if sufficient pressure is applied across the barrier; otherwise, liposomes clog the pores. The precise outcome depends on trans-barrier flux and/or on relative vesicle vs. pore size. Lipid vesicles applied on the skin behave accordingly. Mixed lipid vesicles penetrate the skin if they are sufficiently deformable. If this is the case, they cross inter-cellular constrictions in the organ without significant composition or size modification. To prove this, we labelled vesicles with two different fluorescent markers and applied the suspension on intact murine skin without occlusion. The confocal laser scanning microscopy (CLSM) of the skin then revealed a practically indistinguishable distribution of both labels in the stratum

  6. Controlled deformation of vesicles by flexible structured media

    PubMed Central

    Zhang, Rui; Zhou, Ye; Martínez-González, José A.; Hernández-Ortiz, Juan P.; Abbott, Nicholas L.; de Pablo, Juan J.

    2016-01-01

    Liquid crystalline (LC) materials, such as actin or tubulin networks, are known to be capable of deforming the shape of cells. Here, elements of that behavior are reproduced in a synthetic system, namely, a giant vesicle suspended in a LC, which we view as a first step toward the preparation of active, anisotropic hybrid systems that mimic some of the functionality encountered in biological systems. To that end, we rely on a coupled particle-continuum representation of deformable networks in a nematic LC represented at the level of a Landau–de Gennes free energy functional. Our results indicate that, depending on its elastic properties, the LC is indeed able to deform the vesicle until it reaches an equilibrium, anisotropic shape. The magnitude of the deformation is determined by a balance of elastic and surface forces. For perpendicular anchoring at the vesicle, a Saturn ring defect forms along the equatorial plane, and the vesicle adopts a pancake-like, oblate shape. For degenerate planar anchoring at the vesicle, two boojum defects are formed at the poles of the vesicle, which adopts an elongated, spheroidal shape. During the deformation, the volume of the topological defects in the LC shrinks considerably as the curvature of the vesicle increases. These predictions are confirmed by our experimental observations of spindle-like shapes in experiments with giant unilamellar vesicles with planar anchoring. We find that the tension of the vesicle suppresses vesicle deformation, whereas anchoring strength and large elastic constants promote shape anisotropy. PMID:27532056

  7. Polymerization of hydrogels inside self-assembled block copolymer vesicles.

    PubMed

    Gaspard, Jeffery; Hahn, Mariah S; Silas, James A

    2009-11-17

    Block copolymer vesicles are powerful tools for investigating cell adhesion since they display the fluid, deformable, semipermeable membrane properties of lipid vesicles while having greater chemical and mechanical stability. The aim of the present study was to fabricate block copolymer vesicles containing hydrogel interiors in order to extend achievable vesicle properties and, thereby, their range of cell-like behaviors. Block copolymer vesicles based on poly(butadiene-b-ethylene oxide) were demonstrated to compartmentalize and retain acrylamide solutions through particle dialysis and to allow for subsequent in situ hydrogel polymerization. Small molecule leakage studies of the resulting particles indicated that the cross-link density of the hydrogel interiors had minimal impact on vesicle permeability to small molecules (<430 Da) relative to vesicle membrane properties. In contrast, particle deformation analyses indicated that initial vesicle surface approach and adhesion was dominated by its membrane properties, whereas its ultimate deformation was primarily governed by the hydrogel interior. Thus, the hydrogel-containing vesicles allowed orthogonal control of particle surface and mechanical properties. Analysis of particle behavior in terms of Gibb's free energy minimization indicated that vesicle adhesion energy, membrane tension, and internal osmotic pressure dominated particle adhesion and deformation. Combined, the present work demonstrates the potential for designing compartmentalized, hierarchical polymer-based cell mimics with broadly tunable dynamic-mechanical properties and surface properties.

  8. Extracellular Vesicles in Renal Diseases: More than Novel Biomarkers?

    PubMed Central

    Le, Thu H.

    2016-01-01

    Extracellular vesicles from the urine and circulation have gained significant interest as potential diagnostic biomarkers in renal diseases. Urinary extracellular vesicles contain proteins from all sections of the nephron, whereas most studied circulating extracellular vesicles are derived from platelets, immune cells, and the endothelium. In addition to their diagnostic role as markers of kidney and vascular damage, extracellular vesicles may have functional significance in renal health and disease by facilitating communication between cells and protecting against kidney injury and bacterial infection in the urinary tract. However, the current understanding of extracellular vesicles has derived mostly from studies with very small numbers of patients or in vitro data. Moreover, accurate assessment of these vesicles remains a challenge, in part because of a lack of consensus in the methodologies to measure extracellular vesicles and the inability of most techniques to capture the entire size range of these vesicles. However, newer techniques and standardized protocols to improve the detection of extracellular vesicles are in development. A clearer understanding of the composition and biology of extracellular vesicles will provide insights into their pathophysiologic, diagnostic, and therapeutic roles. PMID:26251351

  9. High affinity 3H-Phe uptake by brush border membrane vesicles from whole larvae of Aedes aegypti (AaBMVw)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Brush border membrane vesicles from whole Aedes aegypti larvae (AaBBMVw) are confirmed to be valid preparations for membrane transport studies. The Abdul-Rauf and Ellar method was used to isolate AaBBMVw that were frozen, stored for several months, transported to a distant site, thawed and used to s...

  10. Focus on Extracellular Vesicles: Development of Extracellular Vesicle-Based Therapeutic Systems

    PubMed Central

    Ohno, Shin-ichiro; Drummen, Gregor P. C.; Kuroda, Masahiko

    2016-01-01

    Many types of cells release phospholipid membrane vesicles thought to play key roles in cell-cell communication, antigen presentation, and the spread of infectious agents. Extracellular vesicles (EVs) carry various proteins, messenger RNAs (mRNAs), and microRNAs (miRNAs), like a “message in a bottle” to cells in remote locations. The encapsulated molecules are protected from multiple types of degradative enzymes in body fluids, making EVs ideal for delivering drugs. This review presents an overview of the potential roles of EVs as natural drugs and novel drug-delivery systems. PMID:26861303

  11. Adhesive interactions between vesicles in the strong adhesion limit

    PubMed Central

    Ramachandran, Arun; Anderson, Travers H.; Leal, L. Gary; Israelachvili, Jacob N.

    2010-01-01

    We consider the adhesive interaction energy between a pair of vesicles in the strong adhesion limit, in which bending forces play a negligible role in determining vesicle shape compared to forces due to membrane stretching. Although force-distance or energy distance relationships characterizing adhesive interactions between fluid bilayers are routinely measured using the surface forces apparatus, the atomic force microscope and the biomembrane force probe, the interacting bilayers in these methods are supported on surfaces (e.g. mica sheet) and cannot be deformed. However, it is known that in a suspension, vesicles composed of the same bilayer can deform by stretching or bending, and can also undergo changes in volume. Adhesively interacting vesicles can thus form flat regions in the contact zone, which will result in an enhanced interaction energy as compared to rigid vesicles. The focus of this paper is to examine the magnitude of the interaction energy between adhesively interacting, deformed vesicles relative to free, undeformed vesicles as a function of the intervesicle separation. The modification of the intervesicle interaction energy due to vesicle deformability can be calculated knowing the undeformed radius of the vesicles, R0, the bending modulus kb, the area expansion modulus Ka, and the adhesive minimum WP(0) and separation DP(0) in the energy of interaction between two flat bilayers, which can be obtained from the force-distance measurements made using the above supported-bilayer methods. For vesicles with constant volumes, we show that adhesive potentials between non-deforming bilayers such as ∣WP(0)∣∼5×10−4mJ/m2, which are ordinarily considered weak in colloidal physics literature, can result in significantly deep (>10×) energy minima due to increase in vesicle area and flattening in the contact region. If the osmotic expulsion of water across the vesicles driven by the tense, stretched membrane in the presence of an osmotically active

  12. Effect of Ca2+ on Vesicle Fusion on Solid Surface: An In vitro Model of Protein-Accelerated Vesicle Fusion

    NASA Astrophysics Data System (ADS)

    Shinozaki, Youichi; Siitonen, Ari M.; Sumitomo, Koji; Furukawa, Kazuaki; Torimitsu, Keiichi

    2008-07-01

    Lipid vesicle fusion is an important reaction in the cell. Calcium ions (Ca2+) participate in various important biological events including the fusion of vesicles with cell membranes in cells. We studied the effect of Ca2+ on the fusion of egg yolk phosphatidylcholine/brain phosphatidylserine (eggPC/brainPS) lipid vesicles on a mica substrate with fast scanning atomic force microscopy (AFM). When unattached and unfused lipid vesicles on mica were rinsed away, discrete patches of fused vesicles were observed under high Ca2+ concentrations. At 0 mM Ca2+, lipid vesicles were fused on mica and formed continuous supported lipid bilayers (SLBs) covering almost the entire mica surface. The effect of Ca2+ on SLB formation was offset by a Ca2+ chelating agent. When lipid vesicles were added during AFM observation, vesicles fused on mica and covered almost all areas even under high Ca2+ concentrations. These results indicate that force between AFM tip and vesicles overcomes the Ca2+-reduced fusion of lipid vesicles.

  13. Covalent attachment of lipid vesicles to a fluid supported bilayer allows observation of DNA-mediated vesicle interactions

    PubMed Central

    van Lengerich, Bettina; Rawle, Robert J.; Boxer, Steven G.

    2010-01-01

    Specific membrane interactions such as lipid vesicle docking and fusion can be mediated by synthetic DNA-lipid conjugates as a model for the protein-driven processes that are ubiquitous in biological systems. Here we present a method of tethering vesicles to a supported lipid bilayer that allows simultaneous deposition of cognate vesicle partners displaying complementary DNA, resulting in well-mixed populations of tethered vesicles that are laterally mobile. Vesicles are covalently attached to a supporting lipid bilayer using a DNA-templated click reaction; then DNA-mediated interactions between tethered vesicles are triggered by spiking the salt concentration. These interactions, such as docking and fusion, can then be observed for individual vesicles as they collide on the surface. The architecture of this new system also permits control over the number of lipid anchors that tether the vesicle to the supporting bilayer. The diffusion coefficient of tethered vesicles anchored by two lipids is approximately 1.6-fold slower than that of vesicles anchored only with a single lipid, consistent with a simple physical model. PMID:20180548

  14. 3D Probed Lipid Dynamics in Small Unilamellar Vesicles.

    PubMed

    Chao, Meng-Hsuan; Lin, Yen-Ting; Dhenadhayalan, Namasivayam; Lee, Hsin-Lung; Lee, Hsin-Yen; Lin, King-Chuen

    2017-04-01

    Single-molecule fluorescence correlation spectroscopy overcomes the resolution barrier of optical microscopy (10≈-20 nm) and is utilized to look into lipid dynamics in small unilamellar vesicles (SUVs; diameter < 100 nm). The fluorescence trajectories of lipid-like tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine (DiD) in the membrane bilayers are acquired at a single-molecule level. The autocorrelation analysis yields the kinetic information on lipid organization, oxygen transport, and lateral diffusion in SUVs' membrane. First, the isomerization feasibility may be restricted by the addition of cholesterols, which form structure conjugation with DiD chromophore. Second, the oxygen transport is prevented from the ultrasmall cluster and cholesterol-rich regions, whereas it can pass through the membrane region with liquid-disordered phase (Ld ) and defects. Third, by analyzing 2D spectra correlating the lipid diffusion coefficient and triplet-state lifetime, the heterogeneity in lipid bilayer can be precisely visualized such as lipid domain with different phases, the defects of lipid packing, and DiD-induced "bouquet" ultrasmall clusters.

  15. Isolation of Chlamydia trachomatis and membrane vesicles derived from host and bacteria

    PubMed Central

    Frohlich, Kyla; Hua, Ziyu; Wang, Jin; Shen, Li

    2012-01-01

    The study of intracellular bacteria and nanometer-size membrane vesicles within infected host cells poses an important challenge as it is difficult to identify each distinct population in the context of the complex populations generated from active host-pathogen interactions. Here, suspension cultures of L929 cells infected with the prevalent obligate intracellular bacterium Chlamydia trachomatis strain F/Cal-IC-13 are utilized for the large scale preparation and isolation of natural membrane vesicles and bacterial forms. Cell lysis with nitrogen cavitation in combination with differential centrifugation, OptiPrep™ density gradient separation, and immunoenrichment using anti-chlamydial lipopolysaccharide antibodies and MagnaBind beads allows for the isolation of both productive and persistent bacterial forms, as well as membrane vesicles derived from the host and pathogen. We have evaluated these populations by electron microscopy and Western blot analysis for identification of biomarkers. In addition, purified persistent forms of C. trachomatis induced by ampicillin display adenosine-5'-triphosphate (ATP) transport activity, suggesting that ampicillin-induced persistent C. trachomatis organisms, at least in part, rely upon host ATP as an energy source. Importantly, several chlamydial cytotoxic and/or secreted proteins are demonstrated to be associated with these vesicles, supporting the idea that membrane vesicles are generated by Chlamydia as a means of carrying and delivering virulence factors necessary for pathogenesis. The ability to produce large-scale infections and generate distinct bacteria and host-derived populations for biochemical analysis, while reducing the burdens of time and cost have implications in all areas of chlamydiology. These protocols can be applied to other strains of C. trachomatis or other intracellular bacteria. PMID:22960504

  16. Gram-negative and Gram-positive bacterial extracellular vesicles.

    PubMed

    Kim, Ji Hyun; Lee, Jaewook; Park, Jaesung; Gho, Yong Song

    2015-04-01

    Like mammalian cells, Gram-negative and Gram-positive bacteria release nano-sized membrane vesicles into the extracellular environment either in a constitutive manner or in a regulated manner. These bacterial extracellular vesicles are spherical bilayered proteolipids enriched with bioactive proteins, lipids, nucleic acids, and virulence factors. Recent progress in this field supports the critical pathophysiological functions of these vesicles in both bacteria-bacteria and bacteria-host interactions. This review provides an overview of the current understanding on Gram-negative and Gram-positive bacterial extracellular vesicles, especially regarding the biogenesis, components, and functions in poly-species communities. We hope that this review will stimulate additional research in this emerging field of bacterial extracellular vesicles and contribute to the development of extracellular vesicle-based diagnostic tools and effective vaccines against pathogenic Gram-negative and Gram-positive bacteria.

  17. C-type natriuretic peptide-modified lipid vesicles: fabrication and use for the treatment of brain glioma.

    PubMed

    Wu, Jia-Shuan; Mu, Li-Min; Bu, Ying-Zi; Liu, Lei; Yan, Yan; Hu, Ying-Jie; Bai, Jing; Zhang, Jing-Ying; Lu, Weiyue; Lu, Wan-Liang

    2017-03-29

    Chemotherapy of brain glioma faces a major obstacle owing to the inability of drug transport across the blood-brain barrier (BBB). Besides, neovasculatures in brain glioma site result in a rapid infiltration, making complete surgical removal virtually impossible. Herein, we reported a novel kind of C-type natriuretic peptide (CNP) modified vinorelbine lipid vesicles for transferring drug across the BBB, and for treating brain glioma along with disrupting neovasculatures. The studies were performed on brain glioma U87-MG cells in vitro and on glioma-bearing nude mice in vivo. The results showed that the CNP-modified vinorelbine lipid vesicles could transport vinorelbine across the BBB, kill the brain glioma, and destroy neovasculatures effectively. The above mechanisms could be associated with the following aspects, namely, long circulation in the blood; drug transport across the BBB via natriuretic peptide receptor B (NPRB)-mediated transcytosis; elimination of brain glioma cells and disruption of neovasculatures by targeting uptake and cytotoxic injury. Besides, CNP-modified vinorelbine lipid vesicles could induce apoptosis of the glioma cells. The mechanisms could be related to the activations of caspase 8, caspase 3, p53, and reactive oxygen species (ROS), and inhibition of survivin. Hence, CNP-modified lipid vesicles could be used as a carrier material for treating brain glioma and disabling glioma neovasculatures.

  18. Insulin triggers surface-directed trafficking of sequestered GLUT4 storage vesicles marked by Rab10.

    PubMed

    Chen, Yu; Lippincott-Schwartz, Jennifer

    2013-01-01

    Understanding how glucose transporter isoform 4 (GLUT4) redistributes to the plasma membrane during insulin stimulation is a major goal of glucose transporter research. GLUT4 molecules normally reside in numerous intracellular compartments, including specialized storage vesicles and early/recycling endosomes. It is unclear how these diverse compartments respond to insulin stimulation to deliver GLUT4 molecules to the plasma membrane. For example, do they fuse with each other first or remain as separate compartments with different trafficking characteristics? Our recent live cell imaging studies are helping to clarify these issues. Using Rab proteins as specific markers to distinguish between storage vesicles and endosomes containing GLUT4, we demonstrate that it is primarily internal GLUT4 storage vesicles (GSVs) marked by Rab10 that approach and fuse at the plasma membrane and GSVs don't interact with endosomes on their way to the plasma membrane. These new findings add strong support to the model that GSV release from intracellular retention plays a major role in supplying GLUT4 molecules onto the PM under insulin stimulation.

  19. Syntaxin-17 delivers PINK1/parkin-dependent mitochondrial vesicles to the endolysosomal system

    PubMed Central

    McLelland, Gian-Luca

    2016-01-01

    Mitochondria are considered autonomous organelles, physically separated from endocytic and biosynthetic pathways. However, recent work uncovered a PINK1/parkin-dependent vesicle transport pathway wherein oxidized or damaged mitochondrial content are selectively delivered to the late endosome/lysosome for degradation, providing evidence that mitochondria are indeed integrated within the endomembrane system. Given that mitochondria have not been shown to use canonical soluble NSF attachment protein receptor (SNARE) machinery for fusion, the mechanism by which mitochondrial-derived vesicles (MDVs) are targeted to the endosomal compartment has remained unclear. In this study, we identify syntaxin-17 as a core mitochondrial SNARE required for the delivery of stress-induced PINK1/parkin-dependent MDVs to the late endosome/lysosome. Syntaxin-17 remains associated with mature MDVs and forms a ternary SNARE complex with SNAP29 and VAMP7 to mediate MDV–endolysosome fusion in a manner dependent on the homotypic fusion and vacuole protein sorting (HOPS) tethering complex. Syntaxin-17 can be traced to the last eukaryotic common ancestor, hinting that the removal of damaged mitochondrial content may represent one of the earliest vesicle transport routes in the cell. PMID:27458136

  20. Mammalian TRAPPIII Complex positively modulates the recruitment of Sec13/31 onto COPII vesicles

    PubMed Central

    Zhao, Shan; Li, Chun Man; Luo, Xiao Min; Siu, Gavin Ka Yu; Gan, Wen Jia; Zhang, Lin; Wu, William K. K.; Chan, Hsiao Chang; Yu, Sidney

    2017-01-01

    The Transport protein particle (TRAPP) complex is a tethering factor for COPII vesicle. Of three forms of TRAPP (TRAPPI, II and III) complexes identified so far, TRAPPIII has been largely considered to play a role in autophagy. While depletion of TRAPPIII specific subunits caused defects in the early secretory pathway and TRAPPIII might interact with components of the COPII vesicle coat, its exact role remains to be determined. In this study, we studied the function of TRAPPIII in early secretory pathway using a TRAPPIII-specific subunit, TRAPPC12, as starting point. We found that TRAPPC12 was localized to the ER exit sites and ERGIC. In cells deleted with TRAPPC12, ERGIC and to a lesser extent, the Golgi became dispersed. ER-to-Golgi transport was also delayed. TRAPPC12, but not TRAPPC8, bound to Sec13/Sec31A tetramer but each Sec protein alone could not interact with TRAPPC12. TRAPPIII positively modulated the assembly of COPII outer layer during COPII vesicle formation. These results identified a novel function of TRAPPIII as a positive modulator of the outer layer of the COPII coat. PMID:28240221

  1. Dynactin, a conserved, ubiquitously expressed component of an activator of vesicle motility mediated by cytoplasmic dynein

    PubMed Central

    1991-01-01

    Although cytoplasmic dynein is known to attach to microtubules and translocate toward their minus ends, dynein's ability to serve in vitro as a minus end-directed transporter of membranous organelles depends on additional soluble factors. We show here that a approximately 20S polypeptide complex (referred to as Activator I; Schroer, T. A., and M.P. Sheetz. 1991a. J. Cell Biol. 115:1309-1318.) stimulates dynein- mediated vesicle transport. A major component of the activator complex is a doublet of 150-kD polypeptides for which we propose the name dynactin (for dynein activator). The 20S dynactin complex is required for in vitro vesicle motility since depletion of it with a mAb to dynactin eliminates vesicle movement. Cloning of a brain specific isoform of dynactin from chicken reveals a 1,053 amino acid polypeptide composed of two coiled-coil alpha-helical domains interrupted by a spacer. Both this structural motif and the underlying primary sequence are highly conserved in vertebrates with 85% sequence identity within a central 1,000-residue domain of the chicken and rat proteins. As abundant as dynein, dynactin is ubiquitously expressed and appears to be encoded by a single gene that yields at least three alternative isoforms. The probable homologue in Drosophila is the gene Glued, whose protein product shares 50% sequence identity with vertebrate dynactin and whose function is essential for viability of most (and perhaps all) cells in the organism. PMID:1836789

  2. Sequestration of organic cations by acidified hepatic endocytic vesicles and implications for biliary excretion.

    PubMed

    Van Dyke, R W; Faber, E D; Meijer, D K

    1992-04-01

    these drugs, accomplished through partial vesicle alkalization by primaquin, decreased excretion of TC, vecuronium and TBuMA, perhaps reflecting the small functional size of the displaceable organellar drug compartment and/or competition between primaquin and the organic cations for membrane transport processes.

  3. Tension-induced pore formation and leakage in adhering vesicles

    NASA Astrophysics Data System (ADS)

    Lenz, P.; Johnson, J. M.; Chan, Y.-H. M.; Boxer, S. G.

    2006-08-01

    The influence of inclusion-induced tension on pore formation is studied theoretically and experimentally. It is shown that fluorescently labeled lipids can enhance pore formation and induce leakage of adhering vesicles. These effects are more pronounced for smaller vesicles. The theoretical predictions are confirmed by experimental two-color fluorescent data. Finally, the influence of the pore formation dynamics on rupture processes of vesicles is analyzed yielding a new picture of the transition to bilayer disks.

  4. Rotavirus interaction with isolated membrane vesicles.

    PubMed

    Ruiz, M C; Alonso-Torre, S R; Charpilienne, A; Vasseur, M; Michelangeli, F; Cohen, J; Alvarado, F

    1994-06-01

    To gain information about the mechanism of epithelial cell infection by rotavirus, we studied the interaction of bovine rotavirus, RF strain, with isolated membrane vesicles from apical membrane of pig enterocytes. Vesicles were charged with high (quenching) concentrations of either carboxyfluorescein or calcein, and the rate of fluorophore release (dequenching) was monitored as a function of time after mixing with purified virus particles. Purified single-shelled particles and untrypsinized double-shelled ones had no effect. Trypsinized double-shelled virions induced carboxyfluorescein release according to sigmoid curves whose lag period and amplitude were a function of virus concentration and depended on both temperature and pH. The presence of 100 mM salts (Tris Cl, NaCl, or KCl) was required, since there was no reaction in isoosmotic salt-free sorbitol media. Other membrane vesicle preparations such as apical membranes of piglet enterocyte and rat placenta syncytiotrophoblasts, basolateral membranes of pig enterocytes, and the undifferentiated plasma membrane of cultured MA104 cells all gave qualitatively similar responses. Inhibition by a specific monoclonal antibody suggests that the active species causing carboxyfluorescein release is VP5*. Ca2+ (1 mM), but not Mg2+, inhibited the reaction. In situ solubilization of the outer capsid of trypsinized double-shelled particles changed release kinetics from sigmoidal to hyperbolic and was not inhibited by Ca2+. Our results indicate that membrane destabilization caused by trypsinized outer capsid proteins of rotavirus leads to fluorophore release. From the data presented here, a hypothetical model of the interaction of the various states of the viral particles with the membrane lipid phase is proposed. Membrane permeabilization induced by rotavirus may be related to the mechanism of entry of the virus into the host cell.

  5. Integration of computational modeling with membrane transport studies reveals new insights into amino acid exchange transport mechanisms.

    PubMed

    Widdows, Kate L; Panitchob, Nuttanont; Crocker, Ian P; Please, Colin P; Hanson, Mark A; Sibley, Colin P; Johnstone, Edward D; Sengers, Bram G; Lewis, Rohan M; Glazier, Jocelyn D

    2015-06-01

    Uptake of system L amino acid substrates into isolated placental plasma membrane vesicles in the absence of opposing side amino acid (zero-trans uptake) is incompatible with the concept of obligatory exchange, where influx of amino acid is coupled to efflux. We therefore hypothesized that system L amino acid exchange transporters are not fully obligatory and/or that amino acids are initially present inside the vesicles. To address this, we combined computational modeling with vesicle transport assays and transporter localization studies to investigate the mechanisms mediating [(14)C]L-serine (a system L substrate) transport into human placental microvillous plasma membrane (MVM) vesicles. The carrier model provided a quantitative framework to test the 2 hypotheses that l-serine transport occurs by either obligate exchange or nonobligate exchange coupled with facilitated transport (mixed transport model). The computational model could only account for experimental [(14)C]L-serine uptake data when the transporter was not exclusively in exchange mode, best described by the mixed transport model. MVM vesicle isolates contained endogenous amino acids allowing for potential contribution to zero-trans uptake. Both L-type amino acid transporter (LAT)1 and LAT2 subtypes of system L were distributed to MVM, with L-serine transport attributed to LAT2. These findings suggest that exchange transporters do not function exclusively as obligate exchangers.

  6. Preparation and mechanism insight of nuclear envelope-like polymer vesicles for facile loading of biomacromolecules and enhanced biocatalytic activity.

    PubMed

    Zhu, Yunqing; Wang, Fangyingkai; Zhang, Cong; Du, Jianzhong

    2014-07-22

    The facile loading of sensitive and fragile biomacromolecules, such as glucose oxidase, hemoglobin, and ribonucleic acid (RNA), via synthetic vehicles directly in pure aqueous media is an important technical challenge. Inspired by the nucleus pore complex that connects the cell nucleus and the cytoplasm across the nuclear envelope, here we describe the development of a kind of polymeric nuclear envelope-like vesicle (NEV) to address this problem. The NEV is tailored to form the polymer pore complex (70 nm, similar to a nucleus pore complex) within the vesicle membrane based on nanophase segregation, which is confirmed via fluorescence spectrometry and dynamic light scattering (DLS) during self-assembly. This pH-triggered polymer pore complex can mediate the transportation of biomacromolecules across the vesicle membrane. Moreover, the NEVs facilitate the natural consecutive enzyme-catalyzed reactions via the H(+) sponge effect. This simple strategy might also be extended for mimicking other synthetic cell organelles.

  7. Noisy Nonlinear Dynamics of Vesicles in Flow

    NASA Astrophysics Data System (ADS)

    Abreu, David; Seifert, Udo

    2013-06-01

    We present a model for the dynamics of fluid vesicles in linear flow which consistently includes thermal fluctuations and nonlinear coupling between different modes. At the transition between tank treading and tumbling, we predict a trembling motion which is at odds with the known deterministic motions and for which thermal noise is strongly amplified. In particular, highly asymmetric shapes are observed even though the deterministic flow only allows for axisymmetric ones. Our results explain quantitatively recent experimental observations [Levant and Steinberg, Phys. Rev. Lett. 109, 268103 (2012)PRLTAO0031-9007].

  8. Immunological properties of seminal vesicle fluid.

    PubMed

    Veselský, L

    1981-08-01

    Protective significance of some seminal plasma components is described. Lactoferrin is characterized as a primary defense factor against microbial invasion. The agglutinating factor in seminal vesicle fluid may prevent premature elimination of the spermatozoa by leukocytes infiltrating into the female genital tract. The protease inhibitors neutralize the activity of the proteases, thereby protecting the tissues and spermatozoa against proteolytic degradation. Antigens absorbed to spermatozoa during the ejaculation may protect the spermatozoa against the immune system of female reproductive tract. Ejaculated spermatozoa contain immunosuppressive substances that inhibit cell-mediated cytotoxicity as well as lymphocyte response to allogenic cells. These substances may constitute the system that prevents immune damage of spermatozoa.

  9. Replicating vesicles as models of primitive cell growth and division.

    PubMed

    Hanczyc, Martin M; Szostak, Jack W

    2004-12-01

    Primitive cells, lacking the complex bio-machinery present in modern cells, would have had to rely on the self-organizing properties of their components and on interactions with their environment to achieve basic cellular functions such as growth and division. Many bilayer-membrane vesicles, depending on their composition and environment, can exhibit complex morphological changes such as growth, fusion, fission, budding, internal vesicle assembly and vesicle-surface interactions. The rich dynamic properties of these vesicles provide interesting models of how primitive cellular replication might have occurred in response to purely physical and chemical forces.

  10. Floating Escherichia coli by expressing cyanobacterial gas vesicle genes

    NASA Astrophysics Data System (ADS)

    Wang, Tianhe; Kang, Li; Li, Jiaheng; Wu, Wenjie; Zhang, Peiran; Gong, Minghao; Lai, Weihong; Zhang, Chunyan; Chang, Lei; Peng, Yong; Yang, Zhongzhou; Li, Lian; Bao, Yingying; Xu, Haowen; Zhang, Xiaohua; Sui, Zhenghong; Yang, Guanpin; Wang, Xianghong

    2015-02-01

    Gas vesicles are hollow, air-filled polyprotein structures that provide the buoyancy to cells. They are found in a variety of prokaryotes. In this study, we isolated a partial gas vesicle protein gene cluster containing gvpA and gvpC20Ψ from Planktothrix rubescens, and inserted it into an expression vector and expressed it in E. coli. The gas vesicle was developed in bacterial cells, which made bacterial cells to float on medium surface. We also amplified gvpA and gvpC20Ψ separately and synthesized an artificial operon by fusing these two genes with the standardized gene expression controlling elements of E. coli. The artificial operon was expressed in E. coli, forming gas vesicles and floating bacteria cells. Our findings verified that the whole set of genes and the overall structure of gas vesicle gene cluster are not necessary for developing gas vesicles in bacteria cells. Two genes, gvpA and gvpC20Ψ, of the gas vesicle gene cluster are sufficient for synthesizing an artificial operon that can develop gas vesicles in bacteria cells. Our findings provided a wide range of applications including easing the harvest of cultured microalgae and bacteria, as well as enriching and remediating aquatic pollutants by constructing gas vesicles in their cells.

  11. Dynamics of multicomponent vesicles in a viscous fluid

    SciTech Connect

    Sohn, Jin Sun Tseng, Y-H Li Shuwang Voigt, Axel Lowengrub, John S.

    2010-01-01

    We develop and investigate numerically a thermodynamically consistent model of two-dimensional multicomponent vesicles in an incompressible viscous fluid. The model is derived using an energy variation approach that accounts for different lipid surface phases, the excess energy (line energy) associated with surface phase domain boundaries, bending energy, spontaneous curvature, local inextensibility and fluid flow via the Stokes equations. The equations are high-order (fourth order) nonlinear and nonlocal due to incompressibility of the fluid and the local inextensibility of the vesicle membrane. To solve the equations numerically, we develop a nonstiff, pseudo-spectral boundary integral method that relies on an analysis of the equations at small scales. The algorithm is closely related to that developed very recently by Veerapaneni et al. [81] for homogeneous vesicles although we use a different and more efficient time stepping algorithm and a reformulation of the inextensibility equation. We present simulations of multicomponent vesicles in an initially quiescent fluid and investigate the effect of varying the average surface concentration of an initially unstable mixture of lipid phases. The phases then redistribute and alter the morphology of the vesicle and its dynamics. When an applied shear is introduced, an initially elliptical vesicle tank-treads and attains a steady shape and surface phase distribution. A sufficiently elongated vesicle tumbles and the presence of different surface phases with different bending stiffnesses and spontaneous curvatures yields a complex evolution of the vesicle morphology as the vesicle bends in regions where the bending stiffness and spontaneous curvature are small.

  12. 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

  13. 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.

  14. Flavonoid transport mechanisms: how to go, and with whom.

    PubMed

    Zhao, Jian

    2015-09-01

    Subcellular flavonoid transport and its underlying regulatory mechanisms are still poorly understood, but are fascinating research frontiers in plant science. Recent studies support and further extend previous hypotheses indicating that vacuolar sequestration of flavonoids involves vesicle trafficking, membrane transporters, and glutathione S-transferase (GST). However, the question remains to be addressed of how three distinct but nonexclusive mechanisms are functionally integrated into diverse but redundant transport routes for vacuolar sequestration or extracellular secretion of flavonoids. In this review, I highlight recent progress in understanding flavonoid-transporting vesicle behavior and properties, GST and membrane transporter functions and mechanisms, and flavonoid transport substrate specificity and preference.

  15. Evidence that the ZNT3 protein controls the total amount of elemental zinc in synaptic vesicles

    USGS Publications Warehouse

    Linkous, D.H.; Flinn, J.M.; Koh, J.Y.; Lanzirotti, A.; Bertsch, P.M.; Jones, B.F.; Giblin, L.J.; Frederickson, C.J.

    2008-01-01

    The ZNT3 protein decorates the presynaptic vesicles of central neurons harboring vesicular zinc, and deletion of this protein removes staining for zinc. However, it has been unclear whether only histochemically reactive zinc is lacking or if, indeed, total elemental zinc is missing from neurons lacking the Slc30a3 gene, which encodes the ZNT3 protein. The limitations of conventional histochemical procedures have contributed to this enigma. However, a novel technique, microprobe synchrotron X-ray fluorescence, reveals that the normal 2- to 3-fold elevation of zinc concentration normally present in the hippocampal mossy fibers is absent in Slc30a3 knockout (ZNT3) mice. Thus, the ZNT3 protein evidently controls not only the "stainability" but also the actual mass of zinc in mossy-fiber synaptic vesicles. This work thus confirms the metal-transporting role of the ZNT3 protein in the brain. ?? The Histochemical Society, Inc.

  16. Coordinated Movement of Vesicles and Actin Bundles during Nerve Growth Revealed by Superresolution Microscopy.

    PubMed

    Nozumi, Motohiro; Nakatsu, Fubito; Katoh, Kaoru; Igarashi, Michihiro

    2017-02-28

    The growth cone is an essential structure for nerve growth. Although its membrane and cytoskeleton are likely to interact coordinately during nerve growth, the mechanisms are unknown due to their close proximity. Here, we used superresolution microscopy to simultaneously observe vesicles and F-actin in growth cones. We identified a novel vesicular generation mechanism that is independent of clathrin and dependent on endophilin-3- and dynamin-1 and that occurs proximal to the leading edge simultaneously with fascin-1-dependent F-actin bundling. In contrast to conventional clathrin-dependent endocytosis, which occurs distal from the leading edge at the basal surfaces of growth cones, this mechanism was distinctly observed at the apical surface using 3D imaging and was involved in mediating axon growth. Reduced endophilin or fascin inhibited this endocytic mechanism. These results suggest that, at the leading edge, vesicles are coordinately generated and transported with actin bundling during nerve growth.

  17. Evidence That the ZNT3 Protein Controls the Total Amount of Elemental Zinc in Synaptic Vesicles

    SciTech Connect

    Linkous,D.; Flinn, J.; Koh, J.; Lanzirotti, A.; Bertsch, P.; Jones, B.; Giblin, L.; Fredrickson, C.

    2008-01-01

    The ZNT3 protein decorates the presynaptic vesicles of central neurons harboring vesicular zinc, and deletion of this protein removes staining for zinc. However, it has been unclear whether only histochemically reactive zinc is lacking or if, indeed, total elemental zinc is missing from neurons lacking the Slc30a3 gene, which encodes the ZNT3 protein. The limitations of conventional histochemical procedures have contributed to this enigma. However, a novel technique, microprobe synchrotron X-ray fluorescence, reveals that the normal 2- to 3-fold elevation of zinc concentration normally present in the hippocampal mossy fibers is absent in Slc30a3 knockout (ZNT3) mice. Thus, the ZNT3 protein evidently controls not only the 'stainability' but also the actual mass of zinc in mossy-fiber synaptic vesicles. This work thus confirms the metal-transporting role of the ZNT3 protein in the brain.

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

    SciTech Connect

    Hasan, Nazarul; Hu, Chuan

    2010-01-01

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

  19. Lst1p and Sec24p Cooperate in Sorting of the Plasma Membrane Atpase into Copii Vesicles in Saccharomyces cerevisiae

    PubMed Central

    Shimoni, Yuval; Kurihara, Tatsuo; Ravazzola, Mariella; Amherdt, Mylène; Orci, Lelio; Schekman, Randy

    2000-01-01

    Formation of ER-derived protein transport vesicles requires three cytosolic components, a small GTPase, Sar1p, and two heterodimeric complexes, Sec23/24p and Sec13/31p, which comprise the COPII coat. We investigated the role of Lst1p, a Sec24p homologue, in cargo recruitment into COPII vesicles in Saccharomyces cerevisiae. A tagged version of Lst1p was purified and eluted as a heterodimer complexed with Sec23p comparable to the Sec23/24p heterodimer. We found that cytosol from an lst1-null strain supported the packaging of α-factor precursor into COPII vesicles but was deficient in the packaging of Pma1p, the essential plasma membrane ATPase. Supplementation of mutant cytosol with purified Sec23/Lst1p restored Pma1p packaging into the vesicles. When purified COPII components were used in the vesicle budding reaction, Pma1p packaging was optimal with a mixture of Sec23/24p and Sec23/Lst1p; Sec23/Lst1p did not replace Sec23/24p. Furthermore, Pma1p coimmunoprecipitated with Lst1p and Sec24p from vesicles. Vesicles formed with a mixture of Sec23/Lst1p and Sec23/24p were similar morphologically and in their buoyant density, but larger than normal COPII vesicles (87-nm vs. 75-nm diameter). Immunoelectronmicroscopic and biochemical studies revealed both Sec23/Lst1p and Sec23/24p on the membranes of the same vesicles. These results suggest that Lst1p and Sec24p cooperate in the packaging of Pma1p and support the view that biosynthetic precursors of plasma membrane proteins must be sorted into ER-derived transport vesicles. Sec24p homologues may comprise a more complex coat whose combinatorial subunit composition serves to expand the range of cargo to be packaged into COPII vesicles. By changing the geometry of COPII coat polymerization, Lst1p may allow the transport of bulky cargo molecules, polymers, or particles. PMID:11086000

  20. Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles.

    PubMed

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

    2015-01-01

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

  1. Nonenzymatic glycation of phosphatidylethanolamine in erythrocyte vesicles

    SciTech Connect

    Patkowska, M.J.; Horowitz, M.I.

    1986-05-01

    Unsealed inside-out and right-side out vesicles were prepared from human red cells. The vesicles were incubated with D-glucose (/sup 14/C(U)) and sodium cyanoborohydride in phosphate buffer, pH 7.4. After incubation, lipids were extracted with 1-butanol and non-lipid contaminants removed by Sephadex G-25 chromatography. Phosphatidylethanolamine-sorbitol was purified by chromatography on columns of silicic acid and phenylboronate agarose gel. Phospholipase C (B. cereus) liberated phosphoethanolamine-sorbitol (I) which comigrated on TLC with synthetic I prepared by reductive condensation of phosphoethanolamine and D-glucose and also with the product of phospholipase C (B. cereus) hydrolysis of reference phosphatidylethanolamine-sorbitol. Exposure of I to alkaline phosphatase (E. coli) gave P/sub i/ and ethanolamine-sorbitol (II) which comigrated on TLC with synthetic II prepared by reductive condensation of ethanolamine and D-glucose or by phospholipase D hydrolysis of reference phosphatidylethanolamine-sorbitol. These studies demonstrate that vesicular phosphatidylethanolamine can be reductively glycated and illustrate the applicability of both phospholipase C and phospholipase D in characterizing glycated phosphoglycerides.

  2. Rheological properties of a vesicle suspension

    NASA Astrophysics Data System (ADS)

    Guedda, M.; Benlahsen, M.; Misbah, C.

    2014-11-01

    The rheological behavior of a dilute suspension of vesicles in linear shear flow at a finite concentration is analytically examined. In the quasispherical limit, two coupled nonlinear equations that describe the vesicle orientation in the flow and its shape evolution were derived [Phys. Rev. Lett. 96, 028104 (2006), 10.1103/PhysRevLett.96.028104] and serve here as a starting point. Of special interest is to provide, for the first time, an exact analytical prediction of the time-dependent effective viscosity ηeff and normal stress differences N1 and N2. Our results shed light on the effect of the viscosity ratio λ (defined as the inner over the outer fluid viscosities) as the main controlling parameter. It is shown that ηeff,N1 , and N2 either tend to a steady state or describe a periodic time-dependent rheological response, previously reported numerically and experimentally. In particular, the shear viscosity minimum and the cusp singularities of ηeff,N1 , and N2 at the tumbling threshold are brought to light. We also report on rheology properties for an arbitrary linear flow. We were able to obtain a constitutive law in a closed form relating the stress tensor to the strain rate tensor. It is found that the resulting constitutive markedly contrasts with classical laws known for other complex fluids, such as emulsions, capsule suspensions, and dilute polymer solutions (Oldroyd B model). We highlight the main differences between our law and classical laws.

  3. Tetraspanins in extracellular vesicle formation and function.

    PubMed

    Andreu, Zoraida; Yáñez-Mó, María

    2014-01-01

    Extracellular vesicles (EVs) represent a novel mechanism of intercellular communication as vehicles for intercellular transfer of functional membrane and cytosolic proteins, lipids, and RNAs. Microvesicles, ectosomes, shedding vesicles, microparticles, and exosomes are the most common terms to refer to the different kinds of EVs based on their origin, composition, size, and density. Exosomes have an endosomal origin and are released by many different cell types, participating in different physiological and/or pathological processes. Depending on their origin, they can alter the fate of recipient cells according to the information transferred. In the last two decades, EVs have become the focus of many studies because of their putative use as non-invasive biomarkers and their potential in bioengineering and clinical applications. In order to exploit this ability of EVs many aspects of their biology should be deciphered. Here, we review the mechanisms involved in EV biogenesis, assembly, recruitment of selected proteins, and genetic material as well as the uptake mechanisms by target cells in an effort to understand EV functions and their utility in clinical applications. In these contexts, the role of proteins from the tetraspanin superfamily, which are among the most abundant membrane proteins of EVs, will be highlighted.

  4. Bacterial outer membrane vesicles and vaccine applications.

    PubMed

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

    2014-01-01

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

  5. Bacterial Outer Membrane Vesicles and Vaccine Applications

    PubMed Central

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

    2014-01-01

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

  6. A kinetic study of stimulus-induced vesicle recycling in electromotor nerve terminals using labile and stable vesicle markers.

    PubMed

    Agoston, D V; Dowe, G H; Fiedler, W; Giompres, P E; Roed, I S; Walker, J H; Whittaker, V P; Yamaguchi, T

    1986-11-01

    The kinetics of recovery, by recycling electromotor synaptic vesicles, of the biophysical parameters of the reserve population has been studied in perfused blocks of electric organ of Torpedo marmorata prestimulated in vivo, followed by density gradient separation of the extracted vesicles in a zonal rotor using labile (acetylcholine and ATP) and stable (proteoglycan) vesicle markers. Stimulation in vivo at 0.15 Hz for 3.3 h depleted tissue acetylcholine much less than stimulation at 1 Hz for 1 h but nevertheless generated a much larger pool of recycled vesicles that recovered more slowly. At the lower rate of stimulation, recovery of the biophysical characteristics of the reserve population by the recycled vesicles, identified by their content of newly synthesized transmitter, was essentially complete by 8 h. The stable proteoglycan marker was immunochemically assayed and was bimodally distributed in the vesicle-containing portion of the density gradient even in experiments with unstimulated or recovered tissue. The second peak corresponded with that of newly synthesized transmitter and was thus identified as containing the recycled vesicles. Its normalized acetylcholine/proteoglycan ratio was lower than that of the first peak, which is consistent with earlier findings that recycled vesicles, before recovery, are only partially loaded with transmitter. However, as expected, the proportion of total vesicular proteoglycan and acetylcholine associated with the recycled vesicle fraction was very much lower in preparations derived from unstimulated or recovered tissue than in those from recently stimulated tissue.

  7. 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…

  8. Schwannoma, a rare tumor of the seminal vesicle

    PubMed Central

    Carrasquinho, Eduardo; Ferreira, Marco; Afonso, Ana; Ferrito, Fernando

    2011-01-01

    We present a rare case of a schwannoma of the seminal vesicle that occurred in a 43-year-old male with symptoms of the lower urinary tract. Ultrasonography and magnetic resonance imaging documented a solid mass in the patient's left seminal vesicle. A transvesical approach with a transtrigonal midline incision was successfully performed. The microscopic aspect was compatible with schwannoma. PMID:24578861

  9. A new role for myosin II in vesicle fission.

    PubMed

    Flores, Juan A; Balseiro-Gomez, Santiago; Cabeza, Jose M; Acosta, Jorge; Ramirez-Ponce, Pilar; Ales, Eva

    2014-01-01

    An endocytic vesicle is formed from a flat plasma membrane patch by a sequential process of invagination, bud formation and fission. The scission step requires the formation of a tubular membrane neck (the fission pore) that connects the endocytic vesicle with the plasma membrane. Progress in vesicle fission can be measured by the formation and closure of the fission pore. Live-cell imaging and sensitive biophysical measurements have provided various glimpses into the structure and behaviour of the fission pore. In the present study, the role of non-muscle myosin II (NM-2) in vesicle fission was tested by analyzing the kinetics of the fission pore with perforated-patch clamp capacitance measurements to detect single vesicle endocytosis with millisecond time resolution in peritoneal mast cells. Blebbistatin, a specific inhibitor of NM-2, dramatically increased the duration of the fission pore and also prevented closure during large endocytic events. Using the fluorescent markers FM1-43 and pHrodo Green dextran, we found that NM-2 inhibition greatly arrested vesicle fission in a late phase of the scission event when the pore reached a final diameter of ∼ 5 nm. Our results indicate that loss of the ATPase activity of myosin II drastically reduces the efficiency of membrane scission by making vesicle closure incomplete and suggest that NM-2 might be especially relevant in vesicle fission during compound endocytosis.

  10. Microscopic evaluation of vesicles shed by erythrocytes at elevated temperatures.

    PubMed

    Moore, Timothy; Sorokulova, Iryna; Pustovyy, Oleg; Globa, Ludmila; Pascoe, David; Rudisill, Mary; Vodyanoy, Vitaly

    2013-11-01

    The images of human erythrocytes and vesicles were analyzed by a light microscopy system with spatial resolution of better than 90 nm. The samples were observed in an aqueous environment and required no freezing, dehydration, staining, shadowing, marking, or any other manipulation. Temperature elevation resulted in significant concentration increase of structurally transformed erythrocytes (echinocytes) and vesicles in the blood. The process of vesicle separation from spiculated erythrocytes was video recorded in real time. At a temperature of 37°C, mean vesicle concentrations and diameters were found to be 1.50 ± 0.35 × 10(6) vesicles per microliter and 0.365 ± 0.065 μm, respectively. The vesicle concentration increased approximately threefold as the temperature increased from 37 to 40°C. It was estimated that 80% of all vesicles found in the blood are smaller than 0.4 μm. Accurate account of vesicle numbers and dimensions suggest that 86% of the lost erythrocyte material is lost not by vesiculation but by another, as yet, unknown mechanism.

  11. Slow Sedimentation and Deformability of Charged Lipid Vesicles

    PubMed Central

    Rey Suárez, Iván; Leidy, Chad; Téllez, Gabriel; Gay, Guillaume; Gonzalez-Mancera, Andres

    2013-01-01

    The study of vesicles in suspension is important to understand the complicated dynamics exhibited by cells in in vivo and in vitro. We developed a computer simulation based on the boundary-integral method to model the three dimensional gravity-driven sedimentation of charged vesicles towards a flat surface. The membrane mechanical behavior was modeled using the Helfrich Hamiltonian and near incompressibility of the membrane was enforced via a model which accounts for the thermal fluctuations of the membrane. The simulations were verified and compared to experimental data obtained using suspended vesicles labelled with a fluorescent probe, which allows visualization using fluorescence microscopy and confers the membrane with a negative surface charge. The electrostatic interaction between the vesicle and the surface was modeled using the linear Derjaguin approximation for a low ionic concentration solution. The sedimentation rate as a function of the distance of the vesicle to the surface was determined both experimentally and from the computer simulations. The gap between the vesicle and the surface, as well as the shape of the vesicle at equilibrium were also studied. It was determined that inclusion of the electrostatic interaction is fundamental to accurately predict the sedimentation rate as the vesicle approaches the surface and the size of the gap at equilibrium, we also observed that the presence of charge in the membrane increases its rigidity. PMID:23874582

  12. A New Role for Myosin II in Vesicle Fission

    PubMed Central

    Cabeza, Jose M.; Acosta, Jorge; Ramirez-Ponce, Pilar; Ales, Eva

    2014-01-01

    An endocytic vesicle is formed from a flat plasma membrane patch by a sequential process of invagination, bud formation and fission. The scission step requires the formation of a tubular membrane neck (the fission pore) that connects the endocytic vesicle with the plasma membrane. Progress in vesicle fission can be measured by the formation and closure of the fission pore. Live-cell imaging and sensitive biophysical measurements have provided various glimpses into the structure and behaviour of the fission pore. In the present study, the role of non-muscle myosin II (NM-2) in vesicle fission was tested by analyzing the kinetics of the fission pore with perforated-patch clamp capacitance measurements to detect single vesicle endocytosis with millisecond time resolution in peritoneal mast cells. Blebbistatin, a specific inhibitor of NM-2, dramatically increased the duration of the fission pore and also prevented closure during large endocytic events. Using the fluorescent markers FM1-43 and pHrodo Green dextran, we found that NM-2 inhibition greatly arrested vesicle fission in a late phase of the scission event when the pore reached a final diameter of ∼ 5 nm. Our results indicate that loss of the ATPase activity of myosin II drastically reduces the efficiency of membrane scission by making vesicle closure incomplete and suggest that NM-2 might be especially relevant in vesicle fission during compound endocytosis. PMID:24959909

  13. Investigating how vesicle size influences vesicle adsorption on titanium oxide: a competition between steric packing and shape deformation.

    PubMed

    Ferhan, Abdul Rahim; Jackman, Joshua A; Cho, Nam-Joon

    2017-01-18

    Understanding the adsorption behavior of lipid vesicles at solid-liquid interfaces is important for obtaining fundamental insights into soft matter adsorbates as well as for practical applications such as supported lipid bilayer (SLB) fabrication. While the process of SLB formation has been highly scrutinized, less understood are the details of vesicle adsorption without rupture, especially at high surface coverages. Herein, we tackle this problem by employing simultaneous quartz crystal microbalance-dissipation (QCM-D) and localized surface plasmon resonance (LSPR) measurements in order to investigate the effect of vesicle size (84-211 nm diameter) on vesicle adsorption onto a titanium oxide surface. Owing to fundamental differences in the measurement principles of the two techniques as well as a mismatch in probing volumes, it was possible to determine both the lipid mass adsorbed near the sensor surface as well as the total mass of adsorbed lipid and hydrodynamically coupled solvent in the adsorbed vesicle layer as a whole. With increasing vesicle size, the QCM-D frequency signal exhibited monotonic behavior reaching an asymptotic value, whereas the QCM-D energy dissipation signal continued to increase according to the vesicle size. In marked contrast, the LSPR-tracked lipid mass near the sensor surface followed a parabolic trend, with the greatest corresponding measurement response occurring for intermediate-size vesicles. The findings reveal that the maximum extent of adsorbed vesicles contacting a solid surface occurs at an intermediate vesicle size due to the competing influences of vesicle deformation and steric packing. Looking forward, such information can be applied to control the molecular self-assembly of phospholipid assemblies as well as provide the basis for investigating deformable, soft matter adsorbates.

  14. Peroxyl radicals promoted changes in water permeability through gramicidin channels in DPPC and lecithin-PC vesicles.

    PubMed

    Soto, M A; Sotomayor, C P; Lissi, E A

    2003-03-01

    Gramicidin incorporation to DPPC or lecithin-PC large unilamellar vesicles (LUVs) leads to pore formation that, under hyper-osmotic conditions, produces a noticeable increase in the rate of trans-membrane water flow. This pore formation is more efficient in the more fluid lecithin-PC LUVs. Exposure of these vesicles to peroxyl radicals generated in the aerobic thermolysis of 2,2'-azo-bis(2-amidinopropane) (AAPH), changes the physical properties of the bilayer (as sensed employing fluorescent probes), modifies gramicidin molecules (as sensed by the decrease in Trp fluorescence) and notably reduces the transbilayer rate of water outflow. In order to evaluate if this reduced water-transport capacity is due to changes in the membrane due to lipid-peroxidation and/or direct damage to gramicidin channels, results obtained in the oxidable vesicles (lecithin-PC) were compared to those obtained in DPPC vesicles. The data obtained show that most of the water transport efficiency loss can be ascribed to a direct disruption of gramicidin channels by AAPH derived peroxyl radicals.

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

    PubMed

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

    2008-06-17

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

  16. Critical dynamics of vesicle stretching transition in elongational flow.

    PubMed

    Kantsler, Vasiliy; Segre, Enrico; Steinberg, Victor

    2008-07-25

    We present results on the stretching of single tubular vesicles in an elongation flow toward dumbbell shapes, and on their relaxation. A critical strain rate epsilonc exists; for strain rates epsilonvesicle remains tubular but fluctuates, though its steady state extension increases with the strain rate epsilon. Above epsilonc, first a shape transition to dumbbell occurs, and then high order shape modes become unstable, leading to a pearling state. We have quantitatively characterized the transition and found a scaling of epsilonc with the system parameters. A remarkable feature of vesicle tube behavior around the critical point is a slowdown of the vesicle relaxation to the final extended state in the vesicle stretching. Such feature is similar to that found in continuous phase transitions and to the critical effects recently observed for polymer molecules near the coil-stretch transition in elongation flow.

  17. Placental Extracellular Vesicles and Feto-Maternal Communication

    PubMed Central

    Tong, M.; Chamley, L.W.

    2015-01-01

    The human placenta is an anatomically unique structure that extrudes a variety of extracellular vesicles into the maternal blood (including syncytial nuclear aggregates, microvesicles, and nanovesicles). Large quantities of extracellular vesicles are produced by the placenta in both healthy and diseased pregnancies. Since their first description more than 120 years ago, placental extracellular vesicles are only now being recognized as important carriers for proteins, lipids, and nucleic acids, which may play a crucial role in feto-maternal communication. Here, we summarize the current literature on the cargos of placental extracellular vesicles and the known effects of such vesicles on maternal cells/systems, especially those of the maternal immune and vascular systems. PMID:25635060

  18. Human placental coated vesicles contain receptor-bound transferrin.

    PubMed Central

    Booth, A G; Wilson, M J

    1981-01-01

    Human placental coated vesicles have been purified by a method involving sucrose-density-gradient centrifugation and treatment with wheat-germ agglutinin. These preparations were free of contamination by placental microvillus fragments. Crossed immunoelectrophoresis demonstrated that the coated vesicles contained a single serum protein, which was identified as transferrin. This transferrin was only observed after the vesicles were treated with a non-ionic detergent, and its behaviour during crossed hydrophobic-interaction immunoelectrophoresis suggested that a large proportion of it was receptor-bound. No other serum proteins, including immunoglobulin G, could be detected in these preparations. Receptor-bound transferrin was the only antigen common to placental coated vesicles and microvilli, implying that other plasma-membrane proteins are excluded from the region of membrane involved in coated-vesicle formation. Images PLATE 2 PLATE 1 Fig. 1. Fig. 2. Fig. 3. PMID:6272755

  19. Interaction of a potyviral VPg with anionic phospholipid vesicles

    SciTech Connect

    Rantalainen, Kimmo I.; Christensen, Peter A.; Hafren, Anders; Otzen, Daniel E.; Kalkkinen, Nisse; Maekinen, Kristiina

    2009-12-05

    The viral genome-linked protein (VPg) of Potato virus A (PVA) is a multifunctional protein that belongs to a class of intrinsically disordered proteins. Typically, this type of protein gains a more stable structure upon interactions or posttranslational modifications. In a membrane lipid strip overlay binding assay, PVA VPg was found to bind phosphatidylserine (PS), but not phosphatidylcholine (PC). According to circular dichroism spectroscopy, the secondary structure of PVA VPg was stabilized upon interactions with PS and phosphatidylglycerol (PG), but not with PC vesicles. It is possible that this stabilization favored the formation of alpha-helical structures. Limited tryptic digestion showed that the interaction with anionic vesicles protected certain, otherwise accessible, trypsin cleavage sites. An electron microscopy study revealed that interaction with VPg substantially increased the vesicle diameter and caused the formation of pore or plaque-like electron dense spots on the vesicle surface, which gradually led to disruption of the vesicles.

  20. A light-scattering characterization of membrane vesicles.

    PubMed Central

    Selser, J C; Yeh, Y; Baskin, R J

    1976-01-01

    A technique has been developed in this paper which enables quasi-elastic laser light scattering to be used to accurately and quantitatively measure the average vesicle diffusion coefficient and the relative dispersion in the diffusion coefficient about this average for dilute polydisperse vesicle suspensions. This technique relies on a theoretical analysis of a modified form of the Z-averaged diffusion coefficient. This modified Z-averaged diffusion coefficient explicitly incorporates vesicle size, structure, and polydispersity in a description of the scattered light autocorrelation spectrum. Light-scattering experiments were performed on a dilute, lobster sarcoplasmic reticulum vesicle suspension and the measured average diffusion coefficient and the diffusion coefficient relative dispersion about this average were determined with accuracies of 2 and 10%, respectively. A comparison of vesicle size inferred from light-scattering results was made with size results from electron microscopic analysis of the same sample. Images FIGURE 2 FIGURE 3 FIGURE 4 PMID:1252585

  1. Redox-Reactive Membrane Vesicles produced by Shewanella

    SciTech Connect

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

    2008-06-01

    Dissimilatory iron reducing bacteria produce and release membrane vesicles with diameters ranging from 50 to 250 nm. The vesicles, which arise from the outer membrane of these Gram-negative bacteria, lack DNA but contain proteins that catalyze the reduction of ferric iron and other multivalent heavy metals and radionuclides. This enzymatic process results in the formation of nano-size biogenic mineral assemblages that resemble nanofossils. Under low-shear conditions, membrane vesicles are commonly tethered to intact cells by electrically conductive filaments known as bacterial nanowires. The functional role of membrane vesicles and associated nanowires is not known, but the potential for mineralized vesicles that morphologically resemble nanofossils to serve as paleontological indicators of early life on earth and as biosignatures of like on other planets is recognized.

  2. Charge-reversal instability in mixed bilayer vesicles

    NASA Astrophysics Data System (ADS)

    Chen, Yi; Nelson, Philip

    2000-08-01

    Bilayer vesicles form readily from mixtures of charged and neutral surfactants. When such a mixed vesicle binds an oppositely charged object, its membrane partially demixes: the adhesion zone recruits more charged surfactants from the rest of the membrane. Given an unlimited supply of adhering objects one might expect the vesicle to remain attractive until it was completely covered. Contrary to this expectation, we show that a vesicle can instead exhibit adhesion saturation, partitioning spontaneously into an attractive zone with definite area fraction, and a repulsive zone. The latter zone rejects additional incoming objects because counterions on the interior of the vesicle migrate there, effectively reversing the membrane's charge. The effect is strongest at high surface charge densities, low ionic strength, and with thin, impermeable membranes. Adhesion saturation in such a situation has recently been observed experimentally [H. Aranda-Espinoza et al., Science 285, 394 (1999)].

  3. Translocation of an Incompressible Vesicle through a Pore

    PubMed Central

    Shojaei, Hamid R.; Muthukumar, Murugappan

    2016-01-01

    We have derived the free energy landscape for the translocation of a single vesicle through a narrow pore by accounting for bending and stretching of the vesicle, and the deformation of the vesicle by the pore. Emergence of a free energy barrier for translocation is a general result, and the magnitude of the barrier is calculated in terms of the various material parameters. The extent of the reduction in the barrier by the presence of an external constant force is calculated. Using the Fokker–Planck formalism, we have calculated the average translocation time corresponding to the various free energy landscapes representing different parameter sets. The dependencies of the average translocation time on the strength of the external force, vesicle size, bending and stretching moduli of the vesicle, and radius and length of the pore are derived, and the computed results are discussed. PMID:27089012

  4. Army Prisoner Population Prediction Study (AP3).

    DTIC Science & Technology

    1983-06-01

    portion represents incarceration , the type confinement facility, and the length of sentences for offenders. It includes "good time" accrual and...34Prevalence and Recidivism Index Ar- rests: A Feedback Model," Law and Society Review, Vol 16, No 2, 1981 Blumstein, A. and Nagin, D., "On the...Optimum Use of Incarceration for Crime Control," Operations Research, Vol 26, No 3, 1978 Blumsteln, A. and Moitra, S., "An Analysis of the Time Series of

  5. ATP-binding cassette-like transporters are involved in the transport of lignin precursors across plasma and vacuolar membranes

    SciTech Connect

    Miao, Y.C.; Liu, C.

    2010-12-28

    Lignin is a complex biopolymer derived primarily from the condensation of three monomeric precursors, the monolignols. The synthesis of monolignols occurs in the cytoplasm. To reach the cell wall where they are oxidized and polymerized, they must be transported across the cell membrane. However, the molecular mechanisms underlying the transport process are unclear. There are conflicting views about whether the transport of these precursors occurs by passive diffusion or is an energized active process; further, we know little about what chemical forms are required. Using isolated plasma and vacuolar membrane vesicles prepared from Arabidopsis, together with applying different transporter inhibitors in the assays, we examined the uptake of monolignols and their derivatives by these native membrane vesicles. We demonstrate that the transport of lignin precursors across plasmalemma and their sequestration into vacuoles are ATP-dependent primary-transport processes, involving ATP-binding cassette-like transporters. Moreover, we show that both plasma and vacuolar membrane vesicles selectively transport different forms of lignin precursors. In the presence of ATP, the inverted plasma membrane vesicles preferentially take up monolignol aglycones, whereas the vacuolar vesicles are more specific for glucoconjugates, suggesting that the different ATP-binding cassette-like transporters recognize different chemical forms in conveying them to distinct sites, and that glucosylation of monolignols is necessary for their vacuolar storage but not required for direct transport into the cell wall in Arabidopsis.

  6. 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.

  7. Aggregation of phospholipid vesicles by water-soluble polymers.

    PubMed Central

    Meyuhas, D; Nir, S; Lichtenberg, D

    1996-01-01

    Water-soluble polymers such as dextran and polyethylene glycol are known to induce aggregation and size growth of phospholipid vesicles. The present study addresses the dependence of these processes on vesicle size and concentration, polymer molecular weight, temperature, and compartmentalization of the vesicles and polymers, using static and dynamic light scattering. Increasing the molecular weight of the polymers resulted in a reduction of the concentration of polymer needed for induction of aggregation of small unilamellar vesicles. The aggregation was fully reversible (by dilution), within a few seconds, up to a polymer concentration of at least 20 wt %. At relatively low phosphatidylcholine (PC) concentrations (up to approximately 1 mM), increasing the PC concentration resulted in faster kinetics of aggregation and reduced the threshold concentration of polymer required for rapid aggregation (CA). At higher PC concentrations, CA was only slightly dependent on the concentration of PC and was approximately equal to the overlapping concentration of the polymer (C*). The extent of aggregation was similar at 37 and 4 degrees C. Aggregation of large unilamellar vesicles required a lower polymer concentration, probably because aggregation occurs in a secondary minimum (without surface contact). In contrast to experiments in which the polymers were added directly to the vesicles, dialysis of the vesicles against polymer-containing solutions did not induce aggregation. Based on this result, it appears that exclusion of polymer from the hydration sphere of vesicles and the consequent depletion of polymer molecules from clusters of aggregated vesicles play the central role in the induction of reversible vesicle aggregation. The results of all the other experiments are consistent with this conclusion. PMID:8913598

  8. Microfluidic filtration system to isolate extracellular vesicles from blood.

    PubMed

    Davies, Ryan T; Kim, Junho; Jang, Su Chul; Choi, Eun-Jeong; Gho, Yong Song; Park, Jaesung

    2012-12-21

    Extracellular vesicles are released by various cell types, particularly tumor cells, and may be potential targets for blood-based cancer diagnosis. However, studies performed on blood-borne vesicles to date have been limited by lack of effective, standardized purification strategies. Using in situ prepared nanoporous membranes, we present a simple strategy employing a microfluidic filtration system to isolate vesicles from whole blood samples. This method can be applied to purify nano-sized particles from blood allowing isolation of intact extracellular vesicles, avoiding the need for laborious and potentially damaging centrifugation steps or overly specific antibody-based affinity purification. Porous polymer monoliths were integrated as membranes into poly(methyl methacrylate) microfluidic chips by benchtop UV photopolymerization through a mask, allowing precise positioning of membrane elements while preserving simplicity of device preparation. Pore size could be manipulated by changing the ratio of porogenic solvent to prepolymer solution, and was tuned to a size proper for extraction of vesicles. Using the membrane as a size exclusion filter, we separated vesicles from cells and large debris by injecting whole blood under pressure through the microfluidic device. To enhance isolation purity, DC electrophoresis was employed as an alternative driving force to propel particles across the filter and increase the separation efficiency of vesicles from proteins. From the whole blood of melanoma-grown mice, we isolated extracellular vesicles and performed RT-PCR to verify their contents of RNA. Melan A mRNA derived from melanoma tumor cells were found enriched in filtered samples, confirming the recovery of vesicles via their cargo. This filtration system can be incorporated into other on-chip processes enabling integrated sample preparation for the downstream analysis of blood-based extracellular vesicles.

  9. Vesicle dynamics during the atmospheric entry heating of cosmic spherules

    NASA Astrophysics Data System (ADS)

    Genge, M. J.

    2017-03-01

    Cosmic spherules are unique igneous objects that form by melting due to gas drag heating during atmospheric entry heating. Vesicles are an important component of many cosmic spherules since they suggest their precursors had finite volatile contents. Vesicle abundances in spherules decrease through the series porphyritic, glassy, barred, to cryptocrystalline spherules. Anomalous hollow spherules, with large off-center vesicles occur in both porphyritic and glassy spheres. Numerical simulation of the dynamic behavior of vesicles during atmospheric flight is presented that indicates vesicles rapidly migrate due to deceleration and separate from nonporphyritic particles. Modest rotation rates of tens of radians s-1 are, however, sufficient to impede loss of vesicles and may explain the presence of small solitary vesicles in barred, cryptocrystalline and glassy spherules. Rapid rotation at spin rates of several thousand radians s-1 are required to concentrate vesicles at the rotational axis and leads to rapid growth by coalescence and either separation or retention depending on the orientation of the rotational axis. Complex rapid rotations that concentrate vesicles in the core of particles are proposed as a mechanism for the formation of hollow spherules. High vesicle contents in porphyritic spherules suggest volatile-rich precursors; however, calculation of volatile retention indicates these have lost >99.9% of volatiles to degassing prior to melting. The formation of hollow spherules, by rapid spin, necessarily implies preatmospheric rotations of several thousand radians s-1. These particles are suggested to represent immature dust, recently released from parent bodies, in which rotations have not been slowed by magnetic damping.

  10. Comparative transcriptomic analysis of human and Drosophila extracellular vesicles

    PubMed Central

    Lefebvre, Fabio Alexis; Benoit Bouvrette, Louis Philip; Perras, Lilyanne; Blanchet-Cohen, Alexis; Garnier, Delphine; Rak, Janusz; Lécuyer, Éric

    2016-01-01

    Extracellular vesicles (EVs) are membrane-enclosed nanoparticles containing specific repertoires of genetic material. In mammals, EVs can mediate the horizontal transfer of various cargos and signaling molecules, notably miRNA and mRNA species. Whether this form of intercellular communication prevails in other metazoans remains unclear. Here, we report the first parallel comparative morphologic and transcriptomic characterization of EVs from Drosophila and human cellular models. Electronic microscopy revealed that human and Drosophila cells release similar EVs with diameters ranging from 30 to 200 nm, which contain complex populations of transcripts. RNA-seq identified abundant ribosomal RNAs, related pseudogenes and retrotransposons in human and Drosophila EVs. Vault RNAs and Y RNAs abounded in human samples, whereas small nucleolar RNAs involved in pseudouridylation were most prevalent in Drosophila EVs. Numerous mRNAs were identified, largely consisting of exonic sequences displaying full-length read coverage and enriched for translation and electronic transport chain functions. By analogy with human systems, these sizeable similarities suggest that EVs could potentially enable RNA-mediated intercellular communication in Drosophila. PMID:27282340

  11. Coxsackievirus B transmission and possible new roles for extracellular vesicles.

    PubMed

    Inal, Jameel M; Jorfi, Samireh

    2013-02-01

    Coxsackievirus B1, a member of the Picornaviridae family is a non-enveloped single-stranded RNA virus associated with human diseases including myocarditis and pancreatitis. Infection of the intestinal mucosa, lined by polarized epithelial cells, requires interaction of coxsackievirus with apically located DAF (decay-accelerating factor) before transport to the basolaterally located CAR (coxsackie and adenovirus receptor), where entry is mediated by endocytosis. As with many other non-enveloped viruses, coxsackievirus has to induce lysis of host cells in order to perpetuate infection. However, recent evidence indicates that virus spread to secondary sites is not only achieved by a lytic mechanism and a non-lytic cell-cell strategy has been suggested for coxsackievirus B3. A physical interaction between infected and non-infected cells has been shown to be an efficient mechanism for retroviral transmission and one type of extracellular vesicle, the exosome, has been implicated in HIV-1 transmission. HIV-1 also takes advantage of depolymerization of actin for spread between T-cells. Calpain-mediated depolymerization of the actin cytoskeleton, as a result of increases in intracellular calcium concentration during coxsackievirus infection, would result in a release of host cell-derived microvesicles. If so, we speculate that maybe such microvesicles, increasingly recognized as major vehicles mediating intercellular communication, could play a role in the intercellular transmission of non-enveloped viruses.

  12. Extracellular Vesicles in Chronic Obstructive Pulmonary Disease

    PubMed Central

    Kadota, Tsukasa; Fujita, Yu; Yoshioka, Yusuke; Araya, Jun; Kuwano, Kazuyoshi; Ochiya, Takahiro

    2016-01-01

    Chronic obstructive pulmonary disease (COPD) is characterized by the progression of irreversible airflow limitation and is a leading cause of morbidity and mortality worldwide. Although several crucial mechanisms of COPD pathogenesis have been studied, the precise mechanism remains unknown. Extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies, are released from almost all cell types and are recognized as novel cell–cell communication tools. They have been shown to carry and transfer a wide variety of molecules, such as microRNAs, messenger RNAs, and proteins, which are involved in physiological functions and the pathology of various diseases. Recently, EVs have attracted considerable attention in pulmonary research. In this review, we summarize the recent findings of EV-mediated COPD pathogenesis. We also discuss the potential clinical usefulness of EVs as biomarkers and therapeutic agents for the treatment of COPD. PMID:27801806

  13. Gas Vesicle Nanoparticles for Antigen Display

    PubMed Central

    DasSarma, Shiladitya; DasSarma, Priya

    2015-01-01

    Microorganisms like the halophilic archaeon Halobacterium sp. NRC-1 produce gas-filled buoyant organelles, which are easily purified as protein nanoparticles (called gas vesicles or GVNPs). GVNPs are non-toxic, exceptionally stable, bioengineerable, and self-adjuvanting. A large gene cluster encoding more than a dozen proteins has been implicated in their biogenesis. One protein, GvpC, found on the exterior surface of the nanoparticles, can accommodate insertions near the C-terminal region and results in GVNPs displaying the inserted sequences on the surface of the nanoparticles. Here, we review the current state of knowledge on GVNP structure and biogenesis as well as available studies on immunogenicity of pathogenic viral, bacterial, and eukaryotic proteins and peptides displayed on the nanoparticles. Recent improvements in genetic tools for bioengineering of GVNPs are discussed, along with future opportunities and challenges for development of vaccines and other applications. PMID:26350601

  14. 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.

  15. Glioblastoma extracellular vesicles: reservoirs of potential biomarkers

    PubMed Central

    Redzic, Jasmina S; Ung, Timothy H; Graner, Michael W

    2014-01-01

    Glioblastoma multiforme (GBM) is the most frequent and most devastating of the primary central nervous system tumors, with few patients living beyond 2 years postdiagnosis. The damage caused by the disease and our treatments for the patients often leave them physically and cognitively debilitated. Generally, GBMs appear after very short clinical histories and are discovered by imaging (using magnetic resonance imaging [MRI]), and the diagnosis is validated by pathology, following surgical resection. The treatment response and diagnosis of tumor recurrence are also tracked by MRI, but there are numerous problems encountered with these monitoring modalities, such as ambiguous interpretation and forms of pseudoprogression. Diagnostic, prognostic, and predictive biomarkers would be an immense boon in following treatment schemes and in determining recurrence, which often requires an invasive intracranial biopsy to verify imaging data. Extracellular vesicles (EVs) are stable, membrane-enclosed, virus-sized particles released from either the cell surface or from endosomal pathways that lead to the systemic release of EVs into accessible biofluids, such as serum/plasma, urine, cerebrospinal fluid, and saliva. EVs carry a wide variety of proteins, nucleic acids, lipids, and other metabolites, with many common features but with enough individuality to be able to identify the cell of origin of the vesicles. These components, if properly interrogated, could allow for the identification of tumor-derived EVs in biofluids, indicating tumor progression, relapse, or treatment failure. That knowledge would allow clinicians to continue with treatment regimens that were actually effective or to change course if the therapies were failing. Here, we review the features of GBM EVs, in terms of EV content and activities that may lead to the use of EVs as serially accessible biomarkers for diagnosis and treatment response in neuro-oncology. PMID:24634586

  16. Synaptic vesicle chips to assay botulinum neurotoxins

    PubMed Central

    2005-01-01

    BoNTs (botulinum neurotoxins), considered to be the most toxic of all biological substances, inhibit neurotransmission through proteolytic cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins [VAMP (vesicle-associated membrane protein, or synaptobrevin), SNAP-25 (25 kDa synaptosome-associated protein) or syntaxin]. Expansion in the use of BoNTs as therapeutic and cosmetic agents, and the potential threat they constitute as biological weapons, underlines the need for rapid and sensitive in vitro assays. Here, we present new automatized bioassays to detect VAMP cleavage by BoNT/B and F. Western blotting and SPR (surface plasmon resonance) methods revealed that BoNT/B and F totally cleave their substrate on immunoisolated SVs (synaptic vesicles). Real-time monitoring of the immunocapture of native SVs from crude lysates on SPR sensor chips enabled the detection of picogram amounts of different SV proteins. Pre-incubation of a membrane fraction containing SVs with BoNT specifically inhibited capture by anti-VAMP antibodies, and amounts as low as 0.1 pg of BoNT/B were detected. This automated SPR assay is approx. 200 times more sensitive, and 25 times more rapid, than the in vivo BoNT/B test currently used. Moreover, the method can be performed using a few thousand cultured neurons and constitutes a new screening assay for inhibitors. Our data indicate that native VAMP is an optimal substrate for in vitro BoNT assays that can be monitored by SPR. PMID:16011482

  17. 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.

  18. Heterogeneity in motor driven transport

    NASA Astrophysics Data System (ADS)

    Tabei, Ali

    2015-03-01

    I will discuss quantitative analysis of particle tracking data for motor driven vesicles inside an insulin secreting cell. We use this method to study the dynamical and structural heterogeneity inside the cell. I will discuss our effort to explain the origin of observed heterogeneity in intracellular transport. Finally, I will explain how analyzing directional correlations in transport trajectories reveals self-similarity in the diffusion media.

  19. Emerging roles of extracellular vesicles in neurodegenerative disorders: focus on HIV-associated neurological complications

    PubMed Central

    Hu, Guoku; Yang, Lu; Cai, Yu; Niu, Fang; Mezzacappa, Frank; Callen, Shannon; Fox, Howard S; Buch, Shilpa

    2016-01-01

    Exosomes are membrane-enriched extracellular vesicles with a proposed diameter in the range of 30–100 nm. They are released during both normal homeostasis as well as under pathological conditions by most cell types. In recent years, there has been robust interest in the study of these vesicles as conduits for the delivery of information between cells in both analogous as well as disparate tissues. Their ability to transport specialized cargo including signaling mediators, proteins, messenger RNA and miRNAs characterizes these vesicles as primary facilitators of cell-to-cell communication and regulation. Exosomes have also been demonstrated to have important roles in the field of cancer biology and metastasis. More recently, their role in several neurodegenerative disorders has been gaining increased momentum as these particles have been shown to promote the spread of toxic factors such as amyloid beta and prions, adding further validity to their role as important regulators of disease pathogenesis. This review briefly summarizes current findings and thoughts on exosome biology in the context of neurodegenerative disorders and the manipulation of these particles for the development of potential therapeutic strategies. PMID:27882942

  20. Extracellular vesicles as modulators of cell-to-cell communication in the healthy and diseased brain

    PubMed Central

    Pegtel, D. M.; Peferoen, L.; Amor, S.

    2014-01-01

    Homeostasis relies heavily on effective cell-to-cell communication. In the central nervous system (CNS), probably more so than in other organs, such communication is crucial to support and protect neurons especially during ageing, as well as to control inflammation, remove debris and infectious agents. Emerging evidence indicates that extracellular vesicles (EVs) including endosome-derived exosomes and fragments of the cellular plasma membrane play a key role in intercellular communication by transporting messenger RNA, microRNA (miRNA) and proteins. In neurodegenerative diseases, secreted vesicles not only remove misfolded proteins, but also transfer aggregated proteins and prions and are thus thought to perpetuate diseases by ‘infecting’ neighbouring cells with these pathogenic proteins. Conversely, in other CNS disorders signals from stressed cells may help control inflammation and inhibit degeneration. EVs may also reflect the status of the CNS and are present in the cerebrospinal fluid indicating that exosomes may act as biomarkers of disease. That extracellular RNA and in particular miRNA, can be transferred by EV also indicates that these vesicles could be used as carriers to specifically target the CNS to deliver immune modulatory drugs, neuroprotective agents and anti-cancer drugs. Here, we discuss the recent evidence indicating the potential role of exosomes in neurological disorders and how knowledge of their biology may enable a Trojan-horse approach to deliver drugs into the CNS and treat neurodegenerative and other disorders of the CNS. PMID:25135977

  1. Structure of yeast Ape1 and its role in autophagic vesicle formation

    PubMed Central

    Su, Ming-Yuan; Peng, Wen-Hsin; Ho, Meng-Ru; Su, Shih-Chieh; Chang, Yuan-Chih; Chen, Guang-Chao; Chang, Chung-I

    2015-01-01

    In Saccharomyces cerevisiae, a constitutive biosynthetic transport pathway, termed the cytoplasm-to-vacuole targeting (Cvt) pathway, sequesters precursor aminopeptidase I (prApe1) dodecamers in the form of a large complex into a Cvt vesicle using autophagic machinery, targeting it into the vacuole (the yeast lysosome) where it is proteolytically processed into its mature form, Ape1, by removal of an amino-terminal 45-amino acid propeptide. prApe1 is thought to serve as a scaffolding cargo critical for the assembly of the Cvt vesicle by presenting the propeptide to mediate higher-ordered complex formation and autophagic receptor recognition. Here we report the X-ray crystal structure of Ape1 at 2.5 Å resolution and reveal its dodecameric architecture consisting of dimeric and trimeric units, which associate to form a large tetrahedron. The propeptide of prApe1 exhibits concentration-dependent oligomerization and forms a stable tetramer. Structure-based mutagenesis demonstrates that disruption of the inter-subunit interface prevents dodecameric assembly and vacuolar targeting in vivo despite the presence of the propeptide. Furthermore, by examining the vacuolar import of propeptide-fused exogenous protein assemblies with different quaternary structures, we found that 3-dimensional spatial distribution of propeptides presented by a scaffolding cargo is essential for the assembly of the Cvt vesicle for vacuolar delivery. This study describes a molecular framework for understanding the mechanism of Cvt or autophagosomal biogenesis in selective macroautophagy. PMID:26208681

  2. Human Eosinophil Leukocytes Express Protein Disulfide Isomerase in Secretory Granules and Vesicles: Ultrastructural Studies.

    PubMed

    Dias, Felipe F; Amaral, Kátia B; Carmo, Lívia A S; Shamri, Revital; Dvorak, Ann M; Weller, Peter F; Melo, Rossana C N

    2014-06-01

    Protein disulfide isomerase (PDI) has fundamental roles in the oxidative folding of proteins in the endoplasmic reticulum (ER) of eukaryotic cells. The study of this molecule has been attracting considerable attention due to its association with other cell functions and human diseases. In leukocytes, such as neutrophils, PDI is involved with cell adhesion, signaling and inflammation. However, the expression of PDI in other leukocytes, such as eosinophils, important cells in inflammatory, allergic and immunomodulatory responses, remains to be defined. Here we used different approaches to investigate PDI expression within human eosinophils. Western blotting and flow cytometry demonstrated high PDI expression in both unstimulated and CCL11/eotaxin-1-stimulated eosinophils, with similar levels in both conditions. By using an immunogold electron microscopy technique that combines better epitope preservation and secondary Fab-fragments of antibodies linked to 1.4-nm gold particles for optimal access to microdomains, we identified different intracellular sites for PDI. In addition to predictable strong PDI labeling at the nuclear envelope, other unanticipated sites, such as secretory granules, lipid bodies and vesicles, including large transport vesicles (eosinophil sombrero vesicles), were also labeled. Thus, we provide the first identification of PDI in human eosinophils, suggesting that this molecule may have additional/specific functions in these leukocytes.

  3. Development, characterization, and skin delivery studies of related ultradeformable vesicles: transfersomes, ethosomes, and transethosomes

    PubMed Central

    Ascenso, Andreia; Raposo, Sara; Batista, Cátia; Cardoso, Pedro; Mendes, Tiago; Praça, Fabíola Garcia; Bentley, Maria Vitória Lopes Badra; Simões, Sandra

    2015-01-01

    Ultradeformable vesicles (UDV) have recently become a promising tool for the development of improved and innovative dermal and transdermal therapies. The aim of this work was to study three related UDV: transfersomes, ethosomes, and transethosomes for the incorporation of actives of distinct polarities, namely, vitamin E and caffeine, and to evaluate the effect of the carrier on skin permeation and penetration. These actives were incorporated in UDV formulations further characterized for vesicles imaging by transmission electron microscopy; mean vesicle size and polydispersity index by photon correlation spectroscopy; zeta potential by laser-Doppler anemometry; deformability by pressure-driven transport; and incorporation efficiency (IE) after actives quantification by high-performance liquid chromatography. Topical delivery studies were performed in order to compare UDV formulations regarding the release, skin permeation, and penetration profiles. All UDV formulations showed size values within the expected range, except transethosomes prepared by “transfersomal method”, for which size was smaller than 100 nm in contrast to that obtained for vesicles prepared by “ethosomal method”. Zeta potential was negative and higher for formulations containing sodium cholate. The IE was much higher for vitamin E- than caffeine-loaded UDV as expected. For flux measurements, the following order was obtained: transethosomes (TE) > ethosomes (E) ≥ transfersomes (T). This result was consistent with the release and skin penetration profiles for Vitamin E-loaded UDV. However, the releasing results were totally the opposite for caffeine-loaded UDV, which might be explained by the solubility and thermodynamic activity of this active in each formulation instead of the UDV deformability attending to the higher non-incorporated fraction of caffeine. Anyway, a high skin penetration and permeation for all caffeine-loaded UDV were obtained. Transethosomes were more deformable than

  4. Development, characterization, and skin delivery studies of related ultradeformable vesicles: transfersomes, ethosomes, and transethosomes.

    PubMed

    Ascenso, Andreia; Raposo, Sara; Batista, Cátia; Cardoso, Pedro; Mendes, Tiago; Praça, Fabíola Garcia; Bentley, Maria Vitória Lopes Badra; Simões, Sandra

    2015-01-01

    Ultradeformable vesicles (UDV) have recently become a promising tool for the development of improved and innovative dermal and transdermal therapies. The aim of this work was to study three related UDV: transfersomes, ethosomes, and transethosomes for the incorporation of actives of distinct polarities, namely, vitamin E and caffeine, and to evaluate the effect of the carrier on skin permeation and penetration. These actives were incorporated in UDV formulations further characterized for vesicles imaging by transmission electron microscopy; mean vesicle size and polydispersity index by photon correlation spectroscopy; zeta potential by laser-Doppler anemometry; deformability by pressure-driven transport; and incorporation efficiency (IE) after actives quantification by high-performance liquid chromatography. Topical delivery studies were performed in order to compare UDV formulations regarding the release, skin permeation, and penetration profiles. All UDV formulations showed size values within the expected range, except transethosomes prepared by "transfersomal method", for which size was smaller than 100 nm in contrast to that obtained for vesicles prepared by "ethosomal method". Zeta potential was negative and higher for formulations containing sodium cholate. The IE was much higher for vitamin E- than caffeine-loaded UDV as expected. For flux measurements, the following order was obtained: transethosomes (TE) > ethosomes (E) ≥ transfersomes (T). This result was consistent with the release and skin penetration profiles for Vitamin E-loaded UDV. However, the releasing results were totally the opposite for caffeine-loaded UDV, which might be explained by the solubility and thermodynamic activity of this active in each formulation instead of the UDV deformability attending to the higher non-incorporated fraction of caffeine. Anyway, a high skin penetration and permeation for all caffeine-loaded UDV were obtained. Transethosomes were more deformable than ethosomes

  5. Bmp4 from the optic vesicle specifies murine retina formation.

    PubMed

    Huang, Jie; Liu, Ying; Oltean, Alina; Beebe, David C

    2015-06-01

    Previous studies of mouse embryos concluded that after the optic vesicle evaginates from the ventral forebrain and contacts the surface ectoderm, signals from the ectoderm specify the distal region of the optic vesicle to become retina and signals from the optic vesicle induce the lens. Germline deletion of Bmp4 resulted in failure of lens formation. We performed conditional deletion of Bmp4 from the optic vesicle to test the function of Bmp4 in murine eye development. The optic vesicle evaginated normally and contacted the surface ectoderm. Lens induction did not occur. The optic cup failed to form and the expression of retina-specific genes decreased markedly in the distal optic vesicle. Instead, cells in the prospective retina expressed genes characteristic of the retinal pigmented epithelium. We conclude that Bmp4 is required for retina specification in mice. In the absence of Bmp4, formation of the retinal pigmented epithelium is the default differentiation pathway of the optic vesicle. Differences in the signaling pathways required for specification of the retina and retinal pigmented epithelium in chicken and mouse embryos suggest major changes in signaling during the evolution of the vertebrate eye.

  6. The dynamics of adhesion of a pair of vesicles

    NASA Astrophysics Data System (ADS)

    Walter, Johann; Leal, L. Gary

    2012-11-01

    Adhesive interactions within a suspension of vesicles, such as many personal care products, vectors for drug delivery or artificial blood, can lead to aggregation of the vesicles and dramatic changes to the properties of the suspension. We study the adhesion of a pair of unilamellar, charged vesicles under flow, in the presence of a non-adsorbing polymer or micelle creating a depletion attraction force between the vesicles. 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 (resistance to bending and area increase are both taken into account). The dynamics of the drainage process are studied. At steady state, the adhesion energy is found to depend greatly on the ability of the vesicles to increase their surface area. Finally, when the vesicles are separated in an elongational flow, different behaviors are observed depending on the deformability of the vesicles: an increase of the film thickness with a constant contact area, or peeling-off phenomenon where the contact area decreases at constant film thickness.

  7. Epsin1 modulates synaptic vesicle retrieval capacity at CNS synapses

    PubMed Central

    Kyung, Jae Won; Bae, Jae Ryul; Kim, Dae-Hwan; Song, Woo Keun; Kim, Sung Hyun

    2016-01-01

    Synaptic vesicle retrieval is an essential process for continuous maintenance of neural information flow after synaptic transmission. Epsin1, originally identified as an EPS15-interacting protein, is a major component of clathrin-mediated endocytosis. However, the role of Epsin1 in synaptic vesicle endocytosis at CNS synapses remains elusive. Here, we showed significantly altered synaptic vesicle endocytosis in neurons transfected with shRNA targeting Epsin1 during/after neural activity. Endocytosis was effectively restored by introducing shRNA-insensitive Epsin1 into Epsin1-depleted neurons. Domain studies performed on neurons in which domain deletion mutants of Epsin1 were introduced after Epsin1 knockdown revealed that ENTH, CLAP, and NPFs are essential for synaptic vesicle endocytosis, whereas UIMs are not. Strikingly, the efficacy of the rate of synaptic vesicle retrieval (the “endocytic capacity”) was significantly decreased in the absence of Epsin1. Thus, Epsin1 is required for proper synaptic vesicle retrieval and modulates the endocytic capacity of synaptic vesicles. PMID:27557559

  8. Observations of Calcium Dynamics in Cortical Secretory Vesicles

    PubMed Central

    Raveh, Adi; Valitsky, Michael; Shani, Liora; Coorssen, Jens R.; Blank, Paul S.; Zimmerberg, Joshua; Rahamimoff, Rami

    2012-01-01

    SUMMARY Calcium (Ca2+) dynamics were evaluated in fluorescently labeled sea urchin secretory vesicles using confocal microscopy. 71% of the vesicles examined exhibited one or more transient increases in the fluorescence signal that was damped in time. The detection of transient increases in signal was dependent upon the affinity of the fluorescence indicator; the free Ca2+ concentration in the secretory vesicles was estimated to be in the range of ~10 – 100 μM. Non-linear stochastic analysis revealed the presence of extra variance in the Ca2+ dependent fluorescence signal. This noise process increased linearly with the amplitude of the Ca2+ signal. Both the magnitude and spatial properties of this noise process were dependent upon the activity of vesicle p-type (Cav2.1) Ca2+ channels. Blocking the p-type Ca2+ channels with ω-agatoxin decreased signal variance, and altered the spatial noise pattern within the vesicle. These fluorescence signal properties are consistent with vesicle Ca2+ dynamics and not simply due to obvious physical properties such as gross movement artifacts or pH driven changes in Ca2+ indicator fluorescence. The results suggest that the free Ca2+ content of cortical secretory vesicles is dynamic; this property may modulate the exocytotic fusion process. PMID:22831912

  9. 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

  10. Removal of Vesicle Structures From Transmission Electron Microscope Images

    PubMed Central

    Jensen, Katrine Hommelhoff; Sigworth, Fred J.; Brandt, Sami Sebastian

    2016-01-01

    In this paper, we address the problem of imaging membrane proteins for single-particle cryo-electron microscopy reconstruction of the isolated protein structure. More precisely, we propose a method for learning and removing the interfering vesicle signals from the micrograph, prior to reconstruction. In our approach, we estimate the subspace of the vesicle structures and project the micrographs onto the orthogonal complement of this subspace. We construct a 2d statistical model of the vesicle structure, based on higher order singular value decomposition (HOSVD), by considering the structural symmetries of the vesicles in the polar coordinate plane. We then propose to lift the HOSVD model to a novel hierarchical model by summarizing the multidimensional HOSVD coefficients by their principal components. Along with the model, a solid vesicle normalization scheme and model selection criterion are proposed to make a compact and general model. The results show that the vesicle structures are accurately separated from the background by the HOSVD model that is also able to adapt to the asymmetries of the vesicles. This is a promising result and suggests even wider applicability of the proposed approach in learning and removal of statistical structures. PMID:26642456

  11. Dynamics of vesicles in a wall-bounded shear flow.

    PubMed

    Abkarian, M; Viallat, A

    2005-08-01

    We report a detailed study of the behavior (shapes, experienced forces, velocities) of giant lipid vesicles subjected to a shear flow close to a wall. Vesicle buoyancy, size, and reduced volume were separately varied. We show that vesicles are deformed by the flow and exhibit a tank-treading motion with steady orientation. Their shapes are characterized by two nondimensional parameters: the reduced volume and the ratio of the shear stress with the hydrostatic pressure. We confirm the existence of a force, able to lift away nonspherical buoyant vesicles from the substrate. We give the functional variation and the value of this lift force (up to 150 pN in our experimental conditions) as a function of the relevant physical parameters: vesicle-substrate distance, wall shear rate, viscosity of the solution, vesicle size, and reduced volume. Circulating deformable cells disclosing a nonspherical shape also experience this force of viscous origin, which contributes to take them away from the endothelium and should be taken into account in studies on cell adhesion in flow chambers, where cells membrane and the adhesive substrate are in relative motion. Finally, the kinematics of vesicles along the flow direction can be described in a first approximation with a model of rigid spheres.

  12. Photosynthetic vesicle architecture and constraints on efficient energy harvesting.

    PubMed

    Sener, Melih; Strümpfer, Johan; Timney, John A; Freiberg, Arvi; Hunter, C Neil; Schulten, Klaus

    2010-07-07

    Photosynthetic chromatophore vesicles found in some purple bacteria constitute one of the simplest light-harvesting systems in nature. The overall architecture of chromatophore vesicles and the structural integration of vesicle function remain poorly understood despite structural information being available on individual constituent proteins. An all-atom structural model for an entire chromatophore vesicle is presented, which improves upon earlier models by taking into account the stoichiometry of core and antenna complexes determined by the absorption spectrum of intact vesicles in Rhodobacter sphaeroides, as well as the well-established curvature-inducing properties of the dimeric core complex. The absorption spectrum of low-light-adapted vesicles is shown to correspond to a light-harvesting-complex 2 to reaction center ratio of 3:1. A structural model for a vesicle consistent with this stoichiometry is developed and used in the computation of excitonic properties. Considered also is the packing density of antenna and core complexes that is high enough for efficient energy transfer and low enough for quinone diffusion from reaction centers to cytochrome bc(1) complexes.

  13. Sar1 promotes vesicle budding from the endoplasmic reticulum but not Golgi compartments

    PubMed Central

    1994-01-01

    Two new members (Sar1a and Sar1b) of the SAR1 gene family have been identified in mammalian cells. Using immunoelectron microscopy, Sar1 was found to be restricted to the transitional region where the protein was enriched 20-40-fold in vesicular carriers mediating ER to Golgi traffic. Biochemical analysis revealed that Sar1 was essential for an early step in vesicle budding. A Sar1-specific antibody potently inhibited export of vesicular stomatitis virus glycoprotein (VSV-G) from the ER in vitro. Consistent with the role of guanine nucleotide exchange in Sar1 function, a trans-dominant mutant (Sar1a[T39N]) with a preferential affinity for GDP also strongly inhibited vesicle budding from the ER. In contrast, Sar1 was not found to be required for the transport of VSV-G between sequential Golgi compartments, suggesting that components active in formation of vesicular carriers mediating ER to Golgi traffic may differ, at least in part, from those involved in intra-Golgi transpor