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Sample records for readily releasable vesicles

  1. Cholecystokinin facilitates glutamate release by increasing the number of readily releasable vesicles and releasing probability.

    PubMed

    Deng, Pan-Yue; Xiao, Zhaoyang; Jha, Archana; Ramonet, David; Matsui, Toshimitsu; Leitges, Michael; Shin, Hee-Sup; Porter, James E; Geiger, Jonathan D; Lei, Saobo

    2010-04-14

    Cholecystokinin (CCK), a neuropeptide originally discovered in the gastrointestinal tract, is abundantly distributed in the mammalian brains including the hippocampus. Whereas CCK has been shown to increase glutamate concentration in the perfusate of hippocampal slices and in purified rat hippocampal synaptosomes, the cellular and molecular mechanisms whereby CCK modulates glutamatergic function remain unexplored. Here, we examined the effects of CCK on glutamatergic transmission in the hippocampus using whole-cell recordings from hippocampal slices. Application of CCK increased AMPA receptor-mediated EPSCs at perforant path-dentate gyrus granule cell, CA3-CA3 and Schaffer collateral-CA1 synapses without effects at mossy fiber-CA3 synapses. CCK-induced increases in AMPA EPSCs were mediated by CCK-2 receptors and were not modulated developmentally and transcriptionally. CCK reduced the coefficient of variation and paired-pulse ratio of AMPA EPSCs suggesting that CCK facilitates presynaptic glutamate release. CCK increased the release probability and the number of readily releasable vesicles with no effects on the rate of recovery from vesicle depletion. CCK-mediated increases in glutamate release required the functions of phospholipase C, intracellular Ca(2+) release and protein kinase Cgamma. CCK released endogenously from hippocampal interneurons facilitated glutamatergic transmission. Our results provide a cellular and molecular mechanism to explain the roles of CCK in the brain.

  2. Readily releasable vesicles recycle at the active zone of hippocampal synapses.

    PubMed

    Schikorski, Thomas

    2014-04-08

    During the synaptic vesicle cycle, synaptic vesicles fuse with the plasma membrane and recycle for repeated exo/endocytic events. By using activity-dependent N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino) styryl) pyridinium dibromide dye uptake combined with fast (<1 s) microwave-assisted fixation followed by photoconversion and ultrastructural 3D analysis, we tracked endocytic vesicles over time, "frame by frame." The first retrieved synaptic vesicles appeared 4 s after stimulation, and these endocytic vesicles were located just above the active zone. Second, the retrieved vesicles did not show any sign of a protein coat, and coated pits were not detected. Between 10 and 30 s, large labeled vesicles appeared that had up to 5 times the size of an individual synaptic vesicle. Starting at around 20 s, these large labeled vesicles decreased in number in favor of labeled synaptic vesicles, and after 30 s, labeled vesicles redocked at the active zone. The data suggest that readily releasable vesicles are retrieved as noncoated vesicles at the active zone.

  3. Temperature-Dependent Differences between Readily Releasable and Reserve Pool Vesicles in Chromaffin Cells

    PubMed Central

    Haynes, Christy L.; Siff, Lauren N.; Wightman, R. Mark

    2007-01-01

    Summary Statistical differences between amperometric traces recorded from chromaffin cells using K+ and Ba2+ secretagogues support the assertion that readily releasable pool (RRP) and reserve pool (RP) vesicles can be probed with pool-specific secretagogues. Release from the RRP was evoked by K+ while release from the RP was evoked by Ba2+. Similar temperature-dependent changes in spike area and half-width for both pools suggest that the content of RRP and RP vesicles is similar and packaged in the same way. Differences between the vesicle pools were revealed in the temperature dependence of spike frequency. While the burst spike frequency of the RRP, which is comprised of pre-docked and primed vesicles, increased 2.8% per °C, the RP spike frequency increased 12% per °C. This difference is attributed to a temperature dependent mobilization of the RP. Furthermore, the RP exhibited more foot events at room temperature than the RRP but this difference was not apparent at 37°C. This trend suggests that RP vesicle membranes have a compromised surface tension compared to RRP vesicles. Collectively, the changes of release characteristics with temperature reveal distinctions between the RRP and the RP. PMID:17467077

  4. Readily releasable pool of synaptic vesicles measured at single synaptic contacts.

    PubMed

    Trigo, Federico F; Sakaba, Takeshi; Ogden, David; Marty, Alain

    2012-10-30

    To distinguish between different models of vesicular release in brain synapses, it is necessary to know the number of vesicles of transmitter that can be released immediately at individual synapses by a high-calcium stimulus, the readily releasable pool (RRP). We used direct stimulation by calcium uncaging at identified, single-site inhibitory synapses to investigate the statistics of vesicular release and the size of the RRP. Vesicular release, detected as quantal responses in the postsynaptic neuron, showed an unexpected stochastic variation in the number of quanta from stimulus to stimulus at high intracellular calcium, with a mean of 1.9 per stimulus and a maximum of three or four. The results provide direct measurement of the RRP at single synaptic sites. They are consistent with models in which release proceeds from a small number of vesicle docking sites with an average occupancy around 0.7.

  5. Long-Term Culture of Astrocytes Attenuates the Readily Releasable Pool of Synaptic Vesicles

    PubMed Central

    Kawano, Hiroyuki; Katsurabayashi, Shutaro; Kakazu, Yasuhiro; Yamashita, Yuta; Kubo, Natsuko; Kubo, Masafumi; Okuda, Hideto; Takasaki, Kotaro; Kubota, Kaori; Mishima, Kenichi; Fujiwara, Michihiro; Harata, N. Charles; Iwasaki, Katsunori

    2012-01-01

    The astrocyte is a major glial cell type of the brain, and plays key roles in the formation, maturation, stabilization and elimination of synapses. Thus, changes in astrocyte condition and age can influence information processing at synapses. However, whether and how aging astrocytes affect synaptic function and maturation have not yet been thoroughly investigated. Here, we show the effects of prolonged culture on the ability of astrocytes to induce synapse formation and to modify synaptic transmission, using cultured autaptic neurons. By 9 weeks in culture, astrocytes derived from the mouse cerebral cortex demonstrated increases in β-galactosidase activity and glial fibrillary acidic protein (GFAP) expression, both of which are characteristic of aging and glial activation in vitro. Autaptic hippocampal neurons plated on these aging astrocytes showed a smaller amount of evoked release of the excitatory neurotransmitter glutamate, and a lower frequency of miniature release of glutamate, both of which were attributable to a reduction in the pool of readily releasable synaptic vesicles. Other features of synaptogenesis and synaptic transmission were retained, for example the ability to induce structural synapses, the presynaptic release probability, the fraction of functional presynaptic nerve terminals, and the ability to recruit functional AMPA and NMDA glutamate receptors to synapses. Thus the presence of aging astrocytes affects the efficiency of synaptic transmission. Given that the pool of readily releasable vesicles is also small at immature synapses, our results are consistent with astrocytic aging leading to retarded synapse maturation. PMID:23110166

  6. Superpriming of synaptic vesicles after their recruitment to the readily releasable pool

    PubMed Central

    Lee, Jae Sung; Ho, Won-Kyung; Neher, Erwin; Lee, Suk-Ho

    2013-01-01

    Recruitment of release-competent vesicles during sustained synaptic activity is one of the major factors governing short-term plasticity. During bursts of synaptic activity, vesicles are recruited to a fast-releasing pool from a reluctant vesicle pool through an actin-dependent mechanism. We now show that newly recruited vesicles in the fast-releasing pool do not respond at full speed to a strong Ca2+ stimulus, but require approximately 4 s to mature to a “superprimed” state. Superpriming was found to be altered by agents that modulate the function of unc13 homolog proteins (Munc13s), but not by calmodulin inhibitors or actin-disrupting agents. These findings indicate that recruitment and superpriming of vesicles are regulated by separate mechanisms, which require integrity of the cytoskeleton and activation of Munc13s, respectively. We propose that refilling of the fast-releasing vesicle pool proceeds in two steps, rapid actin-dependent “positional priming,” which brings vesicles closer to Ca2+ sources, followed by slower superpriming, which enhances the Ca2+ sensitivity of primed vesicles. PMID:23980146

  7. A Well-Defined Readily Releasable Pool with Fixed Capacity for Storing Vesicles at Calyx of Held

    PubMed Central

    Mahfooz, Kashif; Singh, Mahendra; Renden, Robert; Wesseling, John F.

    2016-01-01

    The readily releasable pool (RRP) of vesicles is a core concept in studies of presynaptic function. However, operating principles lack consensus definition and the utility for quantitative analysis has been questioned. Here we confirm that RRPs at calyces of Held from 14 to 21 day old mice have a fixed capacity for storing vesicles that is not modulated by Ca2+. Discrepancies with previous studies are explained by a dynamic flow-through pool, established during heavy use, containing vesicles that are released with low probability despite being immediately releasable. Quantitative analysis ruled out a posteriori explanations for the vesicles with low release probability, such as Ca2+-channel inactivation, and established unexpected boundary conditions for remaining alternatives. Vesicles in the flow-through pool could be incompletely primed, in which case the full sequence of priming steps downstream of recruitment to the RRP would have an average unitary rate of at least 9/s during heavy use. Alternatively, vesicles with low and high release probability could be recruited to distinct types of release sites; in this case the timing of recruitment would be similar at the two types, and the downstream transition from recruited to fully primed would be much faster. In either case, further analysis showed that activity accelerates the upstream step where vesicles are initially recruited to the RRP. Overall, our results show that the RRP can be well defined in the mathematical sense, and support the concept that the defining mechanism is a stable group of autonomous release sites. PMID:27035349

  8. Synaptotagmin-1 and -7 Are Redundantly Essential for Maintaining the Capacity of the Readily-Releasable Pool of Synaptic Vesicles

    PubMed Central

    Burré, Jacqueline; Malenka, Robert C.; Liu, Xinran; Südhof, Thomas C.

    2015-01-01

    In forebrain neurons, Ca2+ triggers exocytosis of readily releasable vesicles by binding to synaptotagmin-1 and -7, thereby inducing fast and slow vesicle exocytosis, respectively. Loss-of-function of synaptotagmin-1 or -7 selectively impairs the fast and slow phase of release, respectively, but does not change the size of the readily-releasable pool (RRP) of vesicles as measured by stimulation of release with hypertonic sucrose, or alter the rate of vesicle priming into the RRP. Here we show, however, that simultaneous loss-of-function of both synaptotagmin-1 and -7 dramatically decreased the capacity of the RRP, again without altering the rate of vesicle priming into the RRP. Either synaptotagmin-1 or -7 was sufficient to rescue the RRP size in neurons lacking both synaptotagmin-1 and -7. Although maintenance of RRP size was Ca2+-independent, mutations in Ca2+-binding sequences of synaptotagmin-1 or synaptotagmin-7—which are contained in flexible top-loop sequences of their C2 domains—blocked the ability of these synaptotagmins to maintain the RRP size. Both synaptotagmins bound to SNARE complexes; SNARE complex binding was reduced by the top-loop mutations that impaired RRP maintenance. Thus, synaptotagmin-1 and -7 perform redundant functions in maintaining the capacity of the RRP in addition to nonredundant functions in the Ca2+ triggering of different phases of release. PMID:26437117

  9. Synapsin-dependent reserve pool of synaptic vesicles supports replenishment of the readily releasable pool under intense synaptic transmission.

    PubMed

    Vasileva, Mariya; Horstmann, Heinz; Geumann, Constanze; Gitler, Daniel; Kuner, Thomas

    2012-10-01

    Synapsins are abundant synaptic vesicle (SV)-associated proteins thought to mediate synaptic vesicle mobility and clustering at most synapses. We used synapsin triple knock-out (TKO) mice to examine the morphological and functional consequences of deleting all synapsin isoforms at the calyx of Held, a giant glutamatergic synapse located in the auditory brain stem. Quantitative three-dimensional (3D) immunohistochemistry of entire calyces showed lower amounts of the synaptic vesicle protein vGluT1 while the level of the active zone marker bassoon was unchanged in TKO terminals. Examination of brain lysates by ELISA revealed a strong reduction in abundance of several synaptic vesicle proteins, while proteins of the active zone cytomatrix or postsynaptic density were unaffected. Serial section scanning electron microscopy of large 3D-reconstructed segments confirmed a decrease in the number of SVs to approximately 50% in TKO calyces. Short-term depression tested at stimulus frequencies ranging from 10 to 300 Hz was accelerated only at frequencies above 100 Hz and the time course of recovery from depression was slowed in calyces lacking synapsins. These results reveal that in wild-type synapses, the synapsin-dependent reserve pool contributes to the replenishment of the readily releasable pool (RRP), although accounting only for a small fraction of the SVs that enter the RRP. In conclusion, our results suggest that synapsins may be required for normal synaptic vesicle biogenesis, trafficking and immobilization of synaptic vesicles, yet they are not essential for sustained high-frequency synaptic transmission at the calyx terminal.

  10. Stress and corticosterone increase the readily releasable pool of glutamate vesicles in synaptic terminals of prefrontal and frontal cortex.

    PubMed

    Treccani, G; Musazzi, L; Perego, C; Milanese, M; Nava, N; Bonifacino, T; Lamanna, J; Malgaroli, A; Drago, F; Racagni, G; Nyengaard, J R; Wegener, G; Bonanno, G; Popoli, M

    2014-04-01

    Stress and glucocorticoids alter glutamatergic transmission, and the outcome of stress may range from plasticity enhancing effects to noxious, maladaptive changes. We have previously demonstrated that acute stress rapidly increases glutamate release in prefrontal and frontal cortex via glucocorticoid receptor and accumulation of presynaptic SNARE complex. Here we compared the ex vivo effects of acute stress on glutamate release with those of in vitro application of corticosterone, to analyze whether acute effect of stress on glutamatergic transmission is mediated by local synaptic action of corticosterone. We found that acute stress increases both the readily releasable pool (RRP) of vesicles and depolarization-evoked glutamate release, while application in vitro of corticosterone rapidly increases the RRP, an effect dependent on synaptic receptors for the hormone, but does not induce glutamate release for up to 20 min. These findings indicate that corticosterone mediates the enhancement of glutamate release induced by acute stress, and the rapid non-genomic action of the hormone is necessary but not sufficient for this effect.

  11. Pan-neurexin perturbation results in compromised synapse stability and a reduction in readily releasable synaptic vesicle pool size

    PubMed Central

    Quinn, Dylan P.; Kolar, Annette; Wigerius, Michael; Gomm-Kolisko, Rachel N.; Atwi, Hanine; Fawcett, James P.; Krueger, Stefan R.

    2017-01-01

    Neurexins are a diverse family of cell adhesion molecules that localize to presynaptic specializations of CNS neurons. Heterologous expression of neurexins in non-neuronal cells leads to the recruitment of postsynaptic proteins in contacting dendrites of co-cultured neurons, implicating neurexins in synapse formation. However, isoform-specific knockouts of either all α- or all β-neurexins show defects in synaptic transmission but an unaltered density of glutamatergic synapses, a finding that argues against an essential function of neurexins in synaptogenesis. To address the role of neurexin in synapse formation and function, we disrupted the function of all α- and β-neurexins in cultured hippocampal neurons by shRNA knockdown or by overexpressing a neurexin mutant that is unable to bind to postsynaptic neurexin ligands. We show that neurexin perturbation results in an attenuation of neurotransmitter release that is in large part due to a reduction in the number of readily releasable synaptic vesicles. We also find that neurexin perturbation fails to alter the ability of neurons to form synapses, but rather leads to more frequent synapse elimination. These experiments suggest that neurexins are dispensable for the formation of initial synaptic contacts, but play an essential role in the stabilization and functional maturation of synapses. PMID:28220838

  12. Nicotine enhancement of dopamine release by a calcium-dependent increase in the size of the readily releasable pool of synaptic vesicles.

    PubMed

    Turner, Timothy J

    2004-12-15

    A major factor underlying compulsive tobacco use is nicotine-induced modulation of dopamine release in the mesolimbic reward pathway (Wise and Rompre, 1989). An established biochemical mechanism for nicotine-enhanced dopamine release is by activating presynaptic nicotinic acetylcholine receptors (nAChRs) (Wonnacott, 1997). Prolonged application of 10(-7) to 10(-5) m nicotine to striatal synaptosomes promoted a sustained efflux of [3H]dopamine. This nicotine effect was mediated by non-alpha7 nAChRs, because it was blocked by 5 mum mecamylamine but was resistant to 100 nm alpha-bungarotoxin (alphaBgTx). Dopamine release was diminished by omitting Na+ or by applying peptide calcium channel blockers, indicating that nAChRs trigger release by depolarizing the nerve terminals. However, because alpha7 receptors rapidly desensitize in the continuous presence of agonists, a repetitive stimulation protocol was used to evaluate the possible significance of desensitization. This protocol produced a transient increase in [3H]dopamine released by depolarization and a significant increase in the response to hypertonic solutions that measure the size of the readily releasable pool (RRP) of synaptic vesicles. The nicotine-induced increase in the size of the readily releasable pool was blocked by alphaBgTx and by the calmodulin antagonist calmidazolium, suggesting that Ca2+ entry through alpha7 nAChRs specifically enhances synaptic vesicle mobilization at dopamine terminals. Thus, nicotine enhances dopamine release by two complementary actions mediated by discrete nAChR subtypes and suggest that the alpha7 nAChR-mediated pathway is tightly and specifically coupled to refilling of the RRP of vesicles in dopamine terminals.

  13. CAPS1 stabilizes the state of readily releasable synaptic vesicles to fusion competence at CA3–CA1 synapses in adult hippocampus

    PubMed Central

    Shinoda, Yo; Ishii, Chiaki; Fukazawa, Yugo; Sadakata, Tetsushi; Ishii, Yuki; Sano, Yoshitake; Iwasato, Takuji; Itohara, Shigeyoshi; Furuichi, Teiichi

    2016-01-01

    Calcium-dependent activator protein for secretion 1 (CAPS1) regulates exocytosis of dense-core vesicles in neuroendocrine cells and of synaptic vesicles in neurons. However, the synaptic function of CAPS1 in the mature brain is unclear because Caps1 knockout (KO) results in neonatal death. Here, using forebrain-specific Caps1 conditional KO (cKO) mice, we demonstrate, for the first time, a critical role of CAPS1 in adult synapses. The amplitude of synaptic transmission at CA3–CA1 synapses was strongly reduced, and paired-pulse facilitation was significantly increased, in acute hippocampal slices from cKO mice compared with control mice, suggesting a perturbation in presynaptic function. Morphological analysis revealed an accumulation of synaptic vesicles in the presynapse without any overall morphological change. Interestingly, however, the percentage of docked vesicles was markedly decreased in the Caps1 cKO. Taken together, our findings suggest that CAPS1 stabilizes the state of readily releasable synaptic vesicles, thereby enhancing neurotransmitter release at hippocampal synapses. PMID:27545744

  14. Munc18-1 expression levels control synapse recovery by regulating readily releasable pool size

    PubMed Central

    Toonen, Ruud F. G.; Wierda, Keimpe; Sons, Michèle S.; de Wit, Heidi; Cornelisse, L. Niels; Brussaard, Arjen; Plomp, Jaap J.; Verhage, Matthijs

    2006-01-01

    Prompt recovery after intense activity is an essential feature of most mammalian synapses. Here we show that synapses with reduced expression of the presynaptic gene munc18-1 suffer from increased depression during intense stimulation at glutamatergic, GABAergic, and neuromuscular synapses. Conversely, munc18-1 overexpression makes these synapses recover faster. Concomitant changes in the readily releasable vesicle pool and its refill kinetics were found. The number of vesicles docked at the active zone and the total number of vesicles per terminal correlated with both munc18-1 expression levels and the size of the releasable vesicle pool. These data show that varying expression of a single gene controls synaptic recovery by modulating the number of docked, release-ready vesicles and thereby replenishment of the secretion capacity. PMID:17110441

  15. Is replenishment of the readily releasable pool associated with vesicular movement?

    PubMed

    Bui, Loc; Glavinović, Mladen I

    2014-04-01

    At the excitatory synapse of rat hippocampus the short-term synaptic depression observed during long high-frequency stimulation is associated with slower replenishment of the readily-releasable pool. Given that the replenishment rate is also not [Ca(++)]o sensitive this puts into question a widely held notion that the vesicles-constrained by the cytoskeleton and rendered free from such constraints by Ca(++) entry that renders them more mobile-are important in the replenishment of the readily-releasable pool. This raises a question-Is vesicular replenishment of the readily releasable pool associated with significant movement? To answer this question we evaluated how okadaic acid and staurosporine (compounds known to affect vesicular mobility) influence the replenishment rate. We used patterned stimulation on the Schaffer collateral fiber pathway and recorded the excitatory post-synaptic currents (EPSCs) from rat CA1 neurons, in the absence and presence of these drugs. The parameters of a circuit model with two vesicular pools were estimated by minimizing the squared difference between the ESPC amplitudes and simulated model output. [Ca(2+)]o did not influence the progressive decrease of the replenishment rate during long, high frequency stimulation. Okadaic acid did not significantly affect any parameters of the vesicular storage and release system, including the replenishment rate. Staurosporine reduced the replenishment coupling, but not the replenishment rate, and this is owing to the fact that it also reduces the ability of the readily releasable pool to contain quanta. Moreover, these compounds were ineffective in influencing how the replenishment rate decreases during long, high frequency stimulation. In conclusion at the excitatory synapses of rat hippocampus the replenishment of the readily releasable pool does not appear to be associated with a significant vesicular movement, and during long high frequency stimulation [Ca(++)]o does not influence the

  16. A Three-Pool Model Dissecting Readily Releasable Pool Replenishment at the Calyx of Held

    PubMed Central

    Guo, Jun; Ge, Jian-long; Hao, Mei; Sun, Zhi-cheng; Wu, Xin-sheng; Zhu, Jian-bing; Wang, Wei; Yao, Pan-tong; Lin, Wei; Xue, Lei

    2015-01-01

    Although vesicle replenishment is critical in maintaining exo-endocytosis recycling, the underlying mechanisms are not well understood. Previous studies have shown that both rapid and slow endocytosis recycle into a very large recycling pool instead of within the readily releasable pool (RRP), and the time course of RRP replenishment is slowed down by more intense stimulation. This finding contradicts the calcium/calmodulin-dependence of RRP replenishment. Here we address this issue and report a three-pool model for RRP replenishment at a central synapse. Both rapid and slow endocytosis provide vesicles to a large reserve pool (RP) ~42.3 times the RRP size. When moving from the RP to the RRP, vesicles entered an intermediate pool (IP) ~2.7 times the RRP size with slow RP-IP kinetics and fast IP-RRP kinetics, which was responsible for the well-established slow and rapid components of RRP replenishment. Depletion of the IP caused the slower RRP replenishment observed after intense stimulation. These results establish, for the first time, a realistic cycling model with all parameters measured, revealing the contribution of each cycling step in synaptic transmission. The results call for modification of the current view of the vesicle recycling steps and their roles. PMID:25825223

  17. A three-pool model dissecting readily releasable pool replenishment at the calyx of held.

    PubMed

    Guo, Jun; Ge, Jian-long; Hao, Mei; Sun, Zhi-cheng; Wu, Xin-sheng; Zhu, Jian-bing; Wang, Wei; Yao, Pan-tong; Lin, Wei; Xue, Lei

    2015-03-31

    Although vesicle replenishment is critical in maintaining exo-endocytosis recycling, the underlying mechanisms are not well understood. Previous studies have shown that both rapid and slow endocytosis recycle into a very large recycling pool instead of within the readily releasable pool (RRP), and the time course of RRP replenishment is slowed down by more intense stimulation. This finding contradicts the calcium/calmodulin-dependence of RRP replenishment. Here we address this issue and report a three-pool model for RRP replenishment at a central synapse. Both rapid and slow endocytosis provide vesicles to a large reserve pool (RP) ~42.3 times the RRP size. When moving from the RP to the RRP, vesicles entered an intermediate pool (IP) ~2.7 times the RRP size with slow RP-IP kinetics and fast IP-RRP kinetics, which was responsible for the well-established slow and rapid components of RRP replenishment. Depletion of the IP caused the slower RRP replenishment observed after intense stimulation. These results establish, for the first time, a realistic cycling model with all parameters measured, revealing the contribution of each cycling step in synaptic transmission. The results call for modification of the current view of the vesicle recycling steps and their roles.

  18. 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

  19. Monitoring dopamine release from single living vesicles with nanoelectrodes.

    PubMed

    Wu, Wen-Zhan; Huang, Wei-Hua; Wang, Wei; Wang, Zong-Li; Cheng, Jie-Ke; Xu, Tao; Zhang, Rong-Ying; Chen, Yu; Liu, Jie

    2005-06-29

    Carbon fiber nanoelectrodes (tip diameter = ca. 100 nm) have been first used to monitor real-time dopamine release from single living vesicles of single rat pheochromocytoma (PC12) cells. The experiments show that active and inactive release sites exist on the surface of cells, and the spatial distributions have been differentiated even in the same active release zone. It is first demonstrated that multiple vesicles can sequentially release dopamine at the same site of the cell surface, which possibly plays the main role in the dopamine release from PC12 cells.

  20. Bayesian Inference of Synaptic Quantal Parameters from Correlated Vesicle Release

    PubMed Central

    Bird, Alex D.; Wall, Mark J.; Richardson, Magnus J. E.

    2016-01-01

    Synaptic transmission is both history-dependent and stochastic, resulting in varying responses to presentations of the same presynaptic stimulus. This complicates attempts to infer synaptic parameters and has led to the proposal of a number of different strategies for their quantification. Recently Bayesian approaches have been applied to make more efficient use of the data collected in paired intracellular recordings. Methods have been developed that either provide a complete model of the distribution of amplitudes for isolated responses or approximate the amplitude distributions of a train of post-synaptic potentials, with correct short-term synaptic dynamics but neglecting correlations. In both cases the methods provided significantly improved inference of model parameters as compared to existing mean-variance fitting approaches. However, for synapses with high release probability, low vesicle number or relatively low restock rate and for data in which only one or few repeats of the same pattern are available, correlations between serial events can allow for the extraction of significantly more information from experiment: a more complete Bayesian approach would take this into account also. This has not been possible previously because of the technical difficulty in calculating the likelihood of amplitudes seen in correlated post-synaptic potential trains; however, recent theoretical advances have now rendered the likelihood calculation tractable for a broad class of synaptic dynamics models. Here we present a compact mathematical form for the likelihood in terms of a matrix product and demonstrate how marginals of the posterior provide information on covariance of parameter distributions. The associated computer code for Bayesian parameter inference for a variety of models of synaptic dynamics is provided in the Supplementary Material allowing for quantal and dynamical parameters to be readily inferred from experimental data sets. PMID:27932970

  1. Pregabalin reduces the release of synaptic vesicles from cultured hippocampal neurons.

    PubMed

    Micheva, Kristina D; Taylor, Charles P; Smith, Stephen J

    2006-08-01

    Pregabalin [S-[+]-3-isobutylGABA or (S)-3-(aminomethyl)-5-methylhexanoic acid, Lyrica] is an anticonvulsant and analgesic medication that is both structurally and pharmacologically related to gabapentin (Neurontin; Pfizer Inc., New York, NY). Previous studies have shown that pregabalin reduces the release of neurotransmitters in several in vitro preparations, although the molecular details of these effects are less clear. The present study was performed using living cultured rat hippocampal neurons with the synaptic vesicle fluorescent dye probe FM4-64 to determine details of the action of pregabalin to reduce neurotransmitter release. Our results indicate that pregabalin treatment, at concentrations that are therapeutically relevant, slightly but significantly reduces the emptying of neurotransmitter vesicles from presynaptic sites in living neurons. Dye release is reduced in both glutamic acid decarboxylase (GAD)-immunoreactive and GAD-negative (presumed glutamatergic) synaptic terminals. Furthermore, both calcium-dependent release and hyperosmotic (calcium-independent) dye release are reduced by pregabalin. The effects of pregabalin on dye release are masked in the presence of l-isoleucine, consistent with the fact that both of these compounds have a high binding affinity to the calcium channel alpha(2)-delta protein. The effect of pregabalin is not apparent in the presence of an N-methyl-d-aspartate (NMDA) antagonist [D(-)-2-amino-5-phosphonopentanoic acid], suggesting that pregabalin action depends on NMDA receptor activation. Finally, the action of pregabalin on dye release is most apparent before and early during a train of electrical stimuli when vesicle release preferentially involves the readily releasable pool.

  2. Vesicle-membrane fusion. Observation of simultaneous membrane incorporation and content release.

    PubMed Central

    Woodbury, D J; Hall, J E

    1988-01-01

    Vesicle fusion, the central process of neurotransmitter release and hormonal secretion, is a complex process culminating in simultaneous incorporation of vesicle membrane into the plasma membrane and release of the vesicular contents extracellularly. This report describes simultaneous observation of membrane incorporation and content release using a model system composed of a planar bilayer and dye-filled vesicles. Images FIGURE 1 PMID:2462925

  3. Presynaptic Mechanisms of Lead Neurotoxicity: Effects on Vesicular Release, Vesicle Clustering and Mitochondria Number

    PubMed Central

    McGlothan, Jennifer L.; Stansfield, Kirstie H.; Stanton, Patric K.; Guilarte, Tomás R.

    2015-01-01

    Childhood lead (Pb2+) intoxication is a global public health problem and accounts for 0.6% of the global burden of disease associated with intellectual disabilities. Despite the recognition that childhood Pb2+ intoxication contributes significantly to intellectual disabilities, there is a fundamental lack of knowledge on presynaptic mechanisms by which Pb2+ disrupts synaptic function. In this study, using a well-characterized rodent model of developmental Pb2+ neurotoxicity, we show that Pb2+ exposure markedly inhibits presynaptic vesicular release in hippocampal Schaffer collateral-CA1 synapses in young adult rats. This effect was associated with ultrastructural changes which revealed a reduction in vesicle number in the readily releasable/docked vesicle pool, disperse vesicle clusters in the resting pool, and a reduced number of presynaptic terminals with multiple mitochondria with no change in presynaptic calcium influx. These studies provide fundamental knowledge on mechanisms by which Pb2+ produces profound inhibition of presynaptic vesicular release that contribute to deficits in synaptic plasticity and intellectual development. PMID:26011056

  4. Altered neurotransmitter release, vesicle recycling and presynaptic structure in the pilocarpine model of temporal lobe epilepsy

    PubMed Central

    Upreti, Chirag; Otero, Rafael; Partida, Carlos; Skinner, Frank; Thakker, Ravi; Pacheco, Luis F.; Zhou, Zhen-yu; Maglakelidze, Giorgi; Velíšková, Jana; Velíšek, Libor; Romanovicz, Dwight; Jones, Theresa; Stanton, Patric K.

    2012-01-01

    In searching for persistent seizure-induced alterations in brain function that might be causally related to epilepsy, presynaptic transmitter release has relatively been neglected. To measure directly the long-term effects of pilocarpine-induced status epilepticus on vesicular release and recycling in hippocampal mossy fibre presynaptic boutons, we used (i) two-photon imaging of FM1-43 vesicular release in rat hippocampal slices; and (ii) transgenic mice expressing the genetically encoded pH-sensitive fluorescent reporter synaptopHluorin preferentially at glutamatergic synapses. In this study we found that, 1–2 months after pilocarpine-induced status epilepticus, there were significant increases in mossy fibre bouton size, faster rates of action potential-driven vesicular release and endocytosis. We also analysed the ultrastructure of rat mossy fibre boutons using transmission electron microscopy. Pilocarpine-induced status epilepticus led to a significant increase in the number of release sites, active zone length, postsynaptic density area and number of vesicles in the readily releasable and recycling pools, all correlated with increased release probability. Our data show that presynaptic release machinery is persistently altered in structure and function by status epilepticus, which could contribute to the development of the chronic epileptic state and may represent a potential new target for antiepileptic therapies. PMID:22344585

  5. RIM-binding protein links synaptic homeostasis to the stabilization and replenishment of high release probability vesicles.

    PubMed

    Müller, Martin; Genç, Özgür; Davis, Graeme W

    2015-03-04

    Here we define activities of RIM-binding protein (RBP) that are essential for baseline neurotransmission and presynaptic homeostatic plasticity. At baseline, rbp mutants have a ∼10-fold decrease in the apparent Ca(2+) sensitivity of release that we attribute to (1) impaired presynaptic Ca(2+) influx, (2) looser coupling of vesicles to Ca(2+) influx, and (3) limited access to the readily releasable vesicle pool (RRP). During homeostatic plasticity, RBP is necessary for the potentiation of Ca(2+) influx and the expansion of the RRP. Remarkably, rbp mutants also reveal a rate-limiting stage required for the replenishment of high release probability (p) vesicles following vesicle depletion. This rate slows ∼4-fold at baseline and nearly 7-fold during homeostatic signaling in rbp. These effects are independent of altered Ca(2+) influx and RRP size. We propose that RBP stabilizes synaptic efficacy and homeostatic plasticity through coordinated control of presynaptic Ca(2+) influx and the dynamics of a high-p vesicle pool.

  6. Disruption of adaptor protein 2μ (AP-2μ) in cochlear hair cells impairs vesicle reloading of synaptic release sites and hearing.

    PubMed

    Jung, SangYong; Maritzen, Tanja; Wichmann, Carolin; Jing, Zhizi; Neef, Andreas; Revelo, Natalia H; Al-Moyed, Hanan; Meese, Sandra; Wojcik, Sonja M; Panou, Iliana; Bulut, Haydar; Schu, Peter; Ficner, Ralf; Reisinger, Ellen; Rizzoli, Silvio O; Neef, Jakob; Strenzke, Nicola; Haucke, Volker; Moser, Tobias

    2015-11-03

    Active zones (AZs) of inner hair cells (IHCs) indefatigably release hundreds of vesicles per second, requiring each release site to reload vesicles at tens per second. Here, we report that the endocytic adaptor protein 2μ (AP-2μ) is required for release site replenishment and hearing. We show that hair cell-specific disruption of AP-2μ slows IHC exocytosis immediately after fusion of the readily releasable pool of vesicles, despite normal abundance of membrane-proximal vesicles and intact endocytic membrane retrieval. Sound-driven postsynaptic spiking was reduced in a use-dependent manner, and the altered interspike interval statistics suggested a slowed reloading of release sites. Sustained strong stimulation led to accumulation of endosome-like vacuoles, fewer clathrin-coated endocytic intermediates, and vesicle depletion of the membrane-distal synaptic ribbon in AP-2μ-deficient IHCs, indicating a further role of AP-2μ in clathrin-dependent vesicle reformation on a timescale of many seconds. Finally, we show that AP-2 sorts its IHC-cargo otoferlin. We propose that binding of AP-2 to otoferlin facilitates replenishment of release sites, for example, via speeding AZ clearance of exocytosed material, in addition to a role of AP-2 in synaptic vesicle reformation.

  7. Impact of Lysosome Status on Extracellular Vesicle Content and Release

    PubMed Central

    Eitan, Erez; Suire, Caitlin; Zhang, Shi; Mattson, Mark P.

    2016-01-01

    Extracellular vesicles (EVs) are nanoscale size bubble-like membranous structures released from cells. EVs contain RNA, lipids and proteins and are thought to serve various roles including intercellular communication and removal of misfolded proteins. The secretion of misfolded and aggregated proteins in EVs may be a cargo disposal alternative to the autophagy-lysosomal and ubiquitin-proteasome pathways. In this review we will discuss the importance of lysosome functionality for the regulation of EV secretion and content. Exosomes are a subtype of EVs that are released by the fusion of multivesicular bodies (MVB) with the plasma membrane. MVBs can also fuse with lysosomes, and the trafficking pathway of MVBs can therefore determine whether or not exosomes are released from cells. Here we summarize data from studies of the effects of lysosome inhibition on the secretion of EVs and on the possibility that cells compensate for lysosome malfunction by disposal of potentially toxic cargos in EVs. A better understanding of the molecular mechanisms that regulate trafficking of MVBs to lysosomes and the plasma membrane may advance an understanding of diseases in which pathogenic proteins, lipids or infectious agents accumulate within or outside of cells. PMID:27238186

  8. RIM1 and RIM2 redundantly determine Ca2+ channel density and readily releasable pool size at a large hindbrain synapse

    PubMed Central

    Han, Yunyun; Babai, Norbert; Kaeser, Pascal; Südhof, Thomas C.

    2014-01-01

    The localization and density of voltage-gated Ca2+ channels at active zones are essential for the amount and kinetics of transmitter release at synapses. RIM proteins are scaffolding proteins at the active zone that bind to several other presynaptic proteins, including voltage-gated Ca2+ channel α-subunits. The long isoforms of RIM proteins, which contain NH2-terminal Rab3- and Munc13-interacting domains, as well as a central PDZ domain and two COOH-terminal C2 domains, are encoded by two genes, Rim1 and Rim2. Here, we used the ideal accessibility of the large calyx of Held synapse for direct presynaptic electrophysiology to investigate whether the two Rim genes have redundant, or separate, functions in determining the presynaptic Ca2+ channel density, and the size of a readily releasable vesicle pool (RRP). Quantitative PCR showed that cochlear nucleus neurons, which include calyx of Held generating neurons, express both RIM1 and RIM2. Conditional genetic inactivation of RIM2 at the calyx of Held led to a subtle reduction in presynaptic Ca2+ current density, whereas deletion of RIM1 was ineffective. The release efficiency of brief presynaptic Ca2+ “tail” currents and the RRP were unaffected in conditional single RIM1 and RIM2 knockout (KO) mice, whereas both parameters were strongly reduced in RIM1/2 double KO mice. Thus, despite a somewhat more decisive role for RIM2 in determining presynaptic Ca2+ channel density, RIM1 and RIM2 can overall replace each other's presynaptic functions at a large relay synapse in the hindbrain, the calyx of Held. PMID:25343783

  9. Sustaining rapid vesicular release at active zones: potential roles for vesicle tethering

    PubMed Central

    Hallermann, Stefan; Silver, R. Angus

    2016-01-01

    Rapid information processing in our nervous system relies on high-frequency fusion of transmitter-filled vesicles at chemical synapses. Some sensory synapses possess prominent electron-dense ribbon structures that provide a scaffold for tethering synaptic vesicles at the active zone (AZ), enabling sustained vesicular release. Here, we review functional data indicating that some central and neuromuscular synapses can also sustain vesicle-fusion rates that are comparable to those of ribbon-type sensory synapses. Comparison of the ultrastructure across these different types of synapses, together with recent work showing that cytomatrix proteins can tether vesicles and speed vesicle reloading, suggests that filamentous structures may play a key role in vesicle supply. We discuss potential mechanisms by which vesicle tethering could contribute to sustained high rates of vesicle fusion across ribbon-type, central, and neuromuscular synapses. PMID:23164531

  10. Calcium-induced calcium release supports recruitment of synaptic vesicles in auditory hair cells

    PubMed Central

    Schnee, Michael E.; Ricci, Anthony J.

    2015-01-01

    Hair cells from auditory and vestibular systems transmit continuous sound and balance information to the central nervous system through the release of synaptic vesicles at ribbon synapses. The high activity experienced by hair cells requires a unique mechanism to sustain recruitment and replenishment of synaptic vesicles for continuous release. Using pre- and postsynaptic electrophysiological recordings, we explored the potential contribution of calcium-induced calcium release (CICR) in modulating the recruitment of vesicles to auditory hair cell ribbon synapses. Pharmacological manipulation of CICR with agents targeting endoplasmic reticulum calcium stores reduced both spontaneous postsynaptic multiunit activity and the frequency of excitatory postsynaptic currents (EPSCs). Pharmacological treatments had no effect on hair cell resting potential or activation curves for calcium and potassium channels. However, these drugs exerted a reduction in vesicle release measured by dual-sine capacitance methods. In addition, calcium substitution by barium reduced release efficacy by delaying release onset and diminishing vesicle recruitment. Together these results demonstrate a role for calcium stores in hair cell ribbon synaptic transmission and suggest a novel contribution of CICR in hair cell vesicle recruitment. We hypothesize that calcium entry via calcium channels is tightly regulated to control timing of vesicle fusion at the synapse, whereas CICR is used to maintain a tonic calcium signal to modulate vesicle trafficking. PMID:26510758

  11. Release activity-dependent control of vesicle endocytosis by the synaptic adhesion molecule N-cadherin.

    PubMed

    van Stegen, Bernd; Dagar, Sushma; Gottmann, Kurt

    2017-01-20

    At synapses in the mammalian brain, continuous information transfer requires the long-term maintenance of homeostatic coupling between exo- and endocytosis of synaptic vesicles. Because classical endocytosis is orders of magnitude slower than the millisecond-range exocytosis of vesicles, high frequency vesicle fusion could potentially compromise structural stability of synapses. However, the molecular mechanisms mediating the tight coupling of exo- and endocytosis are largely unknown. Here, we investigated the role of the transsynaptic adhesion molecules N-cadherin and Neuroligin1 in regulating vesicle exo- and endocytosis by using activity-induced FM4-64 staining and by using synaptophysin-pHluorin fluorescence imaging. The synaptic adhesion molecules N-cadherin and Neuroligin1 had distinct impacts on exo- and endocytosis at mature cortical synapses. Expression of Neuroligin1 enhanced vesicle release in a N-cadherin-dependent way. Most intriguingly, expression of N-cadherin enhanced both vesicle exo- and endocytosis. Further detailed analysis of N-cadherin knockout neurons revealed that the boosting of endocytosis by N-cadherin was largely dependent on preceding high levels of vesicle release activity. In summary, regulation of vesicle endocytosis was mediated at the molecular level by N-cadherin in a release activity-dependent manner. Because of its endocytosis enhancing function, N-cadherin might play an important role in the coupling of vesicle exo- and endocytosis.

  12. Release activity-dependent control of vesicle endocytosis by the synaptic adhesion molecule N-cadherin

    PubMed Central

    van Stegen, Bernd; Dagar, Sushma; Gottmann, Kurt

    2017-01-01

    At synapses in the mammalian brain, continuous information transfer requires the long-term maintenance of homeostatic coupling between exo- and endocytosis of synaptic vesicles. Because classical endocytosis is orders of magnitude slower than the millisecond-range exocytosis of vesicles, high frequency vesicle fusion could potentially compromise structural stability of synapses. However, the molecular mechanisms mediating the tight coupling of exo- and endocytosis are largely unknown. Here, we investigated the role of the transsynaptic adhesion molecules N-cadherin and Neuroligin1 in regulating vesicle exo- and endocytosis by using activity-induced FM4–64 staining and by using synaptophysin-pHluorin fluorescence imaging. The synaptic adhesion molecules N-cadherin and Neuroligin1 had distinct impacts on exo- and endocytosis at mature cortical synapses. Expression of Neuroligin1 enhanced vesicle release in a N-cadherin-dependent way. Most intriguingly, expression of N-cadherin enhanced both vesicle exo- and endocytosis. Further detailed analysis of N-cadherin knockout neurons revealed that the boosting of endocytosis by N-cadherin was largely dependent on preceding high levels of vesicle release activity. In summary, regulation of vesicle endocytosis was mediated at the molecular level by N-cadherin in a release activity-dependent manner. Because of its endocytosis enhancing function, N-cadherin might play an important role in the coupling of vesicle exo- and endocytosis. PMID:28106089

  13. Antibody Binding Alters the Characteristics and Contents of Extracellular Vesicles Released by Histoplasma capsulatum

    PubMed Central

    Nakayasu, Ernesto S.; Sobreira, Tiago J. P.; Choi, Hyungwon; Casadevall, Arturo; Nimrichter, Leonardo; Nosanchuk, Joshua D.

    2016-01-01

    ABSTRACT Histoplasma capsulatum produces extracellular vesicles containing virulence-associated molecules capable of modulating host machinery, benefiting the pathogen. Treatment of H. capsulatum cells with monoclonal antibodies (MAbs) can change the outcome of infection in mice. We evaluated the sizes, enzymatic contents, and proteomic profiles of the vesicles released by fungal cells treated with either protective MAb 6B7 (IgG1) or nonprotective MAb 7B6 (IgG2b), both of which bind H. capsulatum heat shock protein 60 (Hsp60). Our results showed that treatment with either MAb was associated with changes in size and vesicle loading. MAb treatments reduced vesicle phosphatase and catalase activities compared to those of vesicles from untreated controls. We identified 1,125 proteins in vesicles, and 250 of these manifested differences in abundance relative to that of proteins in vesicles isolated from yeast cells exposed to Hsp60-binding MAbs, indicating that surface binding of fungal cells by MAbs modified protein loading in the vesicles. The abundance of upregulated proteins in vesicles upon MAb 7B6 treatment was 44.8% of the protein quantities in vesicles from fungal cells treated with MAb 6B7. Analysis of orthologous proteins previously identified in vesicles from other fungi showed that different ascomycete fungi have similar proteins in their extracellular milieu, many of which are associated with virulence. Our results demonstrate that antibody binding can modulate fungal cell responses, resulting in differential loading of vesicles, which could alter fungal cell susceptibility to host defenses. This finding provides additional evidence that antibody binding modulates microbial physiology and suggests a new function for specific immunoglobulins through alterations of fungal secretion. IMPORTANCE Diverse fungal species release extracellular vesicles, indicating that this is a common pathway for the delivery of molecules to the extracellular space. However

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

    PubMed

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

    2012-12-10

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

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

    PubMed Central

    van de Bospoort, Rhea; Farina, Margherita; Schmitz, Sabine K.; de Jong, Arthur; de Wit, Heidi

    2012-01-01

    Neuronal dense-core vesicles (DCVs) contain diverse cargo crucial for brain development and function, but the mechanisms that control their release are largely unknown. We quantified activity-dependent DCV release in hippocampal neurons at single vesicle resolution. DCVs fused preferentially at synaptic terminals. DCVs also fused at extrasynaptic sites but only after prolonged stimulation. In munc13-1/2–null mutant neurons, synaptic DCV release was reduced but not abolished, and synaptic preference was lost. The remaining fusion required prolonged stimulation, similar to extrasynaptic fusion in wild-type neurons. Conversely, Munc13-1 overexpression (M13OE) promoted extrasynaptic DCV release, also without prolonged stimulation. Thus, Munc13-1/2 facilitate DCV fusion but, unlike for synaptic vesicles, are not essential for DCV release, and M13OE is sufficient to produce efficient DCV release extrasynaptically. PMID:23229896

  16. Tunable sustained release properties of "onion-like" phospholipids multilamellar vesicles.

    PubMed

    Douaihy, Christiane Morkos; Koka, Vonda; Mingotaud, Christophe; Gauffre, Fabienne

    2006-11-01

    "Onion-type" multilamellar micro-vesicles of phospholipids (spherulites) were doped with different amounts of a cationic cosurfactant ((-)N-dodecyl-N-methylephedrinium bromide) for the purpose of controlling the sustained release of anionic drugs. Three weak acid probes (methyl red, chlorophenol red, and ibuprofen) were encapsulated in the vesicles as drug models. The kinetics and rate of release were studied by absorption spectroscopy and HPLC. The effect of probe charge (pH above and below pKa of the probes), of cosurfactant concentration and of added salt was investigated. It was found that, above pKa (i.e., when the probes are anionic), the release can be almost totally inhibited by doping the vesicles with 2.4 wt% of cationic cosurfactant. The release properties can even be finely tuned by controlling the amounts of the cosurfactant. Salt and pH effects demonstrate the role of electrostatic interactions in sustaining the release.

  17. What is Rate-Limiting during Sustained Synaptic Activity: Vesicle Supply or the Availability of Release Sites

    PubMed Central

    Neher, Erwin

    2010-01-01

    For some types of synapses the availability of release-ready vesicles is a limiting factor during ongoing activity. Synaptic strength in this case is determined both by the recruitment of such vesicles and the probability of their release during an action potential. Here it is argued that not the availability of vesicles is the limiting factor for recruitment, but rather the availability of specific sites to which vesicles can dock. PMID:21423530

  18. 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.

  19. Release of outer membrane vesicles by Gram-negative bacteria is a novel envelope stress response

    PubMed Central

    McBroom, Amanda J; Kuehn, Meta J

    2007-01-01

    Conditions that impair protein folding in the Gram-negative bacterial envelope cause stress. The destabilizing effects of stress in this compartment are recognized and countered by a number of signal transduction mechanisms. Data presented here reveal another facet of the complex bacterial stress response, release of outer membrane vesicles. Native vesicles are composed of outer membrane and periplasmic material, and they are released from the bacterial surface without loss of membrane integrity. Here we demonstrate that the quantity of vesicle release correlates directly with the level of protein accumulation in the cell envelope. Accumulation of material occurs under stress, and is exacerbated upon impairment of the normal housekeeping and stress-responsive mechanisms of the cell. Mutations that cause increased vesiculation enhance bacterial survival upon challenge with stressing agents or accumulation of toxic misfolded proteins. Preferential packaging of a misfolded protein mimic into vesicles for removal indicates that the vesiculation process can act to selectively eliminate unwanted material. Our results demonstrate that production of bacterial outer membrane vesicles is a fully independent, general envelope stress response. In addition to identifying a novel mechanism for alleviating stress, this work provides physiological relevance for vesicle production as a protective mechanism. PMID:17163978

  20. Membrane vesicle release in bacteria, eukaryotes, and archaea: a conserved yet underappreciated aspect of microbial life.

    PubMed

    Deatherage, Brooke L; Cookson, Brad T

    2012-06-01

    Interaction of microbes with their environment depends on features of the dynamic microbial surface throughout cell growth and division. Surface modifications, whether used to acquire nutrients, defend against other microbes, or resist the pressures of a host immune system, facilitate adaptation to unique surroundings. The release of bioactive membrane vesicles (MVs) from the cell surface is conserved across microbial life, in bacteria, archaea, fungi, and parasites. MV production occurs not only in vitro but also in vivo during infection, underscoring the influence of these surface organelles in microbial physiology and pathogenesis through delivery of enzymes, toxins, communication signals, and antigens recognized by the innate and adaptive immune systems. Derived from a variety of organisms that span kingdoms of life and called by several names (membrane vesicles, outer membrane vesicles [OMVs], exosomes, shedding microvesicles, etc.), the conserved functions and mechanistic strategies of MV release are similar, including the use of ESCRT proteins and ESCRT protein homologues to facilitate these processes in archaea and eukaryotic microbes. Although forms of MV release by different organisms share similar visual, mechanistic, and functional features, there has been little comparison across microbial life. This underappreciated conservation of vesicle release, and the resulting functional impact throughout the tree of life, explored in this review, stresses the importance of vesicle-mediated processes throughout biology.

  1. The Immediately Releasable Pool of Mouse Chromaffin Cell Vesicles Is Coupled to P/Q-Type Calcium Channels via the Synaptic Protein Interaction Site

    PubMed Central

    Álvarez, Yanina D.; Belingheri, Ana Verónica; Perez Bay, Andrés E.; Javis, Scott E.; Tedford, H. William; Zamponi, Gerald; Marengo, Fernando D.

    2013-01-01

    It is generally accepted that the immediately releasable pool is a group of readily releasable vesicles that are closely associated with voltage dependent Ca2+ channels. We have previously shown that exocytosis of this pool is specifically coupled to P/Q Ca2+ current. Accordingly, in the present work we found that the Ca2+ current flowing through P/Q-type Ca2+ channels is 8 times more effective at inducing exocytosis in response to short stimuli than the current carried by L-type channels. To investigate the mechanism that underlies the coupling between the immediately releasable pool and P/Q-type channels we transiently expressed in mouse chromaffin cells peptides corresponding to the synaptic protein interaction site of Cav2.2 to competitively uncouple P/Q-type channels from the secretory vesicle release complex. This treatment reduced the efficiency of Ca2+ current to induce exocytosis to similar values as direct inhibition of P/Q-type channels via ω-agatoxin-IVA. In addition, the same treatment markedly reduced immediately releasable pool exocytosis, but did not affect the exocytosis provoked by sustained electric or high K+ stimulation. Together, our results indicate that the synaptic protein interaction site is a crucial factor for the establishment of the functional coupling between immediately releasable pool vesicles and P/Q-type Ca2+ channels. PMID:23382986

  2. Complexin facilitates exocytosis and synchronizes vesicle release in two secretory model systems

    PubMed Central

    Lin, Ming-Yi; Rohan, Joyce G; Cai, Haijiang; Reim, Kerstin; Ko, Chien-Ping; Chow, Robert H

    2013-01-01

    Complexins (Cplxs) are small, SNARE-associated proteins believed to regulate fast, calcium-triggered exocytosis. However, studies have pointed to either an inhibitory and/or facilitatory role in exocytosis, and the role of Cplxs in synchronizing exocytosis is relatively unexplored. Here, we compare the function of two types of complexin, Cplx 1 and 2, in two model systems of calcium-dependent exocytosis. In mouse neuromuscular junctions (NMJs), we find that lack of Cplx 1 significantly reduces and desynchronizes calcium-triggered synaptic transmission; furthermore, high-frequency stimulation elicits synaptic facilitation, instead of normal synaptic depression, and the degree of facilitation is highly sensitive to the amount of cytoplasmic calcium buffering. In Cplx 2-null adrenal chromaffin cells, we also find decreased and desynchronized evoked release, and identify a significant reduction in the vesicle pool close to the calcium channels (immediately releasable pool, IRP). Viral transduction with either Cplx 1 or 2 rescues both the size of the evoked response and the synchronicity of release, and it restores the IRP size. Our findings in two model systems are mutually compatible and indicate a role of Cplx 1 and 2 in facilitating vesicle priming, and also lead to the new hypothesis that Cplxs may synchronize vesicle release by promoting coupling between secretory vesicles and calcium channels. PMID:23401610

  3. Intracellular calcium stores drive slow non-ribbon vesicle release from rod photoreceptors

    PubMed Central

    Chen, Minghui; Križaj, David; Thoreson, Wallace B.

    2014-01-01

    Rods are capable of greater slow release than cones contributing to overall slower release kinetics. Slow release in rods involves Ca2+-induced Ca2+ release (CICR). By impairing release from ribbons, we found that unlike cones where release occurs entirely at ribbon-style active zones, slow release from rods occurs mostly at ectopic, non-ribbon sites. To investigate the role of CICR in ribbon and non-ribbon release from rods, we used total internal reflection fluorescence microscopy as a tool for visualizing terminals of isolated rods loaded with fluorescent Ca2+ indicator dyes and synaptic vesicles loaded with dextran-conjugated pH-sensitive rhodamine. We found that rather than simply facilitating release, activation of CICR by ryanodine triggered release directly in rods, independent of plasma membrane Ca2+ channel activation. Ryanodine-evoked release occurred mostly at non-ribbon sites and release evoked by sustained depolarization at non-ribbon sites was mostly due to CICR. Unlike release at ribbon-style active zones, non-ribbon release did not occur at fixed locations. Fluorescence recovery after photobleaching of endoplasmic reticulum (ER)-tracker dye in rod terminals showed that ER extends continuously from synapse to soma. Release of Ca2+ from terminal ER by lengthy depolarization did not significantly deplete Ca2+ from ER in the perikaryon. Collectively, these results indicate that CICR-triggered release at non-ribbon sites is a major mechanism for maintaining vesicle release from rods and that CICR in terminals may be sustained by diffusion of Ca2+ through ER from other parts of the cell. PMID:24550779

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

    PubMed Central

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

    2013-01-01

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

  5. Controlled release application of multilamellar vesicles: a novel drug delivery approach.

    PubMed

    Agnihotri, Sunil A; Soppimath, Kumaresh S; Betageri, Guru V

    2010-02-01

    A novel multilamellar vesicular delivery system was developed for the controlled release application. Multilamellar vesicles were prepared by thin film hydration and converted into proliposomes by freeze-drying. A model drug metoclopramide, a highly hydrophilic drug, was successfully encapsulated into proliposomes. The proliposomes produced were non-sticky, free-flowing powders. The proliposomes were formulated into a unit dosage form by combining with various excipients. The effect of different compositions such as type and concentration of phospholipid or hydrophilic polymer was investigared to optimize the formulation. The formation of multilamellar vesicles was confirmed by observing the process of hydration of proliposomes under an optical microscope. The spherical shape of vesicles was confirmed by transmission electron microscopy (TEM) and mean particle sizes were in the range of 1.3-2.5 microm, as measured by dynamic light scattering technique. Differential scanning calorimetry (DSC) study of formulations was conducted to understand the crystalline nature of drug in the vesicles. The results indicated a molecular level dispersion of drug into proliposomes with encapsulation efficiency up to 43%. Critical formulation parameters were identified to obtain a near zero order in vitro release pattern. Proliposomal formulations produced were suitable as multiparticulate drug delivery systems for the controlled release of a highly hydrophilic molecule.

  6. 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.

  7. Properties of ribbon and non-ribbon release from rod photoreceptors revealed by visualizing individual synaptic vesicles.

    PubMed

    Chen, Minghui; Van Hook, Matthew J; Zenisek, David; Thoreson, Wallace B

    2013-01-30

    Vesicle release from rod photoreceptors is regulated by Ca(2+) entry through L-type channels located near synaptic ribbons. We characterized sites and kinetics of vesicle release in salamander rods by using total internal reflection fluorescence microscopy to visualize fusion of individual synaptic vesicles. A small number of vesicles were loaded by brief incubation with FM1-43 or a dextran-conjugated, pH-sensitive form of rhodamine, pHrodo. Labeled organelles matched the diffraction-limited size of fluorescent microspheres and disappeared rapidly during stimulation. Consistent with fusion, depolarization-evoked vesicle disappearance paralleled electrophysiological release kinetics and was blocked by inhibiting Ca(2+) influx. Rods maintained tonic release at resting membrane potentials near those in darkness, causing depletion of membrane-associated vesicles unless Ca(2+) entry was inhibited. This depletion of release sites implies that sustained release may be rate limited by vesicle delivery. During depolarizing stimulation, newly appearing vesicles approached the membrane at ∼800 nm/s, where they paused for ∼60 ms before fusion. With fusion, vesicles advanced ∼18 nm closer to the membrane. Release events were concentrated near ribbons, but lengthy depolarization also triggered release from more distant non-ribbon sites. Consistent with greater contributions from non-ribbon sites during lengthier depolarization, damaging the ribbon by fluorophore-assisted laser inactivation (FALI) of Ribeye caused only weak inhibition of exocytotic capacitance increases evoked by 200-ms depolarizing test steps, whereas FALI more strongly inhibited capacitance increases evoked by 25 ms steps. Amplifying release by use of non-ribbon sites when rods are depolarized in darkness may improve detection of decrements in release when they hyperpolarize to light.

  8. Properties of Ribbon and Non-Ribbon Release from Rod Photoreceptors Revealed by Visualizing Individual Synaptic Vesicles

    PubMed Central

    Chen, Minghui; Van Hook, Matthew J.; Zenisek, David

    2013-01-01

    Vesicle release from rod photoreceptors is regulated by Ca2+ entry through L-type channels located near synaptic ribbons. We characterized sites and kinetics of vesicle release in salamander rods by using total internal reflection fluorescence microscopy to visualize fusion of individual synaptic vesicles. A small number of vesicles were loaded by brief incubation with FM1–43 or a dextran-conjugated, pH-sensitive form of rhodamine, pHrodo. Labeled organelles matched the diffraction-limited size of fluorescent microspheres and disappeared rapidly during stimulation. Consistent with fusion, depolarization-evoked vesicle disappearance paralleled electrophysiological release kinetics and was blocked by inhibiting Ca2+ influx. Rods maintained tonic release at resting membrane potentials near those in darkness, causing depletion of membrane-associated vesicles unless Ca2+ entry was inhibited. This depletion of release sites implies that sustained release may be rate limited by vesicle delivery. During depolarizing stimulation, newly appearing vesicles approached the membrane at ∼800 nm/s, where they paused for ∼60 ms before fusion. With fusion, vesicles advanced ∼18 nm closer to the membrane. Release events were concentrated near ribbons, but lengthy depolarization also triggered release from more distant non-ribbon sites. Consistent with greater contributions from non-ribbon sites during lengthier depolarization, damaging the ribbon by fluorophore-assisted laser inactivation (FALI) of Ribeye caused only weak inhibition of exocytotic capacitance increases evoked by 200-ms depolarizing test steps, whereas FALI more strongly inhibited capacitance increases evoked by 25 ms steps. Amplifying release by use of non-ribbon sites when rods are depolarized in darkness may improve detection of decrements in release when they hyperpolarize to light. PMID:23365244

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

    PubMed

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

    2010-04-20

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

  10. FK506 induces biphasic Ca2+ release from microsomal vesicles of rat pancreatic acinar cells.

    PubMed

    Ozawa, Terutaka

    2006-07-01

    The effect of the immunosuppressant drug FK506 on microsomal Ca2+ release was investigated in rat pancreatic acinar cells. When FK506 (0.1-200 microM) was added to the microsomal vesicles at a steady state of ATP-dependent 45Ca2+ uptake, FK506 caused a dose-dependent and a biphasic release of 45Ca2+. Almost 10% of total 45Ca2+ uptake was released at FK506 concentrations up to 10 microM (Km=0.47 microM), and 60% of total 45Ca2+ uptake was released at FK506 concentrations over 10 microM (Km=55 microM). Preincubation of the vesicles with cyclic ADP-ribose (cADPR, 0.5 microM) increased the FK506 (< or =10 microM)-induced 45Ca2+ release (Ozawa T, Biochim Biophys Acta 1693: 159-166, 2004). Preincubation with heparin (200 microg/ml) resulted in significant inhibition of the FK506 (30 microM)-induced 45Ca2+ release. Subsequent addition of inositol 1,4,5-trisphosphate (IP3, 5 microM) after FK506 (100 microM)-induced 45Ca2+ release did not cause any release of 45Ca2+. These results indicate that two types of FK506-induced Ca2+ release mechanism operate in the endoplasmic reticulum of rat pancreatic acinar cells: a high-affinity mechanism of Ca2+ release, which involves activation of the ryanodine receptor, and a low-affinity mechanism of Ca2+ release, which involves activation of the IP3 receptor.

  11. Different populations of Wnt-containing vesicles are individually released from polarized epithelial cells

    PubMed Central

    Chen, Qiuhong; Takada, Ritsuko; Noda, Chiyo; Kobayashi, Satoru; Takada, Shinji

    2016-01-01

    Accumulating evidence suggests that exosomes are heterogeneous in molecular composition and physical properties. Here we examined whether epithelial cells secrete a heterogeneous population of exosomes, and if that is the case, whether epithelial cell polarity affects release of different populations of exosomes, especially that of those carrying Wnt. Sucrose-density ultracentrifugation and molecular marker analysis revealed that different populations of exosomes or exosome-like vesicles were released from MDCK cells depending on the cell polarity. Wnt3a associated with these vesicles were detectable in culture media collected from both apical and basolateral sides of the cells. Basolaterally secreted Wnt3a were co-fractionated with a typical exosomal protein TSG101 in fractions having typical exosome densities. In contrast, most of apically secreted Wnt3a, as well as Wnt11, were co-fractionated with CD63 and Hsp70, which are also common to the most exosomes, but recovered in higher density fractions. Wnt3a exhibiting similar floatation behavior to the apically secreted ones were also detectable in the culture media of Wnt3a-expressing L and HEK293 cells. The lipidation of Wnt3a was required for its basolateral secretion in exosomes but was dispensable for the apical one. Thus, epithelial cells release Wnt via distinct populations of vesicles differing in secretion polarity and lipidation dependency. PMID:27765945

  12. 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.

  13. Multistimuli-responsive supramolecular vesicles based on water-soluble pillar[6]arene and SAINT complexation for controllable drug release.

    PubMed

    Cao, Yu; Hu, Xiao-Yu; Li, Yan; Zou, Xiaochun; Xiong, Shuhan; Lin, Chen; Shen, Ying-Zhong; Wang, Leyong

    2014-07-30

    Supramolecular binary vesicles based on the host-guest complexation of water-soluble pillar[6]arene (WP6) and SAINT molecule have been successfully constructed, which showed pH-, Ca(2+)-, and thermal-responsiveness. These supramolecular vesicles can efficiently encapsulate model substrate calcein, which then can be efficiently released either by adjusting the solution pH to acidic condition due to the complete disruption of vesicular structure, or particularly, by adding a certain amount of Ca(2+) due to the Ca(2+)-induced vesicle fusion and accompanied by the structure disruption. More importantly, drug loading and releasing experiments demonstrate that an anticancer drug, DOX, can be successfully encapsulated by the supramolecular vesicles, and the resulting DOX-loaded vesicles exhibit efficient release of the encapsulated DOX with the pH adjustment or the introduction of Ca(2+). Cytotoxicity experiments suggest that the resulting DOX-loaded supramolecular vesicles exhibit comparable therapeutic effect for cancer cells as free DOX and the remarkably reduced damage for normal cells as well. The present multistimuli-responsive supramolecular vesicles have great potential applications in the field of controlled drug delivery. In addition, giant supramolecular vesicles (~3 μm) with large internal volume and good stability can be achieved by increasing the temperature of WP6 ⊃ SAINT vesicular solution, and they might have potential applications for bioimaging.

  14. Antibody Binding Alters the Characteristics and Contents of Extracellular Vesicles Released by Histoplasma capsulatum

    SciTech Connect

    Matos Baltazar, Ludmila; Nakayasu, Ernesto S.; Sobreira, Tiago J. P.; Choi, Hyungwon; Casadevall, Arturo; Nimrichter, Leonardo; Nosanchuk, Joshua D.; Alspaugh, J. Andrew

    2016-03-30

    ABSTRACT

    Histoplasma capsulatumproduces extracellular vesicles containing virulence-associated molecules capable of modulating host machinery, benefiting the pathogen. Treatment ofH. capsulatumcells with monoclonal antibodies (MAbs) can change the outcome of infection in mice. We evaluated the sizes, enzymatic contents, and proteomic profiles of the vesicles released by fungal cells treated with either protective MAb 6B7 (IgG1) or nonprotective MAb 7B6 (IgG2b), both of which bindH. capsulatumheat shock protein 60 (Hsp60). Our results showed that treatment with either MAb was associated with changes in size and vesicle loading. MAb treatments reduced vesicle phosphatase and catalase activities compared to those of vesicles from untreated controls. We identified 1,125 proteins in vesicles, and 250 of these manifested differences in abundance relative to that of proteins in vesicles isolated from yeast cells exposed to Hsp60-binding MAbs, indicating that surface binding of fungal cells by MAbs modified protein loading in the vesicles. The abundance of upregulated proteins in vesicles upon MAb 7B6 treatment was 44.8% of the protein quantities in vesicles from fungal cells treated with MAb 6B7. Analysis of orthologous proteins previously identified in vesicles from other fungi showed that different ascomycete fungi have similar proteins in their extracellular milieu, many of which are associated with virulence. Our results demonstrate that antibody binding can modulate fungal cell responses, resulting in differential loading of vesicles, which could alter fungal cell susceptibility to host defenses. This finding provides additional evidence that antibody binding modulates microbial physiology and suggests a new function for specific immunoglobulins through alterations of fungal secretion.

    IMPORTANCEDiverse fungal species release extracellular vesicles, indicating that this is a

  15. Extracellular superoxide dismutase is present in secretory vesicles of human neutrophils and released upon stimulation.

    PubMed

    Iversen, Marie B; Gottfredsen, Randi H; Larsen, Ulrike G; Enghild, Jan J; Praetorius, Jeppe; Borregaard, Niels; Petersen, Steen V

    2016-08-01

    Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme present in the extracellular matrix (ECM), where it provides protection against oxidative degradation of matrix constituents including type I collagen and hyaluronan. The enzyme is known to associate with macrophages and polymorphonuclear leukocytes (neutrophils) and increasing evidence supports a role for EC-SOD in the development of an inflammatory response. Here we show that human EC-SOD is present at the cell surface of isolated neutrophils as well as stored within secretory vesicles. Interestingly, we find that EC-SOD mRNA is absent throughout neutrophil maturation indicating that the protein is synthesized by other cells and subsequently endocytosed by the neutrophil. When secretory vesicles were mobilized by neutrophil stimulation using formyl-methionyl-leucyl-phenylalanine (fMLF) or phorbol 12-myristate 13-acetate (PMA), the protein was released into the extracellular space and found to associate with DNA released from stimulated cells. The functional consequences were evaluated by the use of neutrophils isolated from wild-type and EC-SOD KO mice, and showed that EC-SOD release significantly reduce the level of superoxide in the extracellular space, but does not affect the capacity to generate neutrophil extracellular traps (NETs). Consequently, our data signifies that EC-SOD released from activated neutrophils affects the redox conditions of the extracellular space and may offer protection against highly reactive oxygen species such as hydroxyl radicals otherwise generated as a result of respiratory burst activity of activated neutrophils.

  16. 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

  17. Vesicle-associated microRNAs are released from blood cells on incubation of blood samples.

    PubMed

    Köberle, Verena; Kakoschky, Bianca; Ibrahim, Ahmed Atef; Schmithals, Christian; Peveling-Oberhag, Jan; Zeuzem, Stefan; Kronenberger, Bernd; Waidmann, Oliver; Pleli, Thomas; Piiper, Albrecht

    2016-03-01

    MicroRNAs (miRNAs) circulating extracellularly in the blood are currently intensively studied as novel disease markers. However, the preanalytical factors influencing the levels of the extracellular miRNAs are still incompletely explored. In particular, it is unknown, whether the incubation of blood samples as occurring in clinical routine can lead to a release of miRNAs from blood cells and thus alter the extracellular miRNA levels before the preparation of serum or plasma from the blood cells. Using a set of marker miRNAs and quantitative RT-PCR, we found that the levels of extracellular miRNA-1, miRNA-16, and miRNA-21 were increased in EDTA and serum collection tubes incubated for 1-3 hours at room temperature and declined thereafter; the levels of the liver-specific miRNA-122 declined monophasically. These events occurred in the absence of significant hemolysis. When the blood was supplemented with Ribonuclease A inhibitor, the levels of miRNA-1, miRNA-16, and miRNA-21 increased substantially during the initial 3 hours of incubation and those of miRNA-122 remained unchanged, indicating that the release of blood cell-derived miRNAs occurred during the initial 3 hours of incubation of the blood tubes, but not at later time points. Separation of 5-hour preincubated blood into vesicle and nonvesicle fractions revealed a selective increase in the portion of vesicle-associated miRNAs. Together, these data indicate that the release of vesicle-associated miRNAs from blood cells can occur in blood samples within the time elapsing in normal clinical practice until their processing without significant hemolysis. This becomes particularly visible on the inhibition of miRNA degradation by Ribonuclease A inhibitor.

  18. Botulinum neurotoxin dose-dependently inhibits release of neurosecretory vesicle-vargeted luciferase from neuronal cells.

    PubMed

    Pathe-Neuschäfer-Rube, Andrea; Neuschäfer-Rube, Frank; Genz, Lara; Püchel, Gerhard P

    2015-01-01

    Botulinum toxin is a bacterial toxin that inhibits neurotransmitter release from neurons and thereby causes a flaccid paralysis. It is used as drug to treat a number of serious ailments and, more frequently, for aesthetic medical interventions. Botulinum toxin for pharmacological applications is isolated from bacterial cultures. Due to partial denaturation of the protein, the specific activity of these preparations shows large variations.Because of its extreme potential toxicity, pharmacological preparations must be carefully tested for their activity. For the current gold standard, the mouse lethality assay, several hundred thousand mice are killed per year. Alternative methods have been developed that suffer from one or more of the following deficits: In vitro enzyme assays test only the activity of the catalytic subunit of the toxin. Enzymatic and cell based immunological assays are specific for just one of the different serotypes. The current study takes a completely different approach that overcomes these limitations: Neuronal cell lines were stably transfected with plasmids coding for luciferases of different species, which were N-terminally tagged with leader sequences that redirect the luciferase into neuro-secretory vesicles. From these vesicles, luciferases were released upon depolarization of the cells. The depolarization-dependent release was efficiently inhibited by of botulinum toxin in a concentration range (1 to 100 pM) that is used in pharmacological preparations. The new assay might thus be an alternative to the mouse lethality assay and the immunological assays already in use.

  19. Action potentials and amphetamine release antipsychotic drug from dopamine neuron synaptic VMAT vesicles

    PubMed Central

    Tucker, Kristal R.; Block, Ethan R.; Levitan, Edwin S.

    2015-01-01

    Based on lysotracker red imaging in cultured hippocampal neurons, antipsychotic drugs (APDs) were proposed to accumulate in synaptic vesicles by acidic trapping and to be released in response to action potentials. Because many APDs are dopamine (DA) D2 receptor (D2R) antagonists, such a mechanism would be particularly interesting if it operated in midbrain DA neurons. Here, the APD cyamemazine (CYAM) is visualized directly by two-photon microscopy in substantia nigra and striatum brain slices. CYAM accumulated slowly into puncta based on vacuolar H+-ATPase activity and dispersed rapidly upon dissipating organelle pH gradients. Thus, CYAM is subject to acidic trapping and released upon deprotonation. In the striatum, Ca2+-dependent reduction of the CYAM punctate signal was induced by depolarization or action potentials. Striatal CYAM overlapped with the dopamine transporter (DAT). Furthermore, parachloroamphetamine (pCA), acting via vesicular monoamine transporter (VMAT), and a charged VMAT, substrate 1-methyl-4-phenylpyridinium (MPP+), reduced striatal CYAM. In vivo CYAM administration and in vitro experiments confirmed that clinically relevant CYAM concentrations result in vesicular accumulation and pCA-dependent release. These results show that some CYAM is in DA neuron VMAT vesicles and suggests a new drug interaction in which amphetamine induces CYAM deprotonation and release as a consequence of the H+ countertransport by VMAT that accompanies vesicular uptake, but not by inducing exchange or acting as a weak base. Therefore, in the striatum, APDs are released with DA in response to action potentials and an amphetamine. This synaptic corelease is expected to enhance APD antagonism of D2Rs where and when dopaminergic transmission occurs. PMID:26216995

  20. Release of Small RNA-containing Exosome-like Vesicles from the Human Filarial Parasite Brugia malayi

    PubMed Central

    Agbedanu, Prince N; Harischandra, Hiruni; Moorhead, Andrew R; Day, Tim A; Bartholomay, Lyric C; Kimber, Michael J

    2015-01-01

    Lymphatic filariasis (LF) is a socio-economically devastating mosquito-borne Neglected Tropical Disease caused by parasitic filarial nematodes. The interaction between the parasite and host, both mosquito and human, during infection, development and persistence is dynamic and delicately balanced. Manipulation of this interface to the detriment of the parasite is a promising potential avenue to develop disease therapies but is prevented by our very limited understanding of the host-parasite relationship. Exosomes are bioactive small vesicles (30–120 nm) secreted by a wide range of cell types and involved in a wide range of physiological processes. Here, we report the identification and partial characterization of exosome-like vesicles (ELVs) released from the infective L3 stage of the human filarial parasite Brugia malayi. Exosome-like vesicles were isolated from parasites in culture media and electron microscopy and nanoparticle tracking analysis were used to confirm that vesicles produced by juvenile B. malayi are exosome-like based on size and morphology. We show that loss of parasite viability correlates with a time-dependent decay in vesicle size specificity and rate of release. The protein cargo of these vesicles is shown to include common exosomal protein markers and putative effector proteins. These Brugia-derived vesicles contain small RNA species that include microRNAs with host homology, suggesting a potential role in host manipulation. Confocal microscopy shows J774A.1, a murine macrophage cell line, internalize purified ELVs, and we demonstrate that these ELVs effectively stimulate a classically activated macrophage phenotype in J774A.1. To our knowledge, this is the first report of exosome-like vesicle release by a human parasitic nematode and our data suggest a novel mechanism by which human parasitic nematodes may actively direct the host responses to infection. Further interrogation of the makeup and function of these bioactive vesicles could seed

  1. 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

  2. C. elegans ciliated sensory neurons release extracellular vesicles that function in animal communication

    PubMed Central

    Wang, Juan; Silva, Malan; Haas, Leonard; Morsci, Natalia; Nguyen, Ken C.Q.; Hall, David H.; Barr, Maureen M.

    2014-01-01

    Summary Cells release extracellular vesicles (ECVs) that play important roles in intercellular communication and may mediate a broad range of physiological and pathological processes. Many fundamental aspects of ECV biogenesis and signaling have yet to be determined, with ECV detection being a challenge and obstacle due to their small size (100nm). We developed an in vivo system to visualize the dynamic release of GFP-labeled ECVs. We show here that specific Caenorhabdidits elegans ciliated sensory neurons shed and release ECVs containing GFP-tagged polycystins LOV-1 and PKD-2. These ECVs are also abundant in the lumen surrounding the cilium. Electron tomography and genetic analysis indicate that ECV biogenesis occurs via budding from the plasma membrane at the ciliary base and not via fusion of multivesicular bodies (MVBs). Intraflagellar transport (IFT) and kinesin-3 KLP-6 are required for environmental release of PKD-2::GFP-containing ECVs. ECVs isolated from wild-type animals induce male tail chasing behavior, while ECVs isolated from klp-6 animals and lacking PKD-2::GFP do not. We conclude that environmentally released ECVs play a role in animal communication and mating related behaviors. PMID:24530063

  3. C. elegans ciliated sensory neurons release extracellular vesicles that function in animal communication.

    PubMed

    Wang, Juan; Silva, Malan; Haas, Leonard A; Morsci, Natalia S; Nguyen, Ken C Q; Hall, David H; Barr, Maureen M

    2014-03-03

    Cells release extracellular vesicles (ECVs) that play important roles in intercellular communication and may mediate a broad range of physiological and pathological processes. Many fundamental aspects of ECV biogenesis and signaling have yet to be determined, with ECV detection being a challenge and obstacle due to the small size (100 nm) of the ECVs. We developed an in vivo system to visualize the dynamic release of GFP-labeled ECVs. We show here that specific Caenorhabdidits elegans ciliated sensory neurons shed and release ECVs containing GFP-tagged polycystins LOV-1 and PKD-2. These ECVs are also abundant in the lumen surrounding the cilium. Electron tomography and genetic analysis indicate that ECV biogenesis occurs via budding from the plasma membrane at the ciliary base and not via fusion of multivesicular bodies. Intraflagellar transport and kinesin-3 KLP-6 are required for environmental release of PKD-2::GFP-containing ECVs. ECVs isolated from wild-type animals induce male tail-chasing behavior, while ECVs isolated from klp-6 animals and lacking PKD-2::GFP do not. We conclude that environmentally released ECVs play a role in animal communication and mating-related behaviors.

  4. Hearing requires otoferlin-dependent efficient replenishment of synaptic vesicles in hair cells.

    PubMed

    Pangrsic, Tina; Lasarow, Livia; Reuter, Kirsten; Takago, Hideki; Schwander, Martin; Riedel, Dietmar; Frank, Thomas; Tarantino, Lisa M; Bailey, Janice S; Strenzke, Nicola; Brose, Nils; Müller, Ulrich; Reisinger, Ellen; Moser, Tobias

    2010-07-01

    Inner hair cell ribbon synapses indefatigably transmit acoustic information. The proteins mediating their fast vesicle replenishment (hundreds of vesicles per s) are unknown. We found that an aspartate to glycine substitution in the C(2)F domain of the synaptic vesicle protein otoferlin impaired hearing by reducing vesicle replenishment in the pachanga mouse model of human deafness DFNB9. In vitro estimates of vesicle docking, the readily releasable vesicle pool (RRP), Ca(2+) signaling and vesicle fusion were normal. Moreover, we observed postsynaptic excitatory currents of variable size and spike generation. However, mutant active zones replenished vesicles at lower rates than wild-type ones and sound-evoked spiking in auditory neurons was sparse and only partially improved during longer interstimulus intervals. We conclude that replenishment does not match the release of vesicles at mutant active zones in vivo and a sufficient standing RRP therefore cannot be maintained. We propose that otoferlin is involved in replenishing synaptic vesicles.

  5. Corticosterone treatment results in enhanced release of peptidergic vesicles in astrocytes via cytoskeletal rearrangements.

    PubMed

    Chatterjee, Sreejata; Sikdar, Sujit K

    2013-12-01

    While the effect of stress on neuronal physiology is widely studied, its effect on the functionality of astrocytes is not well understood. We studied the effect of high doses of stress hormone corticosterone, on two physiological properties of astrocytes, i.e., gliotransmission and interastrocytic calcium waves. To study the release of peptidergic vesicles from astrocytes, hippocampal astrocyte cultures were transfected with a plasmid to express pro-atrial natriuretic peptide (ANP) fused with the emerald green fluorescent protein (ANP.emd). The rate of decrease in fluorescence of ANP.emd on application of ionomycin, a calcium ionophore was monitored. Significant increase in the rate of calcium-dependent exocytosis of ANP.emd was observed with the 100 nM and 1 μM corticosterone treatments for 3 h, which depended on the activation of the glucocorticoid receptor. ANP.emd tagged vesicles exhibited increased mobility in astrocyte culture upon corticosterone treatment. Increasing corticosterone concentrations also resulted in concomitant increase in the calcium wave propagation velocity, initiated by focal ATP application. Corticosterone treatment also resulted in increased GFAP expression and F-actin rearrangements. FITC-Phalloidin immunostaining revealed increased formation of cross linked F-actin networks with the 100 nM and 1 μM corticosterone treatment. Alternatively, blockade of actin polymerization and disruption of microtubules prevented the corticosterone-mediated increase in ANP.emd release kinetics. This study reports for the first time the effect of corticosterone on gliotransmission via modulation of cytoskeletal elements. As ANP acts on both neurons and blood vessels, modulation of its release could have functional implications in neurovascular coupling under pathophysiological conditions of stress.

  6. Characterisation of adipocyte-derived extracellular vesicles released pre- and post-adipogenesis

    PubMed Central

    Connolly, Katherine D.; Guschina, Irina A.; Yeung, Vincent; Clayton, Aled; Draman, Mohd Shazli; Von Ruhland, Christopher; Ludgate, Marian; James, Philip E.; Rees, D. Aled

    2015-01-01

    Extracellular vesicles (EVs) are submicron vesicles released from many cell types, including adipocytes. EVs are implicated in the pathogenesis of obesity-driven cardiovascular disease, although the characteristics of adipocyte-derived EVs are not well described. We sought to define the characteristics of adipocyte-derived EVs before and after adipogenesis, hypothesising that adipogenesis would affect EV structure, molecular composition and function. Using 3T3-L1 cells, EVs were harvested at day 0 and day 15 of differentiation. EV and cell preparations were visualised by electron microscopy and EVs quantified by nanoparticle tracking analysis (NTA). EVs were then assessed for annexin V positivity using flow cytometry; lipid and phospholipid composition using 2D thin layer chromatography and gas chromatography; and vesicular protein content by an immuno-phenotyping assay. Pre-adipogenic cells are connected via a network of protrusions and EVs at both time points display classic EV morphology. EV concentration is elevated prior to adipogenesis, particularly in exosomes and small microvesicles. Parent cells contain higher proportions of phosphatidylserine (PS) and show higher annexin V binding. Both cells and EVs contain an increased proportion of arachidonic acid at day 0. PREF-1 was increased at day 0 whilst adiponectin was higher at day 15 indicating EV protein content reflects the stage of adipogenesis of the cell. Our data suggest that EV production is higher in cells before adipogenesis, particularly in vesicles <300 nm. Cells at this time point possess a greater proportion of PS (required for EV generation) whilst corresponding EVs are enriched in signalling fatty acids, such as arachidonic acid, and markers of adipogenesis, such as PREF-1 and PPARγ. PMID:26609807

  7. Membrane Vesicles Released by Intestinal Epithelial Cells Infected with Rotavirus Inhibit T-Cell Function

    PubMed Central

    Barreto, Alfonso; Rodríguez, Luz-Stella; Rojas, Olga Lucía; Wolf, Marie; Greenberg, Harry B.; Franco, Manuel A.

    2010-01-01

    Abstract Rotavirus (RV) predominantly replicates in intestinal epithelial cells (IEC), and “danger signals” released by these cells may modulate viral immunity. We have recently shown that human model IEC (Caco-2 cells) infected with rhesus-RV release a non-inflammatory group of immunomodulators that includes heat shock proteins (HSPs) and TGF-β1. Here we show that both proteins are released in part in association with membrane vesicles (MV) obtained from filtrated Caco-2 supernatants concentrated by ultracentrifugation. These MV express markers of exosomes (CD63 and others), but not of the endoplasmic reticulum (ER) or nuclei. Larger quantities of proteins associated with MV were released by RV-infected cells than by non-infected cells. VP6 co-immunoprecipitated with CD63 present in these MV, and VP6 co-localized with CD63 in RV-infected cells, suggesting that this viral protein is associated with the MV, and that this association occurs intracellularly. CD63 present in MV preparations from stool samples from 36 children with gastroenteritis due or not due to RV were analyzed. VP6 co-immunoprecipitated with CD63 in 3/8 stool samples from RV-infected children, suggesting that these MV are released by RV-infected cells in vivo. Moreover, fractions that contained MV from RV-infected cells induced death and inhibited proliferation of CD4+ T cells to a greater extent than fractions from non-infected cells. These effects were in part due to TGF-β, because they were reversed by treatment of the T cells with the TGF-β-receptor inhibitor ALK5i. MV from RV-infected and non-infected cells were heterogeneous, with morphologies and typical flotation densities described for exosomes (between 1.10 and 1.18 g/mL), and denser vesicles (>1.24 g/mL). Both types of MV from RV-infected cells were more efficient at inhibiting T-cell function than were those from non-infected cells. We propose that RV infection of IEC releases MV that modulate viral immunity. PMID:21142445

  8. Cationic vesicles based on biocompatible diacyl glycerol-arginine surfactants: physicochemical properties, antimicrobial activity, encapsulation efficiency and drug release.

    PubMed

    Tavano, L; Pinazo, A; Abo-Riya, M; Infante, M R; Manresa, M A; Muzzalupo, R; Pérez, L

    2014-08-01

    Physicochemical characteristics of cationic vesicular systems prepared from biocompatible diacyl glycerol-arginine surfactants are investigated. These systems form stable cationic vesicles by themselves and the average diameter of the vesicles decreases as the alkyl chain length of the surfactant increases. The addition of DPPC also modifies the physicochemical properties of these vesicles. Among the drugs these cationic formulations can encapsulate, we have considered Ciprofloxacin and 5-Fluorouracil (5-FU). We show that the percentage of encapsulated drug depends on both the physicochemical properties of the carrier and the type of drug. The capacity of these systems to carry different molecules was evaluated performing in vitro drug release studies. Finally, the antimicrobial activity of empty and Ciprofloxacin-loaded vesicles against Gram-positive and Gram-negative bacteria has been determined. Three bacteria were tested: Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae. The in vitro drug release from all formulations was effectively delayed. Empty cationic vesicles showed antimicrobial activity and Ciprofloxacin-loaded vesicles showed similar or higher antimicrobial activity than the free drug solution. These results suggest that our formulations represent a great innovation in the pharmaceutical field, due to their dual pharmacological function: one related to the nature of the vehiculated drug and the other related to the innate antibacterial properties of the surfactant-based carriers.

  9. Modeling and measurement of vesicle pools at the cone ribbon synapse: changes in release probability are solely responsible for voltage-dependent changes in release

    PubMed Central

    Thoreson, Wallace B.; Van Hook, Matthew J.; Parmelee, Caitlyn; Curto, Carina

    2015-01-01

    Post-synaptic responses are a product of quantal amplitude (Q), size of the releasable vesicle pool (N), and release probability (P). Voltage-dependent changes in presynaptic Ca2+ entry alter post-synaptic responses primarily by changing P but have also been shown to influence N. With simultaneous whole cell recordings from cone photoreceptors and horizontal cells in tiger salamander retinal slices, we measured N and P at cone ribbon synapses by using a train of depolarizing pulses to stimulate release and deplete the pool. We developed an analytical model that calculates the total pool size contributing to release under different stimulus conditions by taking into account the prior history of release and empirically-determined properties of replenishment. The model provided a formula that calculates vesicle pool size from measurements of the initial post-synaptic response and limiting rate of release evoked by a train of pulses, the fraction of release sites available for replenishment, and the time constant for replenishment. Results of the model showed that weak and strong depolarizing stimuli evoked release with differing probabilities but the same size vesicle pool. Enhancing intraterminal Ca2+ spread by lowering Ca2+ buffering or applying BayK8644 did not increase PSCs evoked with strong test steps showing there is a fixed upper limit to pool size. Together, these results suggest that light-evoked changes in cone membrane potential alter synaptic release solely by changing release probability. PMID:26541100

  10. Modeling and measurement of vesicle pools at the cone ribbon synapse: Changes in release probability are solely responsible for voltage-dependent changes in release.

    PubMed

    Thoreson, Wallace B; Van Hook, Matthew J; Parmelee, Caitlyn; Curto, Carina

    2016-01-01

    Postsynaptic responses are a product of quantal amplitude (Q), size of the releasable vesicle pool (N), and release probability (P). Voltage-dependent changes in presynaptic Ca(2+) entry alter postsynaptic responses primarily by changing P but have also been shown to influence N. With simultaneous whole cell recordings from cone photoreceptors and horizontal cells in tiger salamander retinal slices, we measured N and P at cone ribbon synapses by using a train of depolarizing pulses to stimulate release and deplete the pool. We developed an analytical model that calculates the total pool size contributing to release under different stimulus conditions by taking into account the prior history of release and empirically determined properties of replenishment. The model provided a formula that calculates vesicle pool size from measurements of the initial postsynaptic response and limiting rate of release evoked by a train of pulses, the fraction of release sites available for replenishment, and the time constant for replenishment. Results of the model showed that weak and strong depolarizing stimuli evoked release with differing probabilities but the same size vesicle pool. Enhancing intraterminal Ca(2+) spread by lowering Ca(2+) buffering or applying BayK8644 did not increase PSCs evoked with strong test steps, showing there is a fixed upper limit to pool size. Together, these results suggest that light-evoked changes in cone membrane potential alter synaptic release solely by changing release probability.

  11. Massive release of extracellular vesicles from cancer cells after photodynamic treatment or chemotherapy

    PubMed Central

    Aubertin, Kelly; Silva, Amanda K. A.; Luciani, Nathalie; Espinosa, Ana; Djemat, Aurélie; Charue, Dominique; Gallet, François; Blanc-Brude, Olivier; Wilhelm, Claire

    2016-01-01

    Photodynamic therapy is an emerging cancer treatment that is particularly adapted for localized malignant tumor. The phototherapeutic agent is generally injected in the bloodstream and circulates in the whole organism as a chemotherapeutic agent, but needs light triggering to induce localized therapeutic effects. We found that one of the responses of in vitro and in vivo cancer cells to photodynamic therapy was a massive production and emission of extracellular vesicles (EVs): only 1 hour after the photo-activation, thousands of vesicles per cell were emitted in the extracellular medium. A similar effect has been found after treatment with Doxorubicin (chemotherapy), but far less EVs were produced, even 24 hours after the treatment. Furthermore, we found that the released EVs could transfer extracellular membrane components, drugs and even large intracellular objects to naive target cells. In vivo, photodynamic treatment and chemotherapy increased the levels of circulating EVs several fold, confirming the vast induction of cancer cell vesiculation triggered by anti-cancer therapies. PMID:27752092

  12. Human pyramidal to interneuron synapses are mediated by multi-vesicular release and multiple docked vesicles

    PubMed Central

    Molnár, Gábor; Rózsa, Márton; Baka, Judith; Holderith, Noémi; Barzó, Pál; Nusser, Zoltan; Tamás, Gábor

    2016-01-01

    Classic theories link cognitive abilities to synaptic properties and human-specific biophysical features of synapses might contribute to the unparalleled performance of the human cerebral cortex. Paired recordings and multiple probability fluctuation analysis revealed similar quantal sizes, but 4-times more functional release sites in human pyramidal cell to fast-spiking interneuron connections compared to rats. These connections were mediated on average by three synaptic contacts in both species. Each presynaptic active zone (AZ) contains 6.2 release sites in human, but only 1.6 in rats. Electron microscopy (EM) and EM tomography showed that an AZ harbors 4 docked vesicles in human, but only a single one in rats. Consequently, a Katz’s functional release site occupies ~0.012 μm2 in the human presynaptic AZ and ~0.025 μm2 in the rat. Our results reveal a robust difference in the biophysical properties of a well-defined synaptic connection of the cortical microcircuit of human and rodents. DOI: http://dx.doi.org/10.7554/eLife.18167.001 PMID:27536876

  13. Human pyramidal to interneuron synapses are mediated by multi-vesicular release and multiple docked vesicles.

    PubMed

    Molnár, Gábor; Rózsa, Márton; Baka, Judith; Holderith, Noémi; Barzó, Pál; Nusser, Zoltan; Tamás, Gábor

    2016-08-18

    Classic theories link cognitive abilities to synaptic properties and human-specific biophysical features of synapses might contribute to the unparalleled performance of the human cerebral cortex. Paired recordings and multiple probability fluctuation analysis revealed similar quantal sizes, but 4-times more functional release sites in human pyramidal cell to fast-spiking interneuron connections compared to rats. These connections were mediated on average by three synaptic contacts in both species. Each presynaptic active zone (AZ) contains 6.2 release sites in human, but only 1.6 in rats. Electron microscopy (EM) and EM tomography showed that an AZ harbors 4 docked vesicles in human, but only a single one in rats. Consequently, a Katz's functional release site occupies ~0.012 μm(2) in the human presynaptic AZ and ~0.025 μm(2) in the rat. Our results reveal a robust difference in the biophysical properties of a well-defined synaptic connection of the cortical microcircuit of human and rodents.

  14. Molecular Machines Regulating the Release Probability of Synaptic Vesicles at the Active Zone

    PubMed Central

    Körber, Christoph; Kuner, Thomas

    2016-01-01

    The fusion of synaptic vesicles (SVs) with the plasma membrane of the active zone (AZ) upon arrival of an action potential (AP) at the presynaptic compartment is a tightly regulated probabilistic process crucial for information transfer. The probability of a SV to release its transmitter content in response to an AP, termed release probability (Pr), is highly diverse both at the level of entire synapses and individual SVs at a given synapse. Differences in Pr exist between different types of synapses, between synapses of the same type, synapses originating from the same axon and even between different SV subpopulations within the same presynaptic terminal. The Pr of SVs at the AZ is set by a complex interplay of different presynaptic properties including the availability of release-ready SVs, the location of the SVs relative to the voltage-gated calcium channels (VGCCs) at the AZ, the magnitude of calcium influx upon arrival of the AP, the buffering of calcium ions as well as the identity and sensitivity of the calcium sensor. These properties are not only interconnected, but can also be regulated dynamically to match the requirements of activity patterns mediated by the synapse. Here, we review recent advances in identifying molecules and molecular machines taking part in the determination of vesicular Pr at the AZ. PMID:26973506

  15. 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

  16. Monte Carlo simulation of the effects of vesicle geometry on calcium microdomains and neurotransmitter release

    NASA Astrophysics Data System (ADS)

    Limsakul, Praopim; Modchang, Charin

    2016-07-01

    We investigate the effects of synaptic vesicle geometry on Ca2+ diffusion dynamics in presynaptic terminals using MCell, a realistic Monte Carlo algorithm that tracks individual molecules. By modeling the vesicle as a sphere and an oblate or a prolate spheroid with a reflective boundary, we measure the Ca2+ concentration at various positions relative to the vesicle. We find that the presence of a vesicle as a diffusion barrier modifies the shape of the [Ca2+] microdomain in the vicinity of the vesicle. Ca2+ diffusion dynamics also depend on the distance between the vesicle and the voltage-gated calcium channels (VGCCs) and on the shape of the vesicle. The oblate spheroidal vesicle increases the [Ca2+] up to six times higher than that in the absence of a vesicle, while the prolate spheroidal vesicle can increase the [Ca2+] only 1.4 times. Our results also show that the presence of vesicles that have different geometries can maximally influence the [Ca2+] microdomain when the vesicle is located less than 50 nm from VGCCs.

  17. An Exclusion Zone for Ca2+ Channels around Docked Vesicles Explains Release Control by Multiple Channels at a CNS Synapse

    PubMed Central

    Keller, Daniel; Babai, Norbert; Kochubey, Olexiy; Han, Yunyun; Markram, Henry; Schürmann, Felix; Schneggenburger, Ralf

    2015-01-01

    The spatial arrangement of Ca2+ channels and vesicles remains unknown for most CNS synapses, despite of the crucial importance of this geometrical parameter for the Ca2+ control of transmitter release. At a large model synapse, the calyx of Held, transmitter release is controlled by several Ca2+ channels in a "domain overlap" mode, at least in young animals. To study the geometrical constraints of Ca2+ channel placement in domain overlap control of release, we used stochastic MCell modelling, at active zones for which the position of docked vesicles was derived from electron microscopy (EM). We found that random placement of Ca2+ channels was unable to produce high slope values between release and presynaptic Ca2+ entry, a hallmark of domain overlap, and yielded excessively large release probabilities. The simple assumption that Ca2+ channels can be located anywhere at active zones, except below a critical distance of ~ 30 nm away from docked vesicles ("exclusion zone"), rescued high slope values and low release probabilities. Alternatively, high slope values can also be obtained by placing all Ca2+ channels into a single supercluster, which however results in significantly higher heterogeneity of release probabilities. We also show experimentally that high slope values, and the sensitivity to the slow Ca2+ chelator EGTA-AM, are maintained with developmental maturation of the calyx synapse. Taken together, domain overlap control of release represents a highly organized active zone architecture in which Ca2+ channels must obey a certain distance to docked vesicles. Furthermore, domain overlap can be employed by near-mature, fast-releasing synapses. PMID:25951120

  18. Bassoon and the synaptic ribbon organize Ca²+ channels and vesicles to add release sites and promote refilling.

    PubMed

    Frank, Thomas; Rutherford, Mark A; Strenzke, Nicola; Neef, Andreas; Pangršič, Tina; Khimich, Darina; Fejtova, Anna; Fetjova, Anna; Gundelfinger, Eckart D; Liberman, M Charles; Harke, Benjamin; Bryan, Keith E; Lee, Amy; Egner, Alexander; Riedel, Dietmar; Moser, Tobias

    2010-11-18

    At the presynaptic active zone, Ca²+ influx triggers fusion of synaptic vesicles. It is not well understood how Ca²+ channel clustering and synaptic vesicle docking are organized. Here, we studied structure and function of hair cell ribbon synapses following genetic disruption of the presynaptic scaffold protein Bassoon. Mutant synapses--mostly lacking the ribbon--showed a reduction in membrane-proximal vesicles, with ribbonless synapses affected more than ribbon-occupied synapses. Ca²+ channels were also fewer at mutant synapses and appeared in abnormally shaped clusters. Ribbon absence reduced Ca²+ channel numbers at mutant and wild-type synapses. Fast and sustained exocytosis was reduced, notwithstanding normal coupling of the remaining Ca²+ channels to exocytosis. In vitro recordings revealed a slight impairment of vesicle replenishment. Mechanistic modeling of the in vivo data independently supported morphological and functional in vitro findings. We conclude that Bassoon and the ribbon (1) create a large number of release sites by organizing Ca²+ channels and vesicles, and (2) promote vesicle replenishment.

  19. Bassoon and the synaptic ribbon organize Ca2+ channels and vesicles to add release sites and promote refilling

    PubMed Central

    Frank, T.; Rutherford, M.A.; Strenzke, N.; Neef, A.; Pangršič, T.; Khimich, D.; Fetjova, A.; Gundelfinger, E.D.; Liberman, M.C.; Harke, B.; Bryan, K.E.; Lee, A.; Egner, A.; Riedel, D.; Moser, T.

    2010-01-01

    Summary At the presynaptic active zone, Ca2+ influx triggers fusion of synaptic vesicles. It is not well understood how Ca2+-channel clustering and synaptic vesicle docking are organized. Here we studied structure and function of hair cell ribbon synapses following genetic disruption of the presynaptic scaffold protein Bassoon. Mutant synapses - mostly lacking the ribbon - showed a reduction in membrane-proximal vesicles, with ribbonless synapses affected more than ribbon-occupied synapses. Ca2+-channels were also fewer at mutant synapses and appeared in abnormally shaped clusters. Ribbon absence reduced Ca2+-channel numbers at mutant and wild-type synapses. Fast and sustained exocytosis were reduced notwithstanding normal coupling of the remaining Ca2+-channels to exocytosis. In-vitro recordings revealed a slight impairment of vesicle replenishment. Mechanistic modeling of the in-vivo data independently supported morphological and functional in-vitro findings. We conclude that Bassoon and the ribbon (1) create a large number of release sites by organizing Ca2+-channels and vesicles, and (2) promote vesicle replenishment. PMID:21092861

  20. Quantitative proteomics of extracellular vesicles released from human monocyte-derived macrophages upon β-glucan stimulation.

    PubMed

    Cypryk, Wojciech; Ohman, Tiina; Eskelinen, Eeva-Liisa; Matikainen, Sampsa; Nyman, Tuula A

    2014-05-02

    Fungal infections (mycoses) are common diseases of varying severity that cause problems, especially to immunologically compromised people. Fungi express a variety of pathogen-associated molecular patterns on their surface including β-glucans, which are important immunostimulatory components of fungal cell walls. During stimulatory conditions of infection and colonization, besides intensive intracellular response, human cells actively communicate on the intercellular level by secreting proteins and other biomolecules with several mechanisms. Vesicular secretion remains one of the most important paths for the proteins to exit the cell. Here, we have used high-throughput quantitative proteomics combined with bioinformatics to characterize and quantify vesicle-mediated protein release from β-glucan-stimulated human macrophages differentiated in vitro from primary blood monocytes. We show that β-glucan stimulation induces vesicle-mediated protein secretion. Proteomic study identified 540 distinct proteins from the vesicles, and the identified proteins show a proteomic signature characteristic for their cellular origin. Importantly, we identified several receptors, including cation-dependent mannose-6-phosphate receptor, macrophage scavenger receptor, and P2X7 receptor, that have not been identified from vesicles before. Proteomic data together with detailed pathway and network analysis showed that integrins and their cytoplasmic cargo proteins are highly abundant in extracellular vesicles released upon β-glucan stimulation. In conclusion, the present data provides a solid basis for further studies on the functional role of vesicular protein secretion upon fungal infection.

  1. Lipid-Peptide Vesicle Nanoscale Hybrids for Triggered Drug Release by Mild Hyperthermia in vitro and in vivo

    PubMed Central

    Al-Ahmady, Zahraa S.; Al-Jamal, Wafa’ T.; Bossche, Jeroen V.; Bui, Tam T.; Drake, Alex F.; Mason, A. James; Kostarelos, Kostas

    2012-01-01

    The present study describes leucine zipper peptide-lipid hybrid nanoscale vesicles engineered by self-assembled anchoring of the amphiphilic peptide within the lipid bilayer. These hybrid vesicles aim to combine the advantages of traditional temperature-sensitive liposomes (TSL) with the dissociative, unfolding properties of a temperature-sensitive peptide to optimize drug release under mild hyperthermia, while improving in vivo drug retention. The secondary structure of the peptide and its thermal-responsiveness after anchoring onto liposomes were studied with circular dichroism. In addition, the lipid-peptide vesicles (Lp-peptide) showed a reduction in bilayer fluidity at the inner-core as observed with DPH anisotropy studies, while the opposite effect was observed with ANS probe, indicating peptide interactions with both the head group region and the hydrophobic core. A model drug molecule, doxorubicin, was successfully encapsulated in the Lp-peptide vesicles at higher than 90% efficiency following the remote loading, pH-gradient methodology. The release of doxorubicin from Lp-peptide hybrids in vitro indicated superior serum stability at physiological temperatures compared to lysolipid-containing temperature-sensitive liposomes (LTSL) without affecting the overall thermo-responsive nature of the vesicles at 42 °C. A similar stabilizing effect was observed in vivo after intravenous administration of the Lp-peptide vesicles by measuring 14C-doxorubicin blood kinetics that also led to increased tumor accumulation after 24 hours. We conclude that Lp-peptide hybrid vesicles present a promising new class of TSL that can offer previously unexplored opportunities for the development of clinically-relevant mild hyperthermia-triggered therapeutic modalities. PMID:22857653

  2. Compositional and immunobiological analyses of extracellular vesicles released by Candida albicans.

    PubMed

    Vargas, Gabriele; Rocha, Juliana D B; Oliveira, Debora Leite; Albuquerque, Priscila Costa; Frases, Susana; Santos, Suelen S; Nosanchuk, Joshua Daniel; Gomes, Andre Marco Oliveira; Medeiros, Lia C A S; Miranda, Kildare; Sobreira, Tiago J P; Nakayasu, Ernesto S; Arigi, Emma A; Casadevall, Arturo; Guimaraes, Allan J; Rodrigues, Marcio L; Freire-de-Lima, Celio Geraldo; Almeida, Igor C; Nimrichter, Leonardo

    2015-03-01

    The release of extracellular vesicles (EV) by fungal organisms is considered an alternative transport mechanism to trans-cell wall passage of macromolecules. Previous studies have revealed the presence of EV in culture supernatants from fungal pathogens, such as Cryptococcus neoformans, Histoplasma capsulatum, Paracoccidioides brasiliensis, Sporothrix schenckii, Malassezia sympodialis and Candida albicans. Here we investigated the size, composition, kinetics of internalization by bone marrow-derived murine macrophages (MO) and dendritic cells (DC), and the immunomodulatory activity of C. albicans EV. We also evaluated the impact of EV on fungal virulence using the Galleria mellonella larvae model. By transmission electron microscopy and dynamic light scattering, we identified two populations ranging from 50 to 100 nm and 350 to 850 nm. Two predominant seroreactive proteins (27 kDa and 37 kDa) and a group of polydispersed mannoproteins were observed in EV by immunoblotting analysis. Proteomic analysis of C. albicans EV revealed proteins related to pathogenesis, cell organization, carbohydrate and lipid metabolism, response to stress, and several other functions. The major lipids detected by thin-layer chromatography were ergosterol, lanosterol and glucosylceramide. Short exposure of MO to EV resulted in internalization of these vesicles and production of nitric oxide, interleukin (IL)-12, transforming growth factor-beta (TGF-β) and IL-10. Similarly, EV-treated DC produced IL-12p40, IL-10 and tumour necrosis factor-alpha. In addition, EV treatment induced the up-regulation of CD86 and major histocompatibility complex class-II (MHC-II). Inoculation of G. mellonella larvae with EV followed by challenge with C. albicans reduced the number of recovered viable yeasts in comparison with infected larvae control. Taken together, our results demonstrate that C. albicans EV were immunologically active and could potentially interfere with the host responses in the setting of

  3. Vesicle release probability and pre-primed pool at glutamatergic synapses in area CA1 of the rat neonatal hippocampus

    PubMed Central

    Hanse, Eric; Gustafsson, Bengt

    2001-01-01

    Factors determining the release probability were examined using whole-cell patch-clamp recording and minimal stimulation (10 impulses, 50 Hz) of individual glutamatergic synapses, containing single release sites, on pyramidal neurones in the CA1 region of hippocampal slices from neonatal rats. Release probability following the first action potential in the burst (P1) varied among the synapses from 0 to 0.87 (mean ± s.d.; 0.35 ± 0.28, n = 52) and the average release during the burst (burst pool) varied from 0.4 to 4.1 events (1.7 ± 0.85, n = 52). Heterogeneity in P1 did not co-vary with that of the burst pool. By selecting burst trials during which only one release event occurred, the vesicle release probability (Pves) at a release site could be determined. It was found to vary considerably among the synapses, from 0.04 to 0.94 (0.43 ± 0.28, n = 43). This heterogeneity correlated significantly with that of P1 such that more than half of the variation of P1 could be explained by a variation in Pves. The average number of vesicles directly available for release at the onset of the burst (the pre-primed pool) was estimated as the cumulative release up to that point in the burst where a second release event did not produce higher initial release probability than that found in trials where only one vesicle was released. The average pre-primed pool varied among the synapses from 0.4 to 2.1 (1.03 ± 0.42, n = 43). It co-varied significantly with that of P1 such that it could explain the remaining variation in P1. The difference between the burst pool and the pre-primed pool suggests the presence of a fast (< 100 ms), activity-dependent priming of vesicles. Some synapses (9/52) did not show any initial release (P1 = 0), but release occurred later during the burst (‘low frequency mute synapses’). Their behaviour was explained by an absence of a pre-primed pool. PMID:11230520

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

    PubMed Central

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

    2010-01-01

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

  5. LRRK2 kinase activity regulates synaptic vesicle trafficking and neurotransmitter release through modulation of LRRK2 macro-molecular complex

    PubMed Central

    Cirnaru, Maria D.; Marte, Antonella; Belluzzi, Elisa; Russo, Isabella; Gabrielli, Martina; Longo, Francesco; Arcuri, Ludovico; Murru, Luca; Bubacco, Luigi; Matteoli, Michela; Fedele, Ernesto; Sala, Carlo; Passafaro, Maria; Morari, Michele; Greggio, Elisa; Onofri, Franco; Piccoli, Giovanni

    2014-01-01

    Mutations in Leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains executing several functions, including GTP hydrolysis, kinase activity, and protein binding. Robust evidence suggests that LRRK2 acts at the synaptic site as a molecular hub connecting synaptic vesicles to cytoskeletal elements via a complex panel of protein-protein interactions. Here we investigated the impact of pharmacological inhibition of LRRK2 kinase activity on synaptic function. Acute treatment with LRRK2 inhibitors reduced the frequency of spontaneous currents, the rate of synaptic vesicle trafficking and the release of neurotransmitter from isolated synaptosomes. The investigation of complementary models lacking LRRK2 expression allowed us to exclude potential off-side effects of kinase inhibitors on synaptic functions. Next we studied whether kinase inhibition affects LRRK2 heterologous interactions. We found that the binding among LRRK2, presynaptic proteins and synaptic vesicles is affected by kinase inhibition. Our results suggest that LRRK2 kinase activity influences synaptic vesicle release via modulation of LRRK2 macro-molecular complex. PMID:24904275

  6. Mechanisms and functions of extracellular vesicle release in vivo-What we can learn from flies and worms.

    PubMed

    Beer, Katharina B; Wehman, Ann Marie

    2017-03-04

    Cells from bacteria to man release extracellular vesicles (EVs) that contain signaling molecules like proteins, lipids, and nucleic acids. The content, formation, and signaling roles of these conserved vesicles are diverse, but the physiological relevance of EV signaling in vivo is still debated. Studies in classical genetic model organisms like C. elegans and Drosophila have begun to reveal the developmental and behavioral roles for EVs. In this review, we discuss the emerging evidence for the in vivo signaling roles of EVs. Significant effort has also been made to understand the mechanisms behind the formation and release of EVs, specifically of exosomes derived from exocytosis of multivesicular bodies and of microvesicles derived from plasma membrane budding called ectocytosis. In this review, we detail the impact of flies and worms on understanding the proteins and lipids involved in EV biogenesis and highlight the open questions in the field.

  7. Mechanisms and functions of extracellular vesicle release in vivo—What we can learn from flies and worms

    PubMed Central

    2017-01-01

    ABSTRACT Cells from bacteria to man release extracellular vesicles (EVs) that contain signaling molecules like proteins, lipids, and nucleic acids. The content, formation, and signaling roles of these conserved vesicles are diverse, but the physiological relevance of EV signaling in vivo is still debated. Studies in classical genetic model organisms like C. elegans and Drosophila have begun to reveal the developmental and behavioral roles for EVs. In this review, we discuss the emerging evidence for the in vivo signaling roles of EVs. Significant effort has also been made to understand the mechanisms behind the formation and release of EVs, specifically of exosomes derived from exocytosis of multivesicular bodies and of microvesicles derived from plasma membrane budding called ectocytosis. In this review, we detail the impact of flies and worms on understanding the proteins and lipids involved in EV biogenesis and highlight the open questions in the field. PMID:27689411

  8. Calcium dependence of neurotransmitter release and rate of spontaneous vesicle fusions are altered in Drosophila synaptotagmin mutants.

    PubMed Central

    Littleton, J T; Stern, M; Perin, M; Bellen, H J

    1994-01-01

    Since the demonstration that Ca2+ influx into the presynaptic terminal is essential for neurotransmitter release, there has been much speculation about the Ca2+ receptor responsible for initiating exocytosis. Numerous experiments have shown that the protein, or protein complex, binds multiple Ca2+ ions, resides near the site of Ca2+ influx, and has a relatively low affinity for Ca2+. Synaptotagmin is an integral membrane protein of synaptic vesicles that contains two copies of a domain known to be involved in Ca(2+)-dependent membrane interactions. Synaptotagmin has been shown to bind Ca2+ in vitro with a relatively low affinity. In addition, synaptotagmin has been shown to bind indirectly to Ca2+ channels, positioning the protein close to the site of Ca2+ influx. Recently, a negative regulatory role for synaptotagmin has been proposed, in which it functions as a clamp to prevent fusion of synaptic vesicles with the presynaptic membrane. Release of the clamp would allow exocytosis. Here we present genetic and electrophysiological evidence that synaptotagmin forms a multimeric complex that can function as a clamp in vivo. However, upon nerve stimulation and Ca2+ influx, all synaptotagmin mutations dramatically decrease the ability of Ca2+ to promote release, suggesting that synaptotagmin probably plays a key role in activation of synaptic vesicle fusion. This activity cannot simply be attributed to the removal of a barrier to secretion, as we can electrophysiologically separate the increase in rate of spontaneous vesicle fusion from the decrease in evoked response. We also find that some syt mutations, including those that lack the second Ca(2+)-binding domain, decrease the fourth-order dependence of release on Ca2+ by approximately half, consistent with the hypothesis that a synaptotagmin complex functions as a Ca2+ receptor for initiating exocytosis. Images PMID:7971978

  9. Microdomain [Ca(2+)] Fluctuations Alter Temporal Dynamics in Models of Ca(2+)-Dependent Signaling Cascades and Synaptic Vesicle Release.

    PubMed

    Weinberg, Seth H

    2016-03-01

    Ca(2+)-dependent signaling is often localized in spatially restricted microdomains and may involve only 1 to 100 Ca(2+) ions. Fluctuations in the microdomain Ca(2+) concentration (Ca(2+)) can arise from a wide range of elementary processes, including diffusion, Ca(2+) influx, and association/dissociation with Ca(2+) binding proteins or buffers. However, it is unclear to what extent these fluctuations alter Ca(2+)-dependent signaling. We construct Markov models of a general Ca(2+)-dependent signaling cascade and Ca(2+)-triggered synaptic vesicle release. We compare the hitting (release) time distribution and statistics for models that account for [Ca(2+)] fluctuations with the corresponding models that neglect these fluctuations. In general, when Ca(2+) fluctuations are much faster than the characteristic time for the signaling event, the hitting time distributions and statistics for the models with and without Ca(2+) fluctuation are similar. However, when the timescale of Ca(2+) fluctuations is on the same order as the signaling cascade or slower, the hitting time mean and variability are typically increased, in particular when the average number of microdomain Ca(2+) ions is small, a consequence of a long-tailed hitting time distribution. In a model of Ca(2+)-triggered synaptic vesicle release, we demonstrate the conditions for which [Ca(2+)] fluctuations do and do not alter the distribution, mean, and variability of release timing. We find that both the release time mean and variability can be increased, demonstrating that Ca(2+) fluctuations are an important aspect of microdomain Ca(2+) signaling and further suggesting that Ca(2+) fluctuations in the presynaptic terminal may contribute to variability in synaptic vesicle release and thus variability in neuronal spiking.

  10. Uropathogenic Escherichia coli Releases Extracellular Vesicles That Are Associated with RNA

    PubMed Central

    Blenkiron, Cherie; Simonov, Denis; Muthukaruppan, Anita; Tsai, Peter; Dauros, Priscila; Green, Sasha; Hong, Jiwon; Print, Cristin G.

    2016-01-01

    Background Bacterium-to-host signalling during infection is a complex process involving proteins, lipids and other diffusible signals that manipulate host cell biology for pathogen survival. Bacteria also release membrane vesicles (MV) that can carry a cargo of effector molecules directly into host cells. Supported by recent publications, we hypothesised that these MVs also associate with RNA, which may be directly involved in the modulation of the host response to infection. Methods and Results Using the uropathogenic Escherichia coli (UPEC) strain 536, we have isolated MVs and found they carry a range of RNA species. Density gradient centrifugation further fractionated and characterised the MV preparation and confirmed that the isolated RNA was associated with the highest particle and protein containing fractions. Using a new approach, RNA-sequencing of libraries derived from three different ‘size’ RNA populations (<50nt, 50-200nt and 200nt+) isolated from MVs has enabled us to now report the first example of a complete bacterial MV-RNA profile. These data show that MVs carry rRNA, tRNAs, other small RNAs as well as full-length protein coding mRNAs. Confocal microscopy visualised the delivery of lipid labelled MVs into cultured bladder epithelial cells and showed their RNA cargo labelled with 5-EU (5-ethynyl uridine), was transported into the host cell cytoplasm and nucleus. MV RNA uptake by the cells was confirmed by droplet digital RT-PCR of csrC. It was estimated that 1% of MV RNA cargo is delivered into cultured cells. Conclusions These data add to the growing evidence of pathogenic bacterial MV being associated a wide range of RNAs. It further raises the plausibility for MV-RNA-mediated cross-kingdom communication whereby they influence host cell function during the infection process. PMID:27500956

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

    PubMed Central

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

    2015-01-01

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

  12. Correlative scanning-transmission electron microscopy reveals that a chimeric flavivirus is released as individual particles in secretory vesicles.

    PubMed

    Burlaud-Gaillard, Julien; Sellin, Caroline; Georgeault, Sonia; Uzbekov, Rustem; Lebos, Claude; Guillaume, Jean-Marc; Roingeard, Philippe

    2014-01-01

    The intracellular morphogenesis of flaviviruses has been well described, but flavivirus release from the host cell remains poorly documented. We took advantage of the optimized production of an attenuated chimeric yellow fever/dengue virus for vaccine purposes to study this phenomenon by microscopic approaches. Scanning electron microscopy (SEM) showed the release of numerous viral particles at the cell surface through a short-lived process. For transmission electron microscopy (TEM) studies of the intracellular ultrastructure of the small number of cells releasing viral particles at a given time, we developed a new correlative microscopy method: CSEMTEM (for correlative scanning electron microscopy - transmission electron microscopy). CSEMTEM analysis suggested that chimeric flavivirus particles were released as individual particles, in small exocytosis vesicles, via a regulated secretory pathway. Our morphological findings provide new insight into interactions between flaviviruses and cells and demonstrate that CSEMTEM is a useful new method, complementary to SEM observations of biological events by intracellular TEM investigations.

  13. BDNF Enhances Quantal Neurotransmitter Release and Increases the Number of Docked Vesicles at the Active Zones of Hippocampal Excitatory Synapses

    PubMed Central

    Tyler, William J.; Pozzo-Miller, Lucas D.

    2009-01-01

    Brain-derived neurotrophic factor (BDNF) is emerging as a key mediator of activity-dependent modifications of synaptic strength in the CNS. We investigated the hypothesis that BDNF enhances quantal neurotransmitter release by modulating the distribution of synaptic vesicles within presynaptic terminals using organotypic slice cultures of postnatal rat hippocampus. BDNF specifically increased the number of docked vesicles at the active zone of excitatory synapses on CA1 dendritic spines, with only a small increase in active zone size. In agreement with the hypothesis that an increased docked vesicle density enhances quantal neurotransmitter release, BDNF increased the frequency, but not the amplitude, of AMPA receptor-mediated miniature EPSCs (mEPSCs) recorded from CA1 pyramidal neurons in hippocampal slices. Synapse number, independently estimated from dendritic spine density and electron microscopy measurements, was also increased after BDNF treatment, indicating that the actions of BNDF on mEPSC frequency can be partially attributed to an increased synaptic density. Our results further suggest that all these actions were mediated via tyrosine kinase B (TrkB) receptor activation, established by inhibition of plasma membrane tyrosine kinases with K-252a. These results provide additional evidence of a fundamental role of the BDNF–TrkB signaling cascade in synaptic transmission, as well as in cellular models of hippocampus-dependent learning and memory. PMID:11404410

  14. Osteoblast-released Matrix Vesicles, Regulation of Activity and Composition by Sulfated and Non-sulfated Glycosaminoglycans*

    PubMed Central

    Schmidt, Johannes R.; Kliemt, Stefanie; Preissler, Carolin; Moeller, Stephanie; von Bergen, Martin; Hempel, Ute; Kalkhof, Stefan

    2016-01-01

    Our aging population has to deal with the increasing threat of age-related diseases that impair bone healing. One promising therapeutic approach involves the coating of implants with modified glycosaminoglycans (GAGs) that mimic the native bone environment and actively facilitate skeletogenesis. In previous studies, we reported that coatings containing GAGs, such as hyaluronic acid (HA) and its synthetically sulfated derivative (sHA1) as well as the naturally low-sulfated GAG chondroitin sulfate (CS1), reduce the activity of bone-resorbing osteoclasts, but they also induce functions of the bone-forming cells, the osteoblasts. However, it remained open whether GAGs influence the osteoblasts alone or whether they also directly affect the formation, composition, activity, and distribution of osteoblast-released matrix vesicles (MV), which are supposed to be the active machinery for bone formation. Here, we studied the molecular effects of sHA1, HA, and CS1 on MV activity and on the distribution of marker proteins. Furthermore, we used comparative proteomic methods to study the relative protein compositions of isolated MVs and MV-releasing osteoblasts. The MV proteome is much more strongly regulated by GAGs than the cellular proteome. GAGs, especially sHA1, were found to severely impact vesicle-extracellular matrix interaction and matrix vesicle activity, leading to stronger extracellular matrix formation and mineralization. This study shows that the regulation of MV activity is one important mode of action of GAGs and provides information on underlying molecular mechanisms. PMID:26598647

  15. Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release

    PubMed Central

    1979-01-01

    We describe the design and operation of a machine that freezes biological tissues by contact with a cold metal block, which incorporates a timing circuit that stimulates frog neuromuscular junctions in the last few milliseconds before thay are frozen. We show freeze-fracture replicas of nerve terminals frozen during transmitter discharge, which display synpatic vesicles caught in the act of exocytosis. We use 4-aminopyridine (4-AP) to increase the number of transmitter quanta discharged with each nerve impulse, and show that the number of exocytotic vesicles caught by quick-freezing increases commensurately, indicating that one vesicle undergoes exocytosis for each quantum that is discharged. We perform statistical analyses on the spatial distribution of synaptic vesicle discharge sites along the "active zones" that mark the secretory regions of these nerves, and show that individual vesicles fuse with the plasma membrane independent of one another, as expected from physiological demonstrations that quanta are discharged independently. Thus, the utility of quick- freezing as a technique to capture biological processes as evanescent as synaptic transmission has been established. An appendix describes a new capacitance method to measure freezing rates, which shows that the "temporal resolution" of our quick-freezing technique is 2 ms or better. PMID:38256

  16. BDNF increases release probability and the size of a rapidly recycling vesicle pool within rat hippocampal excitatory synapses

    PubMed Central

    Tyler, William J; Zhang, Xiao-lei; Hartman, Kenichi; Winterer, Jochen; Muller, Wolfgang; Stanton, Patric K; Pozzo-Miller, Lucas

    2006-01-01

    Exerting its actions pre-, post- and peri-synaptically, brain-derived neurotrophic factor (BDNF) is one of the most potent modulators of hippocampal synaptic function. Here, we examined the effects of BDNF on a rapidly recycling pool (RRP) of vesicles within excitatory synapses. First, we estimated vesicular release in hippocampal cultures by performing FM4-64 imaging in terminals impinging on enhanced green fluorescent protein (eGFP)-labelled dendritic spines – a hallmark of excitatory synapses. Consistent with a modulation of the RRP, BDNF increased the evoked destaining rate of FM4-64 only during the initial phase of field stimulation. Multiphoton microscopy in acute hippocampal slices confirmed these observations by selectively imaging the RRP, which was loaded with FM1-43 by hyperosmotic shock. Slices exposed to BDNF showed an increase in the evoked and spontaneous rates of FM1-43 destaining from terminals in CA1 stratum radiatum, mostly representing excitatory terminals of Schaffer collaterals. Variance-mean analysis of evoked EPSCs in CA1 pyramidal neurons further confirmed that release probability is increased in BDNF-treated slices, without changes in the number of independent release sites or average postsynaptic quantal amplitude. Because BDNF was absent during dye loading, imaging, destaining and whole-cell recordings, these results demonstrate that BDNF induces a long-lasting enhancement in the probability of transmitter release at hippocampal excitatory synapses by modulating the RRP. Since the endogenous BDNF scavenger TrkB-IgG prevented the enhancement of FM1-43 destaining rate caused by induction of long-term potentiation in acute hippocampal slices, the modulation of a rapidly recycling vesicle pool may underlie the role of BDNF in hippocampal long-term synaptic plasticity. PMID:16709633

  17. Reversible Recruitment of a Homeostatic Reserve Pool of Synaptic Vesicles Underlies Rapid Homeostatic Plasticity of Quantal Content

    PubMed Central

    Pinter, Martin J.; Rich, Mark M.

    2016-01-01

    Homeostatic regulation is essential for the maintenance of synaptic strength within the physiological range. The current study is the first to demonstrate that both induction and reversal of homeostatic upregulation of synaptic vesicle release can occur within seconds of blocking or unblocking acetylcholine receptors at the mouse neuromuscular junction. Our data suggest that the homeostatic upregulation of release is due to Ca2+-dependent increase in the size of the readily releasable pool (RRP). Blocking vesicle refilling prevented upregulation of quantal content (QC), while leaving baseline release relatively unaffected. This suggested that the upregulation of QC was due to mobilization of a distinct pool of vesicles that were rapidly recycled and thus were dependent on continued vesicle refilling. We term this pool the “homeostatic reserve pool.” A detailed analysis of the time course of vesicle release triggered by a presynaptic action potential suggests that the homeostatic reserve pool of vesicles is normally released more slowly than other vesicles, but the rate of their release becomes similar to that of the major pool during homeostatic upregulation of QC. Remarkably, instead of finding a generalized increase in the recruitment of vesicles into RRP, we identified a distinct homeostatic reserve pool of vesicles that appear to only participate in synchronized release following homeostatic upregulation of QC. Once this small pool of vesicles is depleted by the block of vesicle refilling, homeostatic upregulation of QC is no longer observed. This is the first identification of the population of vesicles responsible for the blockade-induced upregulation of release previously described. SIGNIFICANCE STATEMENT The current study is the first to demonstrate that both the induction and reversal of homeostatic upregulation of synaptic vesicle release can occur within seconds. Our data suggest that homeostatic upregulation of release is due to Ca2+-dependent

  18. Metabolically active extracellular vesicles released from hepatocytes under drug-induced liver-damaging conditions modify serum metabolome and might affect different pathophysiological processes.

    PubMed

    Royo, Felix; Palomo, Laura; Mleczko, Justyna; Gonzalez, Esperanza; Alonso, Cristina; Martínez, Ibon; Pérez-Cormenzana, Miriam; Castro, Azucena; Falcon-Perez, Juan M

    2017-02-15

    Hepatocytes are involved in the endogenous and drug metabolism; many of the enzymes involved in those processes are incorporated into extracellular vesicles and secreted into the bloodstream. Liver-damaging conditions modify the molecular cargo of those vesicles significantly. However, no information about the effect of these hepatic vesicles on the extracellular environment is available. Drug-induced liver damage increases the number of circulating extracellular vesicles and affects the release and content of hepatocyte-derived vesicles. In this work, we evaluated the metabolic effect of these vesicles on the composition of the serum. We performed a targeted ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) metabolomics analysis of serum samples. The samples had been first incubated with hepatic extracellular vesicles from hepatocytes challenged with acetaminophen or diclofenac. The incubation affected the serum levels of 67 metabolites, such as amino acids and different species of lipids. The metabolites included various species of phosphatidylcholines and phosphatidylethanolamines. These compounds are the components of biological membranes; our observations suggest that the vesicles might take part in remodelling and maintenance of the membranes. Alterations in the levels of some other serum metabolites might have deleterious consequences, for example, the tetracosanoic acid with its cardiovascular effects. However, some of the metabolites whose levels were increased, including alpha-linoleic and tauroursodeoxycholic acids, have been reported to have a protective effect. Our targeted metabolomics analysis indicated that the hepatic extracellular vesicles act as nano-metabolic machines supplying the extracellular environment with the means to integrate diverse tissue responses. In conclusion, we show that the hepatic extracellular vesicles are metabolically active and might play a role in the physiopathological response to hepatic insults

  19. Simulated ischaemia induces Ca2+-independent glutamatergic vesicle release through actin filament depolymerization in area CA1 of the hippocampus.

    PubMed

    Andrade, Adriana L; Rossi, David J

    2010-05-01

    Transient, non-catastrophic brain ischaemia can induce either a protected state against subsequent episodes of ischaemia (ischaemic preconditioning) or delayed, selective neuronal death. Altered glutamatergic signalling and altered Ca(2+) homeostasis have been implicated in both processes. Here we use simultaneous patch-clamp recording and Ca(2+) imaging to monitor early changes in glutamate release and cytoplasmic [Ca(2+)] ([Ca(2+)](c)) in an in vitro slice model of hippocampal ischaemia. In slices loaded with the Ca(2+)-sensitive dye Fura-2, ischaemia leads to an early increase in [Ca(2+)](c) that precedes the severe ischaemic depolarization (ID) associated with pan necrosis. The early increase in [Ca(2+)](c) is mediated by influx through the plasma membrane and release from internal stores, and parallels an early increase in vesicular glutamate release that manifests as a fourfold increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs). However, the increase in mEPSC frequency is not prevented by blocking the increase in [Ca(2+)](c), and the early rise in [Ca(2+)](c) is not affected by blocking ionotropic and metabotropic glutamate receptors. Thus, the increase in [Ca(2+)](c) and the increase in glutamate release are independent of each other. Stabilizing actin filaments with jaspamide or phalloidin prevented vesicle release induced by ischaemia. Our results identify several early cellular cascades triggered by ischaemia: Ca(2+) influx, Ca(2+) release from intracellular stores, actin filament depolymerization, and vesicular release of glutamate that depends on actin dynamics but not [Ca(2+)](c). All of these processes precede the catastrophic ID by several minutes, and thus represent potential target mechanisms to influence the outcome of an ischaemic episode.

  20. Hepatocytes release ceramide-enriched pro-inflammatory extracellular vesicles in an IRE1α-dependent manner[S

    PubMed Central

    Kakazu, Eiji; Mauer, Amy S.; Yin, Meng; Malhi, Harmeet

    2016-01-01

    Nonalcoholic steatohepatitis (NASH) is a lipotoxic disease wherein activation of endoplasmic reticulum (ER) stress response and macrophage-mediated hepatic inflammation are key pathogenic features. However, the lipid mediators linking these two observations remain elusive. We postulated that ER stress-regulated release of pro-inflammatory extracellular vesicles (EVs) from lipotoxic hepatocytes may be this link. EVs were isolated from cell culture supernatants of hepatocytes treated with palmitate (PA) to induce lipotoxic ER stress, characterized by immunofluorescence, Western blotting, electron microscopy, and nanoparticle tracking analysis. Sphingolipids were measured by tandem mass spectrometry. EVs were employed in macrophage chemotaxis assays. PA induced significant EV release. Because PA activates ER stress, we used KO hepatocytes to demonstrate that PA-induced EV release was mediated by inositol requiring enzyme 1α (IRE1α)/X-box binding protein-1. PA-induced EVs were enriched in C16:0 ceramide in an IRE1α-dependent manner, and activated macrophage chemotaxis via formation of sphingosine-1-phosphate (S1P) from C16:0 ceramide. This chemotaxis was blocked by sphingosine kinase inhibitors and S1P receptor inhibitors. Lastly, elevated circulating EVs in experimental and human NASH demonstrated increased C16:0 ceramide. PA induces C16:0 ceramide-enriched EV release in an IRE1α-dependent manner. The ceramide metabolite, S1P, activates macrophage chemotaxis, a potential mechanism for the recruitment of macrophages to the liver under lipotoxic conditions. PMID:26621917

  1. Melanosome transfer to keratinocyte in the chicken embryonic skin is mediated by vesicle release associated with Rho-regulated membrane blebbing

    PubMed Central

    Tadokoro, Ryosuke; Murai, Hidetaka; Sakai, Ken-ichiro; Okui, Takahiro; Yokota, Yasuhiro; Takahashi, Yoshiko

    2016-01-01

    During skin pigmentation in amniotes, melanin synthesized in the melanocyte is transferred to keratinocytes by a particle called the melanosome. Previous studies, mostly using dissociated cultured cells, have proposed several different models that explain how the melanosome transfer is achieved. Here, using a technique that labels the plasma membrane of melanocytes within a three-dimensional system that mimics natural tissues, we have visualized the plasma membrane of melanocytes with EGFP in chicken embryonic skin. Confocal time-lapse microscopy reveals that the melanosome transfer is mediated, at least in part, by vesicles produced by plasma membrane. Unexpectedly, the vesicle release is accompanied by the membrane blebbing of melanocytes. Blebs that have encapsulated a melanosome are pinched off to become vesicles, and these melanosome-containing vesicles are finally engulfed by neighboring keratinocytes. For both the membrane blebbing and vesicle release, Rho small GTPase is essential. We further show that the membrane vesicle-mediated melanosome transfer plays a significant role in the skin pigmentation. Given that the skin pigmentation in inter-feather spaces in chickens is similar to that in inter-hair spaces of humans, our findings should have important consequences in cosmetic medicine. PMID:27910904

  2. RIG-I activation induces the release of extracellular vesicles with antitumor activity

    PubMed Central

    Daßler-Plenker, Juliane; Reiners, Katrin S.; van den Boorn, Jasper G.; Hansen, Hinrich P.; Putschli, Bastian; Barnert, Sabine; Schuberth-Wagner, Christine; Schubert, Rolf; Tüting, Thomas; Hallek, Michael; Schlee, Martin; Hartmann, Gunther; Pogge von Strandmann, Elke; Coch, Christoph

    2016-01-01

    ABSTRACT Activation of the innate immune receptor retinoic acid-inducible gene I (RIG-I) by its specific ligand 5′-triphosphate-RNA (3pRNA) triggers antitumor immunity predominantly via NK cell activation and direct apoptosis induction in tumor cells. However, how NK cells are mobilized to attack the tumor cells remains elusive. Here, we show that RIG-I activation induced the secretion of extracellular vesicles (EVs) from melanoma cells, which by themselves revealed antitumor activity in vitro and in vivo. RIG-I-induced EVs from melanoma cells exhibited an increased expression of the NKp30-ligand (BAG6, BAT3) on their surface triggering NK cell-mediated lysis of melanoma cells via activation of the cytotoxicity NK cell-receptor NKp30. Moreover, systemic administration of RIG-I-induced melanoma-EVs showed a potent antitumor activity in a melanoma mouse model in vivo. In conclusion, our data establish a new RIG-I-dependent pathway leading to NK cell-mediated tumor cell killing. PMID:27853642

  3. Activation of metabotropic GABA receptors increases the energy barrier for vesicle fusion.

    PubMed

    Rost, Benjamin R; Nicholson, Patrick; Ahnert-Hilger, Gudrun; Rummel, Andreas; Rosenmund, Christian; Breustedt, Joerg; Schmitz, Dietmar

    2011-09-15

    Neurotransmitter release from presynaptic terminals is under the tight control of various metabotropic receptors. We report here that in addition to the regulation of Ca(2+) channel activity, metabotropic GABA(B) receptors (GABA(B)Rs) at murine hippocampal glutamatergic synapses utilize an inhibitory pathway that directly targets the synaptic vesicle release machinery. Acute application of the GABA(B)R agonist baclofen rapidly and reversibly inhibits vesicle fusion, which occurs independently of the SNAP-25 C-terminus. Using applications of hypertonic sucrose solutions, we find that the size of the readily releasable pool remains unchanged by GABA(B)R activation, but the sensitivity of primed vesicles to hypertonic stimuli appears lowered as the response amplitudes at intermediate sucrose concentrations are smaller and release kinetics are slowed. These data show that presynaptic GABA(B)Rs can inhibit neurotransmitter release directly by increasing the energy barrier for vesicle fusion.

  4. An Active Form of Sphingosine Kinase-1 Is Released in the Extracellular Medium as Component of Membrane Vesicles Shed by Two Human Tumor Cell Lines

    PubMed Central

    Rigogliuso, Salvatrice; Donati, Chiara; Cassarà, Donata; Taverna, Simona; Salamone, Monica; Bruni, Paola; Vittorelli, Maria Letizia

    2010-01-01

    Expression of sphingosine kinase-1 (SphK-1) correlates with a poor survival rate of tumor patients. This effect is probably due to the ability of SphK-1 to be released into the extracellular medium where it catalyzes the biosynthesis of sphingosine-1-phosphate (S1P), a signaling molecule endowed with profound proangiogenic effects. SphK-1 is a leaderless protein which is secreted by an unconventional mechanism. In this paper, we will show that in human hepatocarcinoma Sk-Hep1 cells, extracellular signaling is followed by targeting the enzyme to the cell surface and parallels targeting of FGF-2 to the budding vesicles. We will also show that SphK-1 is present in a catalitycally active form in vesicles shed by SK-Hep1 and human breast carcinoma 8701-BC cells. The enzyme substrate sphingosine is present in shed vesicles where it is produced by neutral ceramidase. Shed vesicles are therefore a site for S1P production in the extracellular medium and conceivably also within host cell following vesicle endocytosis. PMID:20508814

  5. Synaptic vesicle glycoprotein 2C (SV2C) modulates dopamine release and is disrupted in Parkinson disease.

    PubMed

    Dunn, Amy R; Stout, Kristen A; Ozawa, Minagi; Lohr, Kelly M; Hoffman, Carlie A; Bernstein, Alison I; Li, Yingjie; Wang, Minzheng; Sgobio, Carmelo; Sastry, Namratha; Cai, Huaibin; Caudle, W Michael; Miller, Gary W

    2017-03-14

    Members of the synaptic vesicle glycoprotein 2 (SV2) family of proteins are involved in synaptic function throughout the brain. The ubiquitously expressed SV2A has been widely implicated in epilepsy, although SV2C with its restricted basal ganglia distribution is poorly characterized. SV2C is emerging as a potentially relevant protein in Parkinson disease (PD), because it is a genetic modifier of sensitivity to l-DOPA and of nicotine neuroprotection in PD. Here we identify SV2C as a mediator of dopamine homeostasis and report that disrupted expression of SV2C within the basal ganglia is a pathological feature of PD. Genetic deletion of SV2C leads to reduced dopamine release in the dorsal striatum as measured by fast-scan cyclic voltammetry, reduced striatal dopamine content, disrupted α-synuclein expression, deficits in motor function, and alterations in neurochemical effects of nicotine. Furthermore, SV2C expression is dramatically altered in postmortem brain tissue from PD cases but not in Alzheimer disease, progressive supranuclear palsy, or multiple system atrophy. This disruption was paralleled in mice overexpressing mutated α-synuclein. These data establish SV2C as a mediator of dopamine neuron function and suggest that SV2C disruption is a unique feature of PD that likely contributes to dopaminergic dysfunction.

  6. Synaptic vesicle glycoprotein 2C (SV2C) modulates dopamine release and is disrupted in Parkinson disease

    PubMed Central

    Stout, Kristen A.; Ozawa, Minagi; Lohr, Kelly M.; Hoffman, Carlie A.; Bernstein, Alison I.; Li, Yingjie; Wang, Minzheng; Sgobio, Carmelo; Sastry, Namratha; Cai, Huaibin; Caudle, W. Michael

    2017-01-01

    Members of the synaptic vesicle glycoprotein 2 (SV2) family of proteins are involved in synaptic function throughout the brain. The ubiquitously expressed SV2A has been widely implicated in epilepsy, although SV2C with its restricted basal ganglia distribution is poorly characterized. SV2C is emerging as a potentially relevant protein in Parkinson disease (PD), because it is a genetic modifier of sensitivity to l-DOPA and of nicotine neuroprotection in PD. Here we identify SV2C as a mediator of dopamine homeostasis and report that disrupted expression of SV2C within the basal ganglia is a pathological feature of PD. Genetic deletion of SV2C leads to reduced dopamine release in the dorsal striatum as measured by fast-scan cyclic voltammetry, reduced striatal dopamine content, disrupted α-synuclein expression, deficits in motor function, and alterations in neurochemical effects of nicotine. Furthermore, SV2C expression is dramatically altered in postmortem brain tissue from PD cases but not in Alzheimer disease, progressive supranuclear palsy, or multiple system atrophy. This disruption was paralleled in mice overexpressing mutated α-synuclein. These data establish SV2C as a mediator of dopamine neuron function and suggest that SV2C disruption is a unique feature of PD that likely contributes to dopaminergic dysfunction. PMID:28246328

  7. Sequential Drug Release and Enhanced Photothermal and Photoacoustic Effect of Hybrid Reduced Graphene Oxide-Loaded Ultrasmall Gold Nanorod Vesicles for Cancer Therapy

    PubMed Central

    Song, Jibin; Yang, Xiangyu; Jacobson, Orit; Lin, Lisen; Huang, Peng; Niu, Gang; Ma, Qingjie; Chen, Xiaoyuan

    2017-01-01

    We report a hybrid reduced graphene oxide (rGO)-loaded ultrasmall plasmonic gold nanorod vesicle (rGO-AuNRVe) (~65 nm in size) with remarkably amplified photoacoustic (PA) performance and photothermal effects. The hybrid vesicle also exhibits a high loading capacity of doxorubicin (DOX), as both the cavity of the vesicle and the large surface area of the encapsulated rGO can be used for loading DOX, making it an excellent drug carrier. The loaded DOX is released sequentially: near-infrared photothermal heating induces DOX release from the vesicular cavity, and an intracellular acidic environment induces DOX release from the rGO surface. Positron emission tomography imaging showed high passive U87MG tumor accumulation of 64Cu-labeled rGO-AuNRVes (~9.7% ID/g at 24 h postinjection) and strong PA signal in the tumor region. Single intravenous injection of rGO-AuNRVe-DOX followed by low-power-density 808 nm laser irradiation (0.25 W/cm2) revealed effective inhibition of tumor growth due to the combination of chemo- and photothermal therapies. The rGO-AuNRVe-DOX capable of sequential DOX release by laser light and acid environment may have the potential for clinical translation to treat cancer patients with tumors accessible by light. PMID:26308265

  8. [Peculiarities of synaptic vesicle recycling in frog and mouse motor nerve terminals].

    PubMed

    Zefirov, A L; Zakharov, A V; Mukhamedzianov, R D; Petrov, A M

    2008-01-01

    Using electrophysiology and fluorescence microscopy (dye FM1-43), comparative study of neurotransmitter secretion, synaptic vesicle exo-endocytosis, and recycling has been carried out in frog and mouse motor nerve terminals during a long strong stimulation (3 min; 20 imp/s). The obtained data have revealed three synaptic vesicle pools and two recycling ways existing on motor nerve terminals. The strong stimulation induced consecutive depletion of readily releasable, mobilized, and reserve vesicle pools of frog nerve terminals. The exocytosis rate exceeded the endocytosis rate; predominant was the slow synaptic vesicle recycling that replenished the reserve pool. In mouse nerve endings, vesicles of the readily releasable and mobilized pools were only exocytosed, the pools being replenished by fast recycling. At the same time, exo- and endocytosis occurred nearly in parallel and vesicles of the reserve pool did not participate in the neurotransmitter secretion. In is suggested that evolution of motor nerve terminals was directed to a decrease of the vesicle pool size, economic spending, and effective reuse of synaptic vesicles. This is achieved by an increase of endocytosis and recycling rates. These features can provide a long maintenance of a quite level of neurotransmitter secretion in nerve terminals of homoiothermal animals to preserve reliability of synaptic transmission during the high-frequency activity.

  9. 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

  10. Roles of alkaline phosphatase and labile internal mineral in matrix vesicle-mediated calcification. Effect of selective release of membrane-bound alkaline phosphatase and treatment with isosmotic pH 6 buffer.

    PubMed

    Register, T C; McLean, F M; Low, M G; Wuthier, R E

    1986-07-15

    The roles of alkaline phosphatase and labile internal mineral in matrix vesicle-mediated mineralization have been studied by selectively releasing the enzyme from a wide variety of matrix vesicle preparations using treatment with a bacterial phosphatidylinositol-specific phospholipase C and by demineralization of the vesicles using isosmotic pH 6 buffer. Following depletion of 50-90% of the alkaline phosphatase activity or treatment with citrate buffer, the vesicles were tested for their ability to accumulate 45Ca2+ and 32Pi from a synthetic cartilage lymph. Removal of alkaline phosphatase by phospholipase C treatment caused two principal effects, depending on the matrix vesicle preparation. In rapidly mineralizing vesicle fractions which did not require organic phosphate esters (Po) to accumulate mineral ions, release of alkaline phosphatase had only a minor effect. In slowly mineralizing vesicles preparations or those dependent on Po substrates for mineral ion uptake, release of alkaline phosphatase caused significant loss of mineralizing activity. The activity of rapidly calcifying vesicles was shown to be dependent on the presence of labile internal mineral, as demonstrated by major loss in activity when the vesicles were decalcified by various treatments. Ion uptake by demineralized vesicles or those fractionated on sucrose step gradients required Po and was significantly decreased by alkaline phosphatase depletion. Uptake of Pi, however, was not coupled with hydrolysis of the Po substrate. These findings argue against a direct role for alkaline phosphatase as a porter in matrix vesicle Pi uptake, contrary to previous postulates. The results emphasize the importance of internal labile mineral in rapid uptake of mineral ions by matrix vesicles.

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

    SciTech Connect

    Zhang, Xiaojun; Chen, Yuan; Chen, Yong

    2014-03-28

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

  12. 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).

  13. Modeling the influence of short term depression in vesicle release and stochastic calcium channel gating on auditory nerve spontaneous firing statistics.

    PubMed

    Moezzi, Bahar; Iannella, Nicolangelo; McDonnell, Mark D

    2014-01-01

    We propose several modifications to an existing computational model of stochastic vesicle release in inner hair cell ribbon synapses, with the aim of producing simulated auditory nerve fiber spiking data that more closely matches empirical data. Specifically, we studied the inter-spike-interval (ISI) distribution, and long and short term ISI correlations in spontaneous spiking in post-synaptic auditory nerve fibers. We introduced short term plasticity to the pre-synaptic release probability, in a manner analogous to standard stochastic models of cortical short term synaptic depression. This modification resulted in a similar distribution of vesicle release intervals to that estimated from empirical data. We also introduced a biophysical stochastic model of calcium channel opening and closing, but showed that this model is insufficient for generating a match with empirically observed spike correlations. However, by combining a phenomenological model of channel noise and our short term depression model, we generated short and long term correlations in auditory nerve spontaneous activity that qualitatively match empirical data.

  14. Modeling the influence of short term depression in vesicle release and stochastic calcium channel gating on auditory nerve spontaneous firing statistics

    PubMed Central

    Moezzi, Bahar; Iannella, Nicolangelo; McDonnell, Mark D.

    2014-01-01

    We propose several modifications to an existing computational model of stochastic vesicle release in inner hair cell ribbon synapses, with the aim of producing simulated auditory nerve fiber spiking data that more closely matches empirical data. Specifically, we studied the inter-spike-interval (ISI) distribution, and long and short term ISI correlations in spontaneous spiking in post-synaptic auditory nerve fibers. We introduced short term plasticity to the pre-synaptic release probability, in a manner analogous to standard stochastic models of cortical short term synaptic depression. This modification resulted in a similar distribution of vesicle release intervals to that estimated from empirical data. We also introduced a biophysical stochastic model of calcium channel opening and closing, but showed that this model is insufficient for generating a match with empirically observed spike correlations. However, by combining a phenomenological model of channel noise and our short term depression model, we generated short and long term correlations in auditory nerve spontaneous activity that qualitatively match empirical data. PMID:25566047

  15. Membrane Vesicles Released by a hypervesiculating Escherichia coli Nissle 1917 tolR Mutant Are Highly Heterogeneous and Show Reduced Capacity for Epithelial Cell Interaction and Entry

    PubMed Central

    Pérez-Cruz, Carla; Cañas, María-Alexandra; Giménez, Rosa; Badia, Josefa; Mercade, Elena; Aguilera, Laura

    2016-01-01

    Membrane vesicles (MVs) produced by Gram-negative bacteria are being explored for novel clinical applications due to their ability to deliver active molecules to distant host cells, where they can exert immunomodulatory properties. MVs released by the probiotic Escherichia coli Nissle 1917 (EcN) are good candidates for testing such applications. However, a drawback for such studies is the low level of MV isolation from in vitro culture supernatants, which may be overcome by the use of mutants in cell envelope proteins that yield a hypervesiculation phenotype. Here, we confirm that a tolR mutation in EcN increases MV production, as determined by protein, LPS and fluorescent lipid measurements. Transmission electron microscopy (TEM) of negatively stained MVs did not reveal significant differences with wild type EcN MVs. Conversely, TEM observation after high-pressure freezing followed by freeze substitution of bacterial samples, together with cryo-TEM observation of plunge-frozen hydrated isolated MVs showed considerable structural heterogeneity in the EcN tolR samples. In addition to common one-bilayer vesicles (OMVs) and the recently described double-bilayer vesicles (O-IMVs), other types of MVs were observed. Time-course experiments of MV uptake in Caco-2 cells using rhodamine- and DiO-labelled MVs evidenced that EcN tolR MVs displayed reduced internalization levels compared to the wild-type MVs. The low number of intracellular MVs was due to a lower cell binding capacity of the tolR-derived MVs, rather than a different entry pathway or mechanism. These findings indicate that heterogeneity of MVs from tolR mutants may have a major impact on vesicle functionality, and point to the need for conducting a detailed structural analysis when MVs from hypervesiculating mutants are to be used for biotechnological applications. PMID:28036403

  16. Readily integrated, electrically controlled microvalves

    NASA Astrophysics Data System (ADS)

    Song, W. H.; Kwan, J.; Kaigala, G. V.; Hoang, V. N.; Backhouse, C. J.

    2008-04-01

    We present a simple method for fabricating and operating normally open, electrothermally actuated microvalves. These valves are fabricated by placing a gas-permeable elastomeric membrane between two etched glass plates. The reservoirs and channels on one layer are filled with a low melting point polymer (polyethylene glycol, PEG) that exhibits a large volumetric change (of up to 30%) upon phase transition (melting). This volume expansion is used to actuate the membrane and seal the microfluidic channels located in the second etched glass plate. The PEG in the reservoir is heated with integrated patterned platinum-resistive elements. The valve reliably seals the microfluidic channel against external fluid pressures of 10 psi. This valve can be readily integrated with one of the standard technologies for lab-on-a-chip (LOC) fabrication and is suitable for use with the polymerase chain reaction. The novelty of this microvalve lies in the ability to fill dead-end microchannels with a polymer, its self-sealing ability, the ability to remotely actuate the valve by transferring pressure via a microchannel and the compatibility of this microvalve with standard LOC technologies.

  17. A Missense Mutation of the Gene Encoding Synaptic Vesicle Glycoprotein 2A (SV2A) Confers Seizure Susceptibility by Disrupting Amygdalar Synaptic GABA Release

    PubMed Central

    Tokudome, Kentaro; Okumura, Takahiro; Terada, Ryo; Shimizu, Saki; Kunisawa, Naofumi; Mashimo, Tomoji; Serikawa, Tadao; Sasa, Masashi; Ohno, Yukihiro

    2016-01-01

    Synaptic vesicle glycoprotein 2A (SV2A) is specifically expressed in the membranes of synaptic vesicles and modulates action potential-dependent neurotransmitter release. To explore the role of SV2A in the pathogenesis of epileptic disorders, we recently generated a novel rat model (Sv2aL174Q rat) carrying a missense mutation of the Sv2a gene and showed that the Sv2aL174Q rats were hypersensitive to kindling development (Tokudome et al., 2016). Here, we further conducted behavioral and neurochemical studies to clarify the pathophysiological mechanisms underlying the seizure vulnerability in Sv2aL174Q rats. Sv2aL174Q rats were highly susceptible to pentylenetetrazole (PTZ)-induced seizures, yielding a significantly higher seizure scores and seizure incidence than the control animals. Brain mapping analysis of Fos expression, a biological marker of neural excitation, revealed that the seizure threshold level of PTZ region-specifically elevated Fos expression in the amygdala in Sv2aL174Q rats. In vivo microdialysis study showed that the Sv2aL174Q mutation preferentially reduced high K+ (depolarization)-evoked GABA release, but not glutamate release, in the amygdala. In addition, specific control of GABA release by SV2A was supported by its predominant expression in GABAergic neurons, which were co-stained with antibodies against SV2A and glutamate decarboxylase 1. The present results suggest that dysfunction of SV2A by the missense mutation elevates seizure susceptibility in rats by preferentially disrupting synaptic GABA release in the amygdala, illustrating the crucial role of amygdalar SV2A-GABAergic system in epileptogenesis. PMID:27471467

  18. Mesolimbic alpha-, but not beta-adrenoceptors control the accumbal release of dopamine that is derived from reserpine-sensitive storage vesicles.

    PubMed

    Verheij, M M M; Cools, A R

    2009-09-15

    Mesolimbic beta-, but not alpha-adrenoceptors control the accumbal release of dopamine that is derived from alpha-methyl-para-tyrosine-sensitive pools of newly synthesized neurotransmitter. The aim of this study was to investigate which of these adrenoceptors control the accumbal release of dopamine that is derived from reserpine-sensitive pools of previously stored neurotransmitter. Rats, that were divided in low-responders and high-responders to novelty, were pretreated with 1 mg/kg of reserpine before the alpha-adrenergic-agent phentolamine or the beta-adrenergic-agent isoproterenol was locally applied into the nucleus accumbens. The original finding that phentolamine and isoproterenol increased accumbal dopamine levels in low-responders and high-responders was replicated. Reserpine reduced the phentolamine-induced increase of accumbal dopamine in both types of rat. However, phentolamine could still increase accumbal dopamine levels in reserpine-treated high-responders, but not anymore in reserpine-treated low-responders. Reserpine did not reduce the isoproterenol-induced increase of accumbal dopamine in any type of rat. This study demonstrates that mesolimbic alpha-, but not beta-adrenoceptors control the accumbal release of dopamine that is derived from reserpine-sensitive storage vesicles. In addition, these data confirm our previous finding that dopamine can still be released from storage vesicles of reserpinized high-responders, but not of reserpinized low-responders. The collected data underline our notion that alpha- and beta-adrenergic drugs may have therapeutic effects in patients suffering from diseases in which accumbal dopamine is involved.

  19. Cephalosporinases associated with outer membrane vesicles released by Bacteroides spp. protect gut pathogens and commensals against β-lactam antibiotics

    PubMed Central

    Stentz, Régis; Horn, Nikki; Cross, Kathryn; Salt, Louise; Brearley, Charles; Livermore, David M.; Carding, Simon R.

    2015-01-01

    Objectives To identify β-lactamase genes in gut commensal Bacteroides species and to assess the impact of these enzymes, when carried by outer membrane vesicles (OMVs), in protecting enteric pathogens and commensals. Methods A deletion mutant of the putative class A β-lactamase gene (locus tag BT_4507) found in the genome of the human commensal Bacteroides thetaiotaomicron was constructed and a phenotypic analysis performed. A phylogenetic tree was built from an alignment of nine Bacteroides cephalosporinase protein sequences, using the maximum likelihood method. The rate of cefotaxime degradation after incubation with OMVs produced by different Bacteroides species was quantified using a disc susceptibility test. The resistance of Salmonella Typhimurium and Bifidobacterium breve to cefotaxime in liquid culture in the presence of B. thetaiotaomicron OMVs was evaluated by measuring bacterial growth. Results The B. thetaiotaomicron BT_4507 gene encodes a β-lactamase related to the CepA cephalosporinase of Bacteroides fragilis. OMVs produced by B. thetaiotaomicron and several other Bacteroides species, except Bacteroides ovatus, carried surface-associated β-lactamases that could degrade cefotaxime. β-Lactamase-harbouring OMVs from B. thetaiotaomicron protected Salmonella Typhimurium and B. breve from an otherwise lethal dose of cefotaxime. Conclusions The production of membrane vesicles carrying surface-associated β-lactamases by Bacteroides species, which constitute a major part of the human colonic microbiota, may protect commensal bacteria and enteric pathogens, such as Salmonella Typhimurium, against β-lactam antibiotics. PMID:25433011

  20. 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.

  1. 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

  2. Bassoon-disruption slows vesicle replenishment and induces homeostatic plasticity at a CNS synapse

    PubMed Central

    Mendoza Schulz, Alejandro; Jing, Zhizi; María Sánchez Caro, Juan; Wetzel, Friederike; Dresbach, Thomas; Strenzke, Nicola; Wichmann, Carolin; Moser, Tobias

    2014-01-01

    Endbulb of Held terminals of auditory nerve fibers (ANF) transmit auditory information at hundreds per second to bushy cells (BCs) in the anteroventral cochlear nucleus (AVCN). Here, we studied the structure and function of endbulb synapses in mice that lack the presynaptic scaffold bassoon and exhibit reduced ANF input into the AVCN. Endbulb terminals and active zones were normal in number and vesicle complement. Postsynaptic densities, quantal size and vesicular release probability were increased while vesicle replenishment and the standing pool of readily releasable vesicles were reduced. These opposing effects canceled each other out for the first evoked EPSC, which showed unaltered amplitude. We propose that ANF activity deprivation drives homeostatic plasticity in the AVCN involving synaptic upscaling and increased intrinsic BC excitability. In vivo recordings from individual mutant BCs demonstrated a slightly improved response at sound onset compared to ANF, likely reflecting the combined effects of ANF convergence and homeostatic plasticity. Further, we conclude that bassoon promotes vesicular replenishment and, consequently, a large standing pool of readily releasable synaptic vesicles at the endbulb synapse. PMID:24442636

  3. Bassoon-disruption slows vesicle replenishment and induces homeostatic plasticity at a CNS synapse.

    PubMed

    Mendoza Schulz, Alejandro; Jing, Zhizi; Sánchez Caro, Juan María; Wetzel, Friederike; Dresbach, Thomas; Strenzke, Nicola; Wichmann, Carolin; Moser, Tobias

    2014-03-03

    Endbulb of Held terminals of auditory nerve fibers (ANF) transmit auditory information at hundreds per second to bushy cells (BCs) in the anteroventral cochlear nucleus (AVCN). Here, we studied the structure and function of endbulb synapses in mice that lack the presynaptic scaffold bassoon and exhibit reduced ANF input into the AVCN. Endbulb terminals and active zones were normal in number and vesicle complement. Postsynaptic densities, quantal size and vesicular release probability were increased while vesicle replenishment and the standing pool of readily releasable vesicles were reduced. These opposing effects canceled each other out for the first evoked EPSC, which showed unaltered amplitude. We propose that ANF activity deprivation drives homeostatic plasticity in the AVCN involving synaptic upscaling and increased intrinsic BC excitability. In vivo recordings from individual mutant BCs demonstrated a slightly improved response at sound onset compared to ANF, likely reflecting the combined effects of ANF convergence and homeostatic plasticity. Further, we conclude that bassoon promotes vesicular replenishment and, consequently, a large standing pool of readily releasable synaptic vesicles at the endbulb synapse.

  4. Release of HIV-1 sequestered in the vesicles of oral and genital mucosal epithelial cells by epithelial-lymphocyte interaction

    PubMed Central

    Yasen, Aizezi; Herrera, Rossana; Rosbe, Kristina

    2017-01-01

    Oropharyngeal mucosal epithelia of fetuses/neonates/infants and the genital epithelia of adults play a critical role in HIV-1 mother-to-child transmission and sexual transmission of virus, respectively. To study the mechanisms of HIV-1 transmission through mucosal epithelium, we established polarized tonsil, cervical and foreskin epithelial cells. Analysis of HIV-1 transmission through epithelial cells showed that approximately 0.05% of initially inoculated virions transmigrated via epithelium. More than 90% of internalized virions were sequestered in the endosomes of epithelial cells, including multivesicular bodies (MVBs) and vacuoles. Intraepithelial HIV-1 remained infectious for 9 days without viral release. Release of sequestered intraepithelial HIV-1 was induced by the calcium ionophore ionomycin and by cytochalasin D, which increase intracellular calcium and disrupt the cortical actin of epithelial cells, respectively. Cocultivation of epithelial cells containing HIV-1 with activated peripheral blood mononuclear cells and CD4+ T lymphocytes led to the disruption of epithelial cortical actin and spread of virus from epithelial cells to lymphocytes. Treatment of epithelial cells with proinflammatory cytokines tumor necrosis factor-alpha and interferon gamma also induced reorganization of cortical actin and release of virus. Inhibition of MVB formation by small interfering RNA (siRNA)-mediated silencing of its critical protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) expression reduced viral sequestration in epithelial cells and its transmission from epithelial cells to lymphocytes by ~60–70%. Furthermore, inhibition of vacuole formation of epithelial cells by siRNA-inactivated rabankyrin-5 expression also significantly reduced HIV-1 sequestration in epithelial cells and spread of virus from epithelial cells to lymphocytes. Interaction of the intercellular adhesion molecule-1 of epithelial cells with the function-associated antigen-1

  5. Nigrostriatal overabundance of α-synuclein leads to decreased vesicle density and deficits in dopamine release that correlate with reduced motor activity.

    PubMed

    Gaugler, Meret Nora; Genc, Ozgur; Bobela, Wojciech; Mohanna, Safa; Ardah, Mustafa Taleb; El-Agnaf, Omar Mukhtar; Cantoni, Marco; Bensadoun, Jean-Charles; Schneggenburger, Ralf; Knott, Graham W; Aebischer, Patrick; Schneider, Bernard Laurent

    2012-05-01

    α-Synuclein (α-syn) is a presynaptic protein present at most nerve terminals, but its function remains largely unknown. The familial forms of Parkinson's disease associated with multiplications of the α-syn gene locus indicate that overabundance of this protein might have a detrimental effect on dopaminergic transmission. To investigate this hypothesis, we use adeno-associated viral (AAV) vectors to overexpress human α-syn in the rat substantia nigra. Moderate overexpression of either wild-type (WT) or A30P α-syn differs in the motor phenotypes induced, with only the WT form generating hemiparkinsonian impairments. Wild-type α-syn causes a reduction of dopamine release in the striatum that exceeds the loss of dopaminergic neurons, axonal fibers, and the reduction in total dopamine. At the ultrastructural level, the reduced dopamine release corresponds to a decreased density of dopaminergic vesicles and synaptic contacts in striatal terminals. Interestingly, the membrane-binding-deficient A30P mutant does neither notably reduce dopamine release nor it cause ultrastructural changes in dopaminergic axons, showing that α-syn's membrane-binding properties are critically involved in the presynaptic defects. To further determine if the affinity of the protein for membranes determines the extent of motor defects, we compare three forms of α-syn in conditions leading to pronounced degeneration. While membrane-binding α-syns (wild-type and A53T) induce severe motor impairments, an N-terminal deleted form with attenuated affinity for membranes is inefficient in inducing motor defects. Overall, these results demonstrate that α-syn overabundance is detrimental to dopamine neurotransmission at early stages of the degeneration of nigrostriatal dopaminergic axons.

  6. P01.07IMPACT OF EXTRACELLULAR VESICLES RELEASED BY GLIOBLASTOMA CELLS AFTER IRRADIATION ON TUMOR MICROENVIRONMENT

    PubMed Central

    Ding, H.; Pinel, S.; Jouan-Hureaux, V.; Chateau, A.; Boura, C.; Faivre, B.

    2014-01-01

    AIMS: Glioblastoma (GBM) is the most lethal of all human tumors. Ionizing radiation (IR), as a major therapeutic modality, induces multiple types of DNA lesions in cells, therefore causes cell death. However, IR could also affect neighboring unirradiated cells, inducing Bystander Effects as chromosomal aberrations, increased proliferation, etc. Intercellular communication through the release of different components is involved in the mechanism. GBM cells release different soluble factors as well as tumor microvesicles (TMVs) to modify the phenotype of neighboring cells, thus participating in the tumor progression. The present study was designed to investigate in vitro the impact of IR on the communication between tumor cells and endothelial cells in the tumor microenvironment via soluble factors and TMVs. MATERIALS AND METHODS: Two GBM cell lines (T98G, U87) were grown and sham-irradiated (0Gy) or irradiated (2 or 10Gy) using a Clinac iX linear accelerator. Cell culture media (CM) were collected. Filtrate (containing only soluble factors) and TMVs were separated with successive centrifugations and Pierce concentrator. Cell viability was assessed by cell counting using trypan blue. TMVs quantifications were performed by flow cytometry. The effect of CM/Filtrate/TMVs on the global behavior (proliferation, adhesion) of bystander tumor cells or HUVEC was investigated using the xCELLigence system (ACEA). RESULTS: As expected, irradiation caused a loss of cell number in U87 and T98G: 20% at 2Gy and 60% at 10Gy 48h post-IR as compared to untreated cells. Both CM and Filtrate collected from sham-irradiated tumor cells induced a 50% reduction of bystander tumor cells proliferation, while CM and Filtrate recovered from 10Gy-irradiated cells had different influence on the proliferation: herein, the inhibitory properties of CM were less marked than those of Filtrate. The discordant effects between CM and Filtrate led us to investigate the role of TMVs. When quantified using

  7. Lack of Outer Membrane Protein A Enhances the Release of Outer Membrane Vesicles and Survival of Vibrio cholerae and Suppresses Viability of Acanthamoeba castellanii

    PubMed Central

    Valeru, Soni Priya; Shanan, Salah; Alossimi, Haifa; Sandström, Gunnar

    2014-01-01

    Vibrio cholerae, the causative agent of the diarrhoeal disease cholera, survives in aquatic environments. The bacterium has developed a survival strategy to grow and survive inside Acanthamoeba castellanii. It has been shown that V. cholerae expresses outer membrane proteins as virulence factors playing a role in the adherence to interacted host cells. This study examined the role of outer membrane protein A (OmpA) and outer membrane vesicles (OMVs) in survival of V. cholerae alone and during its interaction with A. castellanii. The results showed that an OmpA mutant of V. cholerae survived longer than wild-type V. cholerae when cultivated alone. Cocultivation with A. castellanii enhanced the survival of both bacterial strains and OmpA protein exhibited no effect on attachment, engulfment, and survival inside the amoebae. However, cocultivation of the OmpA mutant of V. cholerae decreased the viability of A. castellanii and this bacterial strain released more OMVs than wild-type V. cholerae. Surprisingly, treatment of amoeba cells with OMVs isolated from the OmpA mutant significantly decreased viable counts of the amoeba cells. In conclusion, the results might highlight a regulating rule for OmpA in survival of V. cholerae and OMVs as a potent virulence factor for this bacterium towards eukaryotes in the environment. PMID:24799908

  8. 1alpha,25(OH)2D3 is an autocrine regulator of extracellular matrix turnover and growth factor release via ERp60 activated matrix vesicle metalloproteinases.

    PubMed

    Boyan, Barbara D; Wong, Kevin L; Fang, Mimi; Schwartz, Zvi

    2007-03-01

    Growth plate chondrocytes produce proteoglycan-rich type II collagen extracellular matrix (ECM). During cell maturation and hypertrophy, ECM is reorganized via a process regulated by 1alpha,25(OH)(2)D(3) and involving matrix metalloproteinases (MMPs), including MMP-3 and MMP-2. 1alpha,25(OH)(2)D(3) regulates MMP incorporation into matrix vesicles (MVs), where they are stored until released. Like plasma membranes (PM), MVs contain the 1alpha,25(OH)(2)D(3)-binding protein ERp60, phospholipase A(2) (PLA(2)), and caveolin-1, but appear to lack nuclear Vitamin D receptors (VDRs). Chondrocytes produce 1alpha,25(OH)(2)D(3) (10(-8)M), which binds ERp60, activating PLA(2), and resulting lysophospholipids lead to MV membrane disorganization, releasing active MMPs. MV MMP-3 activates TGF-beta1 stored in the ECM as large latent TGF-beta1 complexes, consisting of latent TGF-beta1 binding protein, latency associated peptide, and latent TGF-beta1. Others have shown that MMP-2 specifically activates TGF-beta2. TGF-beta1 regulates 1alpha,25(OH)(2)D(3)-production, providing a mechanism for local control of growth factor activation. 1alpha,25(OH)(2)D(3) activates PKCalpha in the PM via ERp60-signaling through PLA(2), lysophospholipid production, and PLCbeta. It also regulates distribution of phospholipids and PKC isoforms between MVs and PMs, enriching the MVs in PKCzeta. Direct activation of MMP-3 in MVs requires ERp60. However, when MVs are treated with 1alpha,25(OH)(2)D(3), PKCzeta activity is decreased and PKCalpha is unaffected, suggesting a more complex feedback mechanism, potentially involving MV lipid signaling.

  9. Cholesterol regulates multiple forms of vesicle endocytosis at a mammalian central synapse.

    PubMed

    Yue, Hai-Yuan; Xu, Jianhua

    2015-07-01

    Endocytosis in synapses sustains neurotransmission by recycling vesicle membrane and maintaining the homeostasis of synaptic membrane. A role of membrane cholesterol in synaptic endocytosis remains controversial because of conflicting observations, technical limitations in previous studies, and potential interference from non-specific effects after cholesterol manipulation. Furthermore, it remains unclear whether cholesterol participates in distinct forms of endocytosis that function under different activity levels. In this study, applying the whole-cell membrane capacitance measurement to monitor endocytosis in real time at the rat calyx of Held terminals, we found that disrupting cholesterol with dialysis of cholesterol oxidase or methyl-β-cyclodextrin impaired three different forms of endocytosis, including slow endocytosis, rapid endocytosis, and endocytosis of the retrievable membrane that exists at the surface before stimulation. The effects were observed when disruption of cholesterol was mild enough not to change Ca(2+) channel current or vesicle exocytosis, indicative of stringent cholesterol requirement in synaptic endocytosis. Extracting cholesterol with high concentrations of methyl-β-cyclodextrin reduced exocytosis, mainly by decreasing the readily releasable pool and the vesicle replenishment after readily releasable pool depletion. Our study suggests that cholesterol is an important, universal regulator in multiple forms of vesicle endocytosis at mammalian central synapses.

  10. Pharmacology meets vesicular trafficking at a central nervous system synapse: pregabalin effects on synaptic vesicle cycling in hippocampal neurons.

    PubMed

    Holz, Ronald W

    2006-08-01

    The contribution by Micheva et al. in this issue of Molecular Pharmacology adds to our understanding of the action of pregabalin, a drug used for treatment of partial seizures and neuropathic pain. The authors examine the effects of pregabalin on presynaptic function of cultured hippocampal neurons using a powerful technique to follow the trafficking of synaptic vesicles in individual boutons. The study revealed that pregabalin reduces the readily releasable pool of synaptic vesicles in an N-methyl-d-aspartate receptor-dependent manner.

  11. 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

  12. 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

  13. RIM determines Ca2+ channel density and vesicle docking at the presynaptic active zone

    PubMed Central

    Han, Yunyun; Kaeser, Pascal S.; Südhof, Thomas C.; Schneggenburger, Ralf

    2012-01-01

    At presynaptic active zones, neurotransmitter release is initiated by the opening of voltage-gated Ca2+ channels close to docked vesicles. The mechanisms that enrich Ca2+ channels at active zones are, however, largely unknown, possibly because of the limited presynaptic accessibility of most synapses. Here, we have established a Cre-lox based conditional knock-out approach at a presynaptically accessible CNS synapse, the calyx of Held, to directly study the functions of RIM proteins. Removal of all RIM1/2 isoforms strongly reduced the presynaptic Ca2+ channel density, revealing a new role of RIM proteins in Ca2+ channel targeting. Removal of RIMs also reduced the readily-releasable pool, paralleled by a similar reduction of the number of docked vesicles, and the Ca2+ channel - vesicle coupling was decreased. Thus, RIM proteins co-ordinately regulate key functions for fast transmitter release: enabling a high presynaptic Ca2+ channel density, and vesicle docking at the active zone. PMID:21262468

  14. Synthetic Polymers from Readily Available Monosaccharides

    NASA Astrophysics Data System (ADS)

    Galbis, J. A.; García-Martín, M. G.

    The low degradability of petroleum-based polymers and the massive use of these materials constitute a serious problem because of the environmental pollution that they can cause. Thus, sustained efforts have been extensively devoted to produce new polymers based on natural renewing resources and with higher degradability. Of the different natural sources, carbohydrates stand out as highly convenient raw materials because they are inexpensive, readily available, and provide great stereochemical diversity. New polymers, analogous to the more accredited technical polymers, but based on chiral monomers, have been synthesized from natural and available sugars. This chapter describes the potential of sugar-based monomers as precursors to a wide variety of macromolecular materials.

  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. Synaptic vesicle pool size, release probability and synaptic depression are sensitive to Ca2+ buffering capacity in the developing rat calyx of Held

    PubMed Central

    Leão, R.M.; von Gersdorff, H.

    2010-01-01

    The calyx of Held, a specialized synaptic terminal in the medial nucleus of the trapezoid body, undergoes a series of changes during postnatal development that prepares this synapse for reliable high frequency firing. These changes reduce short-term synaptic depression during tetanic stimulation and thereby prevent action potential failures during a stimulus train. We measured presynaptic membrane capacitance changes in calyces from young postnatal day 5–7 (p5–7) or older (p10–12) rat pups to examine the effect of calcium buffer capacity on vesicle pool size and the efficiency of exocytosis. Vesicle pool size was sensitive to the choice and concentration of exogenous Ca2+ buffer, and this sensitivity was much stronger in younger animals. Pool size and exocytosis efficiency in p5–7 calyces were depressed by 0.2 mM EGTA to a greater extent than with 0.05 mM BAPTA, even though BAPTA is a 100-fold faster Ca2+ buffer. However, this was not the case for p10–12 calyces. With 5 mM EGTA, exocytosis efficiency was reduced to a much larger extent in young calyces compared to older calyces. Depression of exocytosis using pairs of 10-ms depolarizations was reduced by 0.2 mM EGTA compared to 0.05 mM BAPTA to a similar extent in both age groups. These results indicate a developmentally regulated heterogeneity in the sensitivity of different vesicle pools to Ca2+ buffer capacity. We propose that, during development, a population of vesicles that are tightly coupled to Ca2+ channels expands at the expense of vesicles more distant from Ca2+ channels. PMID:19219302

  17. A new pathogenic Leptospira, not readily cultivated.

    PubMed

    ALEXANDER, A D; STOENNER, H G; WOOD, G E; BYRNE, R J

    1962-04-01

    Alexander, Aaron D. (Walter Reed Army Institute of Research, Washington, D.C.), Herbert G. Stoenner, Garnett E. Wood, and Robert J. Byrne. A new pathogenic Leptospira, not readily cultivated. J. Bacteriol. 83:754-760. 1962.-A pathogenic Leptospira was isolated from water of the Grand River, (S.D.) that differed significantly from other known leptospirae in that it could not be cultivated in conventional leptospiral media. Growth was promoted in Fletcher's medium modified to contain 20% rabbit serum. The isolate, after several serial passages, was lethal for hamsters. It could not be adapted to grow in the chick embryo. Guinea pigs and calves inoculated with the isolate developed febrile and antibody responses but showed no other overt signs of disease. The strain was identified on the basis of cross-agglutination and agglutinin-adsorption tests as a new subserotype of Leptospira naam and was therefore designated as L. naam, subserotype dakotii.

  18. A NEW PATHOGENIC LEPTOSPIRA, NOT READILY CULTIVATED

    PubMed Central

    Alexander, Aaron D.; Stoenner, Herbert G.; Wood, Garnett E.; Byrne, Robert J.

    1962-01-01

    Alexander, Aaron D. (Walter Reed Army Institute of Research, Washington, D.C.), Herbert G. Stoenner, Garnett E. Wood, and Robert J. Byrne. A new pathogenic Leptospira, not readily cultivated. J. Bacteriol. 83:754–760. 1962.—A pathogenic Leptospira was isolated from water of the Grand River, (S.D.) that differed significantly from other known leptospirae in that it could not be cultivated in conventional leptospiral media. Growth was promoted in Fletcher's medium modified to contain 20% rabbit serum. The isolate, after several serial passages, was lethal for hamsters. It could not be adapted to grow in the chick embryo. Guinea pigs and calves inoculated with the isolate developed febrile and antibody responses but showed no other overt signs of disease. The strain was identified on the basis of cross-agglutination and agglutinin-adsorption tests as a new subserotype of Leptospira naam and was therefore designated as L. naam, subserotype dakotii. PMID:13860321

  19. 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).

  20. 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

  1. A Novel Sensitive Method to Measure Catechol-O-Methyltransferase Activity Unravels the Presence of This Activity in Extracellular Vesicles Released by Rat Hepatocytes.

    PubMed

    Casal, Enriqueta; Palomo, Laura; Cabrera, Diana; Falcon-Perez, Juan M

    2016-01-01

    There is a clear need for drug treatments to be selected according to the characteristics of an individual patient, in order to improve efficacy and reduce the number and severity of adverse drug reactions. One of the main enzymes to take into account in pharmacogenomics is catechol O-methyltransferase (COMT), which catalyzes the transfer of a methyl group from S-adenosylmethionine to catechols and catecholamines, like the neurotransmitters dopamine, epinephrine, and norepinephrine. Although, most of this enzyme is associated to intracellular vesicles, recently it has also been detected in extracellular vesicles secreted by hepatocytes and in serum circulating vesicles. COMT has implications in many neurological and psychiatric disorders like Parkinson's disease, chronic fatigue, pain response, schizophrenia, and bipolar disorders. Remarkably, genetic variations of COMT affect its activity and are associated to various human disorders from psychiatric diseases to estrogen-induced cancers. Consequently, the establishment of new methods to evaluate COMT activity is an important aspect to investigate the biology of this drug-metabolizing enzyme. Herein, we have developed a sensitive and selective method to determine COMT activity. We first optimized the activity in rat liver incubated with two different substrates; norepinephrine and dopamine. The enzymatically formed products (normetanephrine and 3-methoxytyramine, respectively) were extracted by solid-phase extraction using weak cation exchange cartridges, chromatographically separated, and detected and quantified using a mass spectrometer. The range of quantitation for both products was from 0.005 to 25 μg/mL. This methodology offers acceptable recovery for both enzymatic products (≥75%) and good accuracy and precision (≤15%). The lower limit of quantifications were 0.01 and 0.005 μM for 3-methoxytyramine and normetanephrine, respectively. Importantly, this sensitive assay was able to detect the presence of

  2. A Novel Sensitive Method to Measure Catechol-O-Methyltransferase Activity Unravels the Presence of This Activity in Extracellular Vesicles Released by Rat Hepatocytes

    PubMed Central

    Casal, Enriqueta; Palomo, Laura; Cabrera, Diana; Falcon-Perez, Juan M.

    2016-01-01

    There is a clear need for drug treatments to be selected according to the characteristics of an individual patient, in order to improve efficacy and reduce the number and severity of adverse drug reactions. One of the main enzymes to take into account in pharmacogenomics is catechol O-methyltransferase (COMT), which catalyzes the transfer of a methyl group from S-adenosylmethionine to catechols and catecholamines, like the neurotransmitters dopamine, epinephrine, and norepinephrine. Although, most of this enzyme is associated to intracellular vesicles, recently it has also been detected in extracellular vesicles secreted by hepatocytes and in serum circulating vesicles. COMT has implications in many neurological and psychiatric disorders like Parkinson's disease, chronic fatigue, pain response, schizophrenia, and bipolar disorders. Remarkably, genetic variations of COMT affect its activity and are associated to various human disorders from psychiatric diseases to estrogen-induced cancers. Consequently, the establishment of new methods to evaluate COMT activity is an important aspect to investigate the biology of this drug-metabolizing enzyme. Herein, we have developed a sensitive and selective method to determine COMT activity. We first optimized the activity in rat liver incubated with two different substrates; norepinephrine and dopamine. The enzymatically formed products (normetanephrine and 3-methoxytyramine, respectively) were extracted by solid-phase extraction using weak cation exchange cartridges, chromatographically separated, and detected and quantified using a mass spectrometer. The range of quantitation for both products was from 0.005 to 25 μg/mL. This methodology offers acceptable recovery for both enzymatic products (≥75%) and good accuracy and precision (≤15%). The lower limit of quantifications were 0.01 and 0.005 μM for 3-methoxytyramine and normetanephrine, respectively. Importantly, this sensitive assay was able to detect the presence of

  3. 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

  4. 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

  5. Streptococcus mutans extracellular DNA is upregulated during growth in biofilms, actively released via membrane vesicles, and influenced by components of the protein secretion machinery.

    PubMed

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

    2014-07-01

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

  6. 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.

  7. Traumatic Brain Injury Impairs Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor Complex Formation and Alters Synaptic Vesicle Distribution in the Hippocampus

    PubMed Central

    Carlson, Shaun W.; Yan, Hong; Ma, Michelle; Li, Youming; Henchir, Jeremy

    2016-01-01

    Abstract Traumatic brain injury (TBI) impairs neuronal function and can culminate in lasting cognitive impairment. While impaired neurotransmitter release has been well established after experimental TBI, little is understood about the mechanisms underlying this consequence. In the synapse, vesicular docking and neurotransmitter release requires the formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Impairments in vesicle docking, and alterations in SNARE complex formation are associated with impaired neurotransmitter release. We hypothesized that TBI reduces SNARE complex formation and disrupts synaptic vesicle distribution in the hippocampus. To examine the effect of TBI on the SNARE complex, rats were subjected to controlled cortical impact (CCI) or sham injury, and the brains were assessed at 6 h, 1 d, one week, two weeks, or four weeks post-injury. Immunoblotting of hippocampal homogenates revealed significantly reduced SNARE complex formation at one week and two weeks post-injury. To assess synaptic vesicles distribution, rats received CCI or sham injury and the brains were processed for transmission electron microscopy at one week post-injury. Synapses in the hippocampus were imaged at 100k magnification, and vesicle distribution was assessed in pre-synaptic terminals at the active zone. CCI resulted in a significant reduction in vesicle number within 150 nm of the active zone. These findings provide the first evidence of TBI-induced impairments in synaptic vesicle docking, and suggest that reductions in the pool of readily releasable vesicles and impaired SNARE complex formation are two novel mechanisms contributing to impaired neurotransmission after TBI. PMID:25923735

  8. Impaired synaptic vesicle recycling contributes to presynaptic dysfunction in lipoprotein lipase-deficient mice.

    PubMed

    Liu, X; Zhang, B; Yang, H; Wang, H; Liu, Y; Huang, A; Liu, T; Tian, X; Tong, Y; Zhou, T; Zhang, T; Xing, G; Xiao, W; Guo, X; Fan, D; Han, X; Liu, G; Zhou, Z; Chui, D

    2014-11-07

    Lipoprotein lipase (LPL) is expressed at high levels in hippocampal neurons, although its function is unclear. We previously reported that LPL-deficient mice have learning and memory impairment and fewer synaptic vesicles in hippocampal neurons, but properties of synaptic activity in LPL-deficient neurons remain unexplored. In this study, we found reduced frequency of miniature excitatory postsynaptic currents (mEPSCs) and readily releasable pool (RRP) size in LPL-deficient neurons, which led to presynaptic dysfunction and plasticity impairment without altering postsynaptic activity. We demonstrated that synaptic vesicle recycling, which is known to play an important role in maintaining the RRP size in active synapses, is impaired in LPL-deficient neurons. Moreover, lipid assay revealed deficient docosahexaenoic acid (DHA) and arachidonic acid (AA) in the hippocampus of LPL-deficient mice; exogenous DHA or AA supplement partially restored synaptic vesicle recycling capability. These results suggest that impaired synaptic vesicle recycling results from deficient DHA and AA and contributes to the presynaptic dysfunction and plasticity impairment in LPL-deficient neurons.

  9. 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

  10. Characterization and Diagnostic Application of Trypanosoma cruzi Trypomastigote Excreted-Secreted Antigens Shed in Extracellular Vesicles Released from Infected Mammalian Cells.

    PubMed

    Bautista-López, Norma L; Ndao, Momar; Camargo, Fabio Vasquez; Nara, Takeshi; Annoura, Takeshi; Hardie, Darryl B; Borchers, Christoph H; Jardim, Armando

    2017-03-01

    Chagas disease, caused by Trypanosoma cruzi, although endemic in many parts of Central and South America, is emerging as a global health threat through the potential contamination of blood supplies. Consequently, in the absence of a gold standard assay for the diagnosis of Chagas disease, additional antigens or strategies are needed. A proteomic analysis of the trypomastigote excreted-secreted antigens (TESA) associated with exosomal vesicles shed by T. cruzi identified ∼80 parasite proteins, with the majority being trans-sialidases. Mass spectrometry analysis of immunoprecipitation products performed using Chagas immune sera showed a marked enrichment in a subset of TESA proteins. Of particular relevance for diagnostic applications were the retrotransposon hot spot (RHS) proteins, which are absent in Leishmania spp., parasites that often confound diagnosis of Chagas disease. Interestingly, serological screens using recombinant RHS showed a robust immunoreactivity with sera from patients with clinical stages of Chagas ranging from asymptomatic to advance cardiomyopathy and this immunoreactivity was comparable to that of crude TESA. More importantly, no cross-reactivity with RHS was detected with sera from patients with malaria, leishmaniasis, toxoplasmosis, or African sleeping sickness, making this protein an attractive reagent for diagnosis of Chagas disease.

  11. Brain metastatic cancer cells release microRNA-181c-containing extracellular vesicles capable of destructing blood-brain barrier.

    PubMed

    Tominaga, Naoomi; Kosaka, Nobuyoshi; Ono, Makiko; Katsuda, Takeshi; Yoshioka, Yusuke; Tamura, Kenji; Lötvall, Jan; Nakagama, Hitoshi; Ochiya, Takahiro

    2015-04-01

    Brain metastasis is an important cause of mortality in breast cancer patients. A key event during brain metastasis is the migration of cancer cells through blood-brain barrier (BBB). However, the molecular mechanism behind the passage through this natural barrier remains unclear. Here we show that cancer-derived extracellular vesicles (EVs), mediators of cell-cell communication via delivery of proteins and microRNAs (miRNAs), trigger the breakdown of BBB. Importantly, miR-181c promotes the destruction of BBB through the abnormal localization of actin via the downregulation of its target gene, PDPK1. PDPK1 degradation by miR-181c leads to the downregulation of phosphorylated cofilin and the resultant activated cofilin-induced modulation of actin dynamics. Furthermore, we demonstrate that systemic injection of brain metastatic cancer cell-derived EVs promoted brain metastasis of breast cancer cell lines and are preferentially incorporated into the brain in vivo. Taken together, these results indicate a novel mechanism of brain metastasis mediated by EVs that triggers the destruction of BBB.

  12. Transcriptome and long noncoding RNA sequencing of three extracellular vesicle subtypes released from the human colon cancer LIM1863 cell line

    PubMed Central

    Chen, Maoshan; Xu, Rong; Ji, Hong; Greening, David W.; Rai, Alin; Izumikawa, Keiichi; Ishikawa, Hideaki; Takahashi, Nobuhiro; Simpson, Richard J.

    2016-01-01

    Previously we reported that LIM1863 colorectal cancer (CRC) cells secrete three distinct extracellular vesicle subtypes – two subpopulations of exosomes (apical EpCAM-Exos and basolateral A33-Exos) and shed microvesicles (sMVs) – with distinct protein and miRNA signatures. Here, we extend our omics approach to understand the fundamental role of LIM1863-derived EVs by performing a comprehensive analysis of their mRNAs and long non-coding RNAs (lncRNAs) using RNA-Seq. We show that 2,389 mRNAs, 317 pseudogene transcripts, 1,028 lncRNAs and 206 short non-coding RNAs selectively distributed to (i.e., are enriched in) LIM1863 EVs, relative to the parent cell. An Ensembl/UniProtKB analysis revealed 1,937 mRNAs encode canonical proteins, 348 isoforms (including splice-variant proteins), and 119 ‘missing proteins’ (i.e., annotated in Ensembl but not UniProtKB). Further dissection of our protein/RNA data revealed that 6/151 observed RNA binding proteins have the potential to interact with ~75% of EV-enriched RNAs. Intriguingly, the co-existence of U1 and U2 ribonucleoproteins and their cognate snRNAs in LIM1863 EVs suggests a possible association of CRC EVs with recipient cell splicing events. Our data reveal several potential lncRNA CRC biomarkers and novel splicing/fusion genes that, collectively, will advance our understanding of EV biology in CRC and accelerate the development of EV-based diagnostics and therapeutics. PMID:27917920

  13. 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.

  14. 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)].

  15. 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.

  16. 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

  17. A Network of Three Types of Filaments Organizes Synaptic Vesicles for Storage, Mobilization, and Docking

    PubMed Central

    Chen, Xiaobing; Reese, Thomas S.

    2016-01-01

    Synaptic transmission between neurons requires precise management of synaptic vesicles. While individual molecular components of the presynaptic terminal are well known, exactly how the molecules are organized into a molecular machine serving the storage and mobilization of synaptic vesicles to the active zone remains unclear. Here we report three filament types associated with synaptic vesicles in glutamatergic synapses revealed by electron microscope tomography in unstimulated, dissociated rat hippocampal neurons. One filament type, likely corresponding to the SNAREpin complex, extends from the active zone membrane and surrounds docked vesicles. A second filament type contacts all vesicles throughout the active zone and pairs vesicles together. On the third filament type, vesicles attach to side branches extending from the long filament core and form vesicle clusters that are distributed throughout the vesicle cloud and along the active zone membrane. Detailed analysis of presynaptic structure reveals how each of the three filament types interacts with synaptic vesicles, providing a means to traffic reserved and recycled vesicles from the cloud of vesicles into the docking position at the active zone. SIGNIFICANCE STATEMENT The formation and release of synaptic vesicles has been extensively investigated. Explanations of the release of synaptic vesicles generally begin with the movement of vesicles from the cloud into the synaptic active zone. However, the presynaptic terminal is filled with filamentous material that would appear to limit vesicular diffusion. Here, we provide a systematic description of three filament types connecting synaptic vesicles. A picture emerges illustrating how the cooperative attachment and release of these three filament types facilitate the movement of vesicles to the active zone to become docked in preparation for release. PMID:26985032

  18. 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.

  19. Challenges posed by extracellular vesicles from eukaryotic microbes

    PubMed Central

    Wolf, Julie M.; Casadevall, Arturo

    2014-01-01

    Extracellular vesicles (EV) produced by eukaryotic microbes play an important role during infection. EV release is thought to benefit microbial invasion by delivering a high concentration of virulence factors to distal host cells or to the cytoplasm of a host cell. EV can significantly impact the outcome of host-pathogen interaction in a cargo-dependent manner. Release of EV from eukaryotic microbes poses unique challenges when compared to their bacterial or archaeal counterparts. Firstly, the membrane-bound organelles within eukaryotes facilitate multiple mechanisms of vesicle generation. Secondly, the fungal cell wall poses a unique barrier between the vesicle release site at the plasma membrane and its destined extracellular environment. This review focuses on these eukaryotic-specific aspects of vesicle synthesis and release. PMID:25460799

  20. 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.

  1. Two distinct secretory vesicle-priming steps in adrenal chromaffin cells.

    PubMed

    Liu, Yuanyuan; Schirra, Claudia; Edelmann, Ludwig; Matti, Ulf; Rhee, JeongSeop; Hof, Detlef; Bruns, Dieter; Brose, Nils; Rieger, Heiko; Stevens, David R; Rettig, Jens

    2010-09-20

    Priming of large dense-core vesicles (LDCVs) is a Ca(2+)-dependent step by which LDCVs enter a release-ready pool, involving the formation of the soluble N-ethyl-maleimide sensitive fusion protein attachment protein (SNAP) receptor complex consisting of syntaxin, SNAP-25, and synaptobrevin. Using mice lacking both isoforms of the calcium-dependent activator protein for secretion (CAPS), we show that LDCV priming in adrenal chromaffin cells entails two distinct steps. CAPS is required for priming of the readily releasable LDCV pool and sustained secretion in the continued presence of high Ca(2+) concentrations. Either CAPS1 or CAPS2 can rescue secretion in cells lacking both CAPS isoforms. Furthermore, the deficit in the readily releasable LDCV pool resulting from CAPS deletion is reversed by a constitutively open form of syntaxin but not by Munc13-1, a priming protein that facilitates the conversion of syntaxin to the open conformation. Our data indicate that CAPS functions downstream of Munc13s but also interacts functionally with Munc13s in the LDCV-priming process.

  2. 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.

  3. 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.

  4. Outer Membrane Vesicles and Soluble Factors Released by Probiotic Escherichia coli Nissle 1917 and Commensal ECOR63 Enhance Barrier Function by Regulating Expression of Tight Junction Proteins in Intestinal Epithelial Cells.

    PubMed

    Alvarez, Carina-Shianya; Badia, Josefa; Bosch, Manel; Giménez, Rosa; Baldomà, Laura

    2016-01-01

    The gastrointestinal epithelial layer forms a physical and biochemical barrier that maintains the segregation between host and intestinal microbiota. The integrity of this barrier is critical in maintaining homeostasis in the body and its dysfunction is linked to a variety of illnesses, especially inflammatory bowel disease. Gut microbes, and particularly probiotic bacteria, modulate the barrier integrity by reducing gut permeability and reinforcing tight junctions. Probiotic Escherichia coli Nissle 1917 (EcN) is a good colonizer of the human gut with proven therapeutic efficacy in the remission of ulcerative colitis in humans. EcN positively modulates the intestinal epithelial barrier through upregulation and redistribution of the tight junction proteins ZO-1, ZO-2 and claudin-14. Upregulation of claudin-14 has been attributed to the secreted protein TcpC. Whether regulation of ZO-1 and ZO-2 is mediated by EcN secreted factors remains unknown. The aim of this study was to explore whether outer membrane vesicles (OMVs) released by EcN strengthen the epithelial barrier. This study includes other E. coli strains of human intestinal origin that contain the tcpC gene, such as ECOR63. Cell-free supernatants collected from the wild-type strains and from the derived tcpC mutants were fractionated into isolated OMVs and soluble secreted factors. The impact of these extracellular fractions on the epithelial barrier was evaluated by measuring transepithelial resistance and expression of several tight junction proteins in T-84 and Caco-2 polarized monolayers. Our results show that the strengthening activity of EcN and ECOR63 does not exclusively depend on TcpC. Both OMVs and soluble factors secreted by these strains promote upregulation of ZO-1 and claudin-14, and down-regulation of claudin-2. The OMVs-mediated effects are TcpC-independent. Soluble secreted TcpC contributes to the upregulation of ZO-1 and claudin-14, but this protein has no effect on the transcriptional

  5. Outer Membrane Vesicles and Soluble Factors Released by Probiotic Escherichia coli Nissle 1917 and Commensal ECOR63 Enhance Barrier Function by Regulating Expression of Tight Junction Proteins in Intestinal Epithelial Cells

    PubMed Central

    Alvarez, Carina-Shianya; Badia, Josefa; Bosch, Manel; Giménez, Rosa; Baldomà, Laura

    2016-01-01

    The gastrointestinal epithelial layer forms a physical and biochemical barrier that maintains the segregation between host and intestinal microbiota. The integrity of this barrier is critical in maintaining homeostasis in the body and its dysfunction is linked to a variety of illnesses, especially inflammatory bowel disease. Gut microbes, and particularly probiotic bacteria, modulate the barrier integrity by reducing gut permeability and reinforcing tight junctions. Probiotic Escherichia coli Nissle 1917 (EcN) is a good colonizer of the human gut with proven therapeutic efficacy in the remission of ulcerative colitis in humans. EcN positively modulates the intestinal epithelial barrier through upregulation and redistribution of the tight junction proteins ZO-1, ZO-2 and claudin-14. Upregulation of claudin-14 has been attributed to the secreted protein TcpC. Whether regulation of ZO-1 and ZO-2 is mediated by EcN secreted factors remains unknown. The aim of this study was to explore whether outer membrane vesicles (OMVs) released by EcN strengthen the epithelial barrier. This study includes other E. coli strains of human intestinal origin that contain the tcpC gene, such as ECOR63. Cell-free supernatants collected from the wild-type strains and from the derived tcpC mutants were fractionated into isolated OMVs and soluble secreted factors. The impact of these extracellular fractions on the epithelial barrier was evaluated by measuring transepithelial resistance and expression of several tight junction proteins in T-84 and Caco-2 polarized monolayers. Our results show that the strengthening activity of EcN and ECOR63 does not exclusively depend on TcpC. Both OMVs and soluble factors secreted by these strains promote upregulation of ZO-1 and claudin-14, and down-regulation of claudin-2. The OMVs-mediated effects are TcpC-independent. Soluble secreted TcpC contributes to the upregulation of ZO-1 and claudin-14, but this protein has no effect on the transcriptional

  6. Magainin 2a - Induced Permeabilization of Phospholipid Vesicles

    DTIC Science & Technology

    1991-01-02

    unilamellar vesicles (SUVs) of phosphatidylserine ( PS ). Addition of peptide to the SUVs causes an initial rapid release of dye, lasting about 100 sec...Measurements 39 VII. Results 40 A. Effect of MGN2a on the Permeability of PS Vesicles 40 B. Effect of Extravesicular Osmotic Pressure on 6CF Release from...Depiction of all-or-none versus graded release mechanisms 34 4. Standard quenching curve for 6CF entrapped within PS SUVs 38 5. MGN2a-induced release

  7. 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.

  8. Estimates for the pool size of releasable quanta at a single central synapse and for the time required to refill the pool.

    PubMed

    Stevens, C F; Tsujimoto, T

    1995-01-31

    Local superfusion of limited dendritic areas with hypertonic or hyperkalemic solutions stimulates the release of quanta from a small population of synapses made on rodent hippocampal neurons maintained in primary culture, and each quantal event can be detected in the postsynaptic neuron. With maintained stimulation, the initial release rate is about 20 quanta per sec per synapse, and this rate declines exponentially to a final low level. These observations can be interpreted as depletion of available quanta and, with this interpretation, a bouton would contain one to two dozen quanta in its readily releasable pool. Tests with a second application of the solution that produces release reveal that the pool of readily releasable quanta is replenished with a time constant of about 10 sec (36 degrees C). The pool of quanta defined in this way may correspond to the population of vesicles docked at the bouton's active zone.

  9. Released fraction and total size of a pool of immediately available transmitter quanta at a calyx synapse.

    PubMed

    Schneggenburger, R; Meyer, A C; Neher, E

    1999-06-01

    The size of a pool of readily releasable vesicles at a giant brainstem synapse, the calyx of Held, was probed with three independent approaches. Using simultaneous pre- and postsynaptic whole-cell recordings, two forms of presynaptic Ca2+ stimuli were applied in rapid succession: uncaging of Ca2+ by flash photolysis and the opening of voltage-gated Ca2+ channels. The ensuing transmitter release showed a nearly complete cross-inhibition between the two stimuli, indicating the depletion of a limited pool of about 700 transmitter quanta. The pool size was confirmed in experiments using enhanced extracellular Ca2+ concentrations, as well as short, high-frequency stimulus trains. The results reveal a surprisingly large pool of functionally available vesicles, of which a fraction of about 0.2 is released by a single presynaptic action potential under physiological conditions.

  10. Characterization of extracellular vesicles in whole blood: Influence of pre-analytical parameters and visualization of vesicle-cell interactions using imaging flow cytometry.

    PubMed

    Fendl, Birgit; Weiss, René; Fischer, Michael B; Spittler, Andreas; Weber, Viktoria

    2016-09-09

    Extracellular vesicles are central players in intercellular communication and are released from the plasma membrane under tightly regulated conditions, depending on the physiological and pathophysiological state of the producing cell. Their heterogeneity requires a spectrum of methods for isolation and characterization, where pre-analytical parameters have profound impact on vesicle analysis, particularly in blood, since sampling, addition of anticoagulants, as well as post-sampling vesicle generation may influence the outcome. Here, we characterized microvesicles directly in whole blood using a combination of flow cytometry and imaging flow cytometry. We assessed the influence of sample agitation, anticoagulation, and temperature on post-sampling vesicle generation, and show that vesicle counts remained stable over time in samples stored without agitation. Storage with gentle rolling mimicking agitation, in contrast, resulted in strong release of platelet-derived vesicles in blood anticoagulated with citrate or heparin, whereas vesicle counts remained stable upon anticoagulation with EDTA. Using imaging flow cytometry, we could visualize microvesicles adhering to blood cells and revealed an anticoagulant-dependent increase in vesicle-cell aggregates over time. We demonstrate that vesicles adhere preferentially to monocytes and granulocytes in whole blood, while no microvesicles could be visualized on lymphocytes. Our data underscore the relevance of pre-analytical parameters in vesicle analysis and demonstrate that imaging flow cytometry is a suitable tool to study the interaction of extracellular vesicles with their target cells.

  11. 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.

  12. 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.

  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. Altered Active Zones, Vesicle Pools, Nerve Terminal Conductivity, and Morphology during Experimental MuSK Myasthenia Gravis

    PubMed Central

    Patel, Vishwendra; Oh, Anne; Voit, Antanina; Sultatos, Lester G.; Babu, Gopal J.; Wilson, Brenda A.; Ho, Mengfei; McArdle, Joseph J.

    2014-01-01

    Recent studies demonstrate reduced motor-nerve function during autoimmune muscle-specific tyrosine kinase (MuSK) myasthenia gravis (MG). To further understand the basis of motor-nerve dysfunction during MuSK-MG, we immunized female C57/B6 mice with purified rat MuSK ectodomain. Nerve-muscle preparations were dissected and neuromuscular junctions (NMJs) studied electrophysiologically, morphologically, and biochemically. While all mice produced antibodies to MuSK, only 40% developed respiratory muscle weakness. In vitro study of respiratory nerve-muscle preparations isolated from these affected mice revealed that 78% of NMJs produced endplate currents (EPCs) with significantly reduced quantal content, although potentiation and depression at 50 Hz remained qualitatively normal. EPC and mEPC amplitude variability indicated significantly reduced number of vesicle-release sites (active zones) and reduced probability of vesicle release. The readily releasable vesicle pool size and the frequency of large amplitude mEPCs also declined. The remaining NMJs had intermittent (4%) or complete (18%) failure of neurotransmitter release in response to 50 Hz nerve stimulation, presumably due to blocked action potential entry into the nerve terminal, which may arise from nerve terminal swelling and thinning. Since MuSK-MG-affected muscles do not express the AChR γ subunit, the observed prolongation of EPC decay time was not due to inactivity-induced expression of embryonic acetylcholine receptor, but rather to reduced catalytic activity of acetylcholinesterase. Muscle protein levels of MuSK did not change. These findings provide novel insight into the pathophysiology of autoimmune MuSK-MG. PMID:25438154

  15. A Molecular Dynamics Simulation of Vesicle Deformation and Rupture in Confined Poiseuille Flow

    NASA Astrophysics Data System (ADS)

    Harman, Alison

    Vesicles are simple structures, but display complex, non-linear dynamics in fluid flow. I investigate the deformation of nanometer-sized vesicles, both fully-inflated and those with excess area, as they travel in tightly confined capillaries. By varying both channel size and flow strength, I simulate vesicles as they transition from steady-state to unstable shapes, and then rupture in strong flow fields. By employing a molecular dynamics model of the vesicle, fluid, and capillary system one is able to rupture the lipid bilayer of these vesicles. This is unique in that most other numerical methods for modelling vesicles are unable to show rupture. The rupture of fully-inflated vesicles is applicable to drug delivery in which the release of the encapsulated medicine needs to be controlled. The deformation and rupture of vesicles with excess area could be applicable to red blood cells which have similar rheological properties.

  16. Membrane vesicles in sea water: heterogeneous DNA content and implications for viral abundance estimates.

    PubMed

    Biller, Steven J; McDaniel, Lauren D; Breitbart, Mya; Rogers, Everett; Paul, John H; Chisholm, Sallie W

    2017-02-01

    Diverse microbes release membrane-bound extracellular vesicles from their outer surfaces into the surrounding environment. Vesicles are found in numerous habitats including the oceans, where they likely have a variety of functional roles in microbial ecosystems. Extracellular vesicles are known to contain a range of biomolecules including DNA, but the frequency with which DNA is packaged in vesicles is unknown. Here, we examine the quantity and distribution of DNA associated with vesicles released from five different bacteria. The average quantity of double-stranded DNA and size distribution of DNA fragments released within vesicles varies among different taxa. Although some vesicles contain sufficient DNA to be visible following staining with the SYBR fluorescent DNA dyes typically used to enumerate viruses, this represents only a small proportion (<0.01-1%) of vesicles. Thus DNA is packaged heterogeneously within vesicle populations, and it appears that vesicles are likely to be a minor component of SYBR-visible particles in natural sea water compared with viruses. Consistent with this hypothesis, chloroform treatment of coastal and offshore seawater samples reveals that vesicles increase epifluorescence-based particle (viral) counts by less than an order of magnitude and their impact is variable in space and time.

  17. 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.

  18. Repository of not readily available documents for project W-320

    SciTech Connect

    Conner, J.C.

    1997-04-18

    The purpose of this document is to provide a readily available source of the technical reports needed for the development of the safety documentation provided for the waste retrieval sluicing system (WRSS), designed to remove the radioactive and chemical sludge from tank 241-C-106, and transport that material to double-shell tank 241-AY-102 via a new, temporary, shielded, encased transfer line.

  19. Efficient synthesis of readily water-soluble sulfonic Acid carbamates.

    PubMed

    Idzik, Krzysztof R; Nödler, Karsten; Licha, Tobias

    2015-04-16

    A series of various readily water-soluble carbamates were synthesized with good yields. These compounds are useful chemical tracers for assessing the cooling progress in a georeservoir during geothermal power plant operation. Acylation of primary amines was carried out as well as using a solution of sodium bicarbonate and without the presence of salt. Products were characterized by 1H-NMR and 13C-NMR. Purity was confirmed through elemental analysis.

  20. 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

  1. Dynamic Partitioning of Synaptic Vesicle Pools by the SNARE-Binding Protein Tomosyn.

    PubMed

    Cazares, Victor A; Njus, Meredith M; Manly, Amanda; Saldate, Johnny J; Subramani, Arasakumar; Ben-Simon, Yoav; Sutton, Michael A; Ashery, Uri; Stuenkel, Edward L

    2016-11-02

    Neural networks engaged in high-frequency activity rely on sustained synaptic vesicle recycling and coordinated recruitment from functionally distinct synaptic vesicle (SV) pools. However, the molecular pathways matching neural activity to SV dynamics and release requirements remain unclear. Here we identify unique roles of SNARE-binding Tomosyn1 (Tomo1) proteins as activity-dependent substrates that regulate dynamics of SV pool partitioning at rat hippocampal synapses. Our analysis is based on monitoring changes in distinct functionally defined SV pools via V-Glut1-pHluorin fluorescence in cultured hippocampal neurons in response to alterations in presynaptic protein expression. Specifically, we find knockdown of Tomo1 facilitates release efficacy from the Readily Releasable Pool (RRP), and regulates SV distribution to the Total Recycling Pool (TRP), which is matched by a decrease in the SV Resting Pool. Notably, these effects were reversed by Tomo1 rescue and overexpression. Further, we identify that these actions of Tomo1 are regulated via activity-dependent phosphorylation by cyclin-dependent kinase 5 (Cdk5). Assessment of molecular interactions that may contribute to these actions identified Tomo1 interaction with the GTP-bound state of Rab3A, an SV GTPase involved in SV targeting and presynaptic membrane tethering. In addition, Tomo1 via Rab3A-GTP was also observed to interact with Synapsin 1a/b cytoskeletal interacting proteins. Finally, our data indicate that Tomo1 regulation of SV pool sizes serves to adapt presynaptic neurotransmitter release to chronic silencing of network activity. Overall, the results establish Tomo1 proteins as central mediators in neural activity-dependent changes in SV distribution among SV pools.

  2. Lipid-targeting peptide probes for extracellular vesicles

    PubMed Central

    Flynn, Aaron D.; Yin, Hang

    2017-01-01

    Extracellular vesicles released from cells are under intense investigation for their roles in cell-cell communication and cancer progression. However, individual vesicles have been difficult to probe as their small size renders them invisible by conventional light microscopy. However, as a consequence of their small size these vesicles possess highly curved lipid membranes that offer an unconventional target for curvature-sensing probes. In this article, we present a strategy for using peptide-based biosensors to detect highly curved membranes and the negatively charged membrane lipid phosphatidylserine, we delineate several assays used to validate curvature- and lipid-targeting mechanisms, and we explore potential applications in probing extracellular vesicles released from sources such as apoptotic cells, cancer cells, or activated platelets. PMID:26909741

  3. Electroformation of Giant Unilamellar Vesicles on Stainless Steel Electrodes

    PubMed Central

    2017-01-01

    Giant unilamellar vesicles (GUVs) are well-established model systems for studying membrane structure and dynamics. Electroformation, also referred to as electroswelling, is one of the most prevalent methods for producing GUVs, as it enables modulation of the lipid hydration process to form relatively monodisperse, defect-free vesicles. Currently, however, it is expensive and time-consuming compared with other methods. In this study, we demonstrate that 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine GUVs can be prepared readily at a fraction of the cost on stainless steel electrodes, such as commercially available syringe needles, without any evidence of lipid oxidation or hydrolysis. PMID:28393132

  4. 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

  5. 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.

  6. Additive effects on the energy barrier for synaptic vesicle fusion cause supralinear effects on the vesicle fusion rate.

    PubMed

    Schotten, Sebastiaan; Meijer, Marieke; Walter, Alexander Matthias; Huson, Vincent; Mamer, Lauren; Kalogreades, Lawrence; ter Veer, Mirelle; Ruiter, Marvin; Brose, Nils; Rosenmund, Christian; Sørensen, Jakob Balslev; Verhage, Matthijs; Cornelisse, Lennart Niels

    2015-04-14

    The energy required to fuse synaptic vesicles with the plasma membrane ('activation energy') is considered a major determinant in synaptic efficacy. From reaction rate theory, we predict that a class of modulations exists, which utilize linear modulation of the energy barrier for fusion to achieve supralinear effects on the fusion rate. To test this prediction experimentally, we developed a method to assess the number of releasable vesicles, rate constants for vesicle priming, unpriming, and fusion, and the activation energy for fusion by fitting a vesicle state model to synaptic responses induced by hypertonic solutions. We show that complexinI/II deficiency or phorbol ester stimulation indeed affects responses to hypertonic solution in a supralinear manner. An additive vs multiplicative relationship between activation energy and fusion rate provides a novel explanation for previously observed non-linear effects of genetic/pharmacological perturbations on synaptic transmission and a novel interpretation of the cooperative nature of Ca(2+)-dependent release.

  7. Spatiotemporal Regulation of Synaptic Vesicle Fusion Sites in Central Synapses.

    PubMed

    Maschi, Dario; Klyachko, Vitaly A

    2017-04-05

    The number and availability of vesicle release sites at the synaptic active zone (AZ) are critical factors governing neurotransmitter release; yet, these fundamental synaptic parameters have remained undetermined. Moreover, how neural activity regulates the spatiotemporal properties of the release sites within individual central synapses is unknown. Here, we combined a nanoscale imaging approach with advanced image analysis to detect individual vesicle fusion events with ∼27 nm localization precision at single hippocampal synapses under physiological conditions. Our results revealed the presence of multiple distinct release sites within individual hippocampal synapses. Release sites were distributed throughout the AZ and underwent repeated reuse. Furthermore, the spatiotemporal properties of the release sites were activity dependent with a reduction in reuse frequency and a shift in location toward the AZ periphery during high-frequency stimulation. These findings have revealed fundamental spatiotemporal properties of individual release sites in small central synapses and their activity-dependent modulation.

  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. 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.

  11. 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.

  12. Shear-stress sensitive lenticular vesicles for targeted drug delivery.

    PubMed

    Holme, Margaret N; Fedotenko, Illya A; Abegg, Daniel; Althaus, Jasmin; Babel, Lucille; Favarger, France; Reiter, Renate; Tanasescu, Radu; Zaffalon, Pierre-Léonard; Ziegler, André; Müller, Bert; Saxer, Till; Zumbuehl, Andreas

    2012-08-01

    Atherosclerosis results in the narrowing of arterial blood vessels and this causes significant changes in the endogenous shear stress between healthy and constricted arteries. Nanocontainers that can release drugs locally with such rheological changes can be very useful. Here, we show that vesicles made from an artificial 1,3-diaminophospholipid are stable under static conditions but release their contents at elevated shear stress. These vesicles have a lenticular morphology, which potentially leads to instabilities along their equator. Using a model cardiovascular system based on polymer tubes and an external pump to represent shear stress in healthy and constricted vessels of the heart, we show that drugs preferentially release from the vesicles in constricted vessels that have high shear stress.

  13. 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.

  14. 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

  15. 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

  16. Macrocyclic peptides self-assemble into robust vesicles with molecular recognition capabilities.

    PubMed

    Jeong, Woo-jin; Lim, Yong-beom

    2014-11-19

    In this study, we developed macrocyclic peptide building blocks that formed self-assembled peptide vesicles with molecular recognition capabilities. Macrocyclic peptides were significantly different from conventional amphiphiles, in that they could self-assemble into vesicles at very high hydrophilic-to-total mass ratios. The flexibility of the hydrophobic self-assembly segment was critical for vesicle formation. The unique features of this peptide vesicle system include a homogeneous size distribution, unusually small size, and robust structural and thermal stability. The peptide vesicles successfully entrapped a hydrophilic model drug, released the payload very slowly, and were internalized by cells in a highly efficient manner. Moreover, the peptide vesicles exhibited molecular recognition capabilities, in that they selectively bound to target RNA through surface-displayed peptides. This study demonstrates that self-assembled peptide vesicles can be used as strong intracellular delivery vehicles that recognize specific biomacromolecular targets.

  17. Transient release kinetics of rod bipolar cells revealed by capacitance measurement of exocytosis from axon terminals in rat retinal slices

    PubMed Central

    Oltedal, Leif; Hartveit, Espen

    2010-01-01

    Presynaptic transmitter release has mostly been studied through measurements of postsynaptic responses, but a few synapses offer direct access to the presynaptic terminal, thereby allowing capacitance measurements of exocytosis. For mammalian rod bipolar cells, synaptic transmission has been investigated in great detail by recording postsynaptic currents in AII amacrine cells. Presynaptic measurements of the dynamics of vesicular cycling have so far been limited to isolated rod bipolar cells in dissociated preparations. Here, we first used computer simulations of compartmental models of morphologically reconstructed rod bipolar cells to adapt the ‘Sine + DC’ technique for capacitance measurements of exocytosis at axon terminals of intact rod bipolar cells in retinal slices. In subsequent physiological recordings, voltage pulses that triggered presynaptic Ca2+ influx evoked capacitance increases that were proportional to the pulse duration. With pulse durations ≤100 ms, the increase saturated at ∼10 fF, corresponding to the size of a readily releasable pool of vesicles. Pulse durations ≥400 ms evoked additional capacitance increases, probably reflecting recruitment from additional pools of vesicles. By using Ca2+ tail current stimuli, we separated Ca2+ influx from Ca2+ channel activation kinetics, allowing us to estimate the intrinsic release kinetics of the readily releasable pool, yielding a time constant of ∼1.1 ms and a maximum release rate of 2–3 vesicles (release site)−1 ms−1. Following exocytosis, we observed endocytosis with time constants ranging from 0.7 to 17 s. Under physiological conditions, it is likely that release will be transient, with the kinetics limited by the activation kinetics of the voltage-gated Ca2+ channels. PMID:20211976

  18. Role for Mycobacterium tuberculosis Membrane Vesicles in Iron Acquisition

    PubMed Central

    Prados-Rosales, Rafael; Weinrick, Brian C.; Piqué, Daniel G.; Jacobs, William R.; Casadevall, Arturo

    2014-01-01

    Mycobacterium tuberculosis releases membrane vesicles packed with molecules that can modulate the immune response. Because environmental conditions often influence the production and content of bacterial vesicles, this study examined M. tuberculosis microvesicles released under iron limitation, a common condition faced by pathogens inside the host. The findings indicate that M. tuberculosis increases microvesicle production in response to iron restriction and that these microvesicles contain mycobactin, which can serve as an iron donor and supports replication of iron-starved mycobacteria. Consequently, the results revealed a role of microvesicles in iron acquisition in M. tuberculosis, which can be critical for survival in the host. PMID:24415729

  19. Binding Isotherms and Time Courses Readily from Magnetic Resonance.

    PubMed

    Xu, Jia; Van Doren, Steven R

    2016-08-16

    Evidence is presented that binding isotherms, simple or biphasic, can be extracted directly from noninterpreted, complex 2D NMR spectra using principal component analysis (PCA) to reveal the largest trend(s) across the series. This approach renders peak picking unnecessary for tracking population changes. In 1:1 binding, the first principal component captures the binding isotherm from NMR-detected titrations in fast, slow, and even intermediate and mixed exchange regimes, as illustrated for phospholigand associations with proteins. Although the sigmoidal shifts and line broadening of intermediate exchange distorts binding isotherms constructed conventionally, applying PCA directly to these spectra along with Pareto scaling overcomes the distortion. Applying PCA to time-domain NMR data also yields binding isotherms from titrations in fast or slow exchange. The algorithm readily extracts from magnetic resonance imaging movie time courses such as breathing and heart rate in chest imaging. Similarly, two-step binding processes detected by NMR are easily captured by principal components 1 and 2. PCA obviates the customary focus on specific peaks or regions of images. Applying it directly to a series of complex data will easily delineate binding isotherms, equilibrium shifts, and time courses of reactions or fluctuations.

  20. 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

  1. 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

  2. Cycling of Dense Core Vesicles Involved in Somatic Exocytosis of Serotonin by Leech Neurons

    PubMed Central

    Trueta, Citlali; Kuffler, Damien P.; De-Miguel, Francisco F.

    2012-01-01

    We studied the cycling of dense core vesicles producing somatic exocytosis of serotonin. Our experiments were made using electron microscopy and vesicle staining with fluorescent dye FM1-43 in Retzius neurons of the leech, which secrete serotonin from clusters of dense core vesicles in a frequency-dependent manner. Electron micrographs of neurons at rest or after 1 Hz stimulation showed two pools of dense core vesicles. A perinuclear pool near Golgi apparatuses, from which vesicles apparently form, and a peripheral pool with vesicle clusters at a distance from the plasma membrane. By contrast, after 20 Hz electrical stimulation 47% of the vesicle clusters were apposed to the plasma membrane, with some omega exocytosis structures. Dense core and small clear vesicles apparently originating from endocytosis were incorporated in multivesicular bodies. In another series of experiments, neurons were stimulated at 20 Hz while bathed in a solution containing peroxidase. Electron micrographs of these neurons contained gold particles coupled to anti-peroxidase antibodies in dense core vesicles and multivesicular bodies located near the plasma membrane. Cultured neurons depolarized with high potassium in the presence of FM1-43 displayed superficial fluorescent spots, each reflecting a vesicle cluster. A partial bleaching of the spots followed by another depolarization in the presence of FM1-43 produced restaining of some spots, other spots disappeared, some remained without restaining and new spots were formed. Several hours after electrical stimulation the FM1-43 spots accumulated at the center of the somata. This correlated with electron micrographs of multivesicular bodies releasing their contents near Golgi apparatuses. Our results suggest that dense core vesicle cycling related to somatic serotonin release involves two steps: the production of clear vesicles and multivesicular bodies after exocytosis, and the formation of new dense core vesicles in the perinuclear

  3. 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.

  4. 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

  5. Physical determinants of vesicle mobility and supply at a central synapse

    PubMed Central

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

    2016-01-01

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

  6. 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.

  7. A model of synaptic vesicle-pool depletion and replenishment can account for the interspike interval distributions and nonrenewal properties of spontaneous spike trains of auditory-nerve fibers.

    PubMed

    Peterson, Adam J; Irvine, Dexter R F; Heil, Peter

    2014-11-05

    In mammalian auditory systems, the spiking characteristics of each primary afferent (type I auditory-nerve fiber; ANF) are mainly determined by a single ribbon synapse in a single receptor cell (inner hair cell; IHC). ANF spike trains therefore provide a window into the operation of these synapses and cells. It was demonstrated previously (Heil et al., 2007) that the distribution of interspike intervals (ISIs) of cat ANFs during spontaneous activity can be modeled as resulting from refractoriness operating on a non-Poisson stochastic point process of excitation (transmitter release events from the IHC). Here, we investigate nonrenewal properties of these cat-ANF spontaneous spike trains, manifest as negative serial ISI correlations and reduced spike-count variability over short timescales. A previously discussed excitatory process, the constrained failure of events from a homogeneous Poisson point process, can account for these properties, but does not offer a parsimonious explanation for certain trends in the data. We then investigate a three-parameter model of vesicle-pool depletion and replenishment and find that it accounts for all experimental observations, including the ISI distributions, with only the release probability varying between spike trains. The maximum number of units (single vesicles or groups of simultaneously released vesicles) in the readily releasable pool and their replenishment time constant can be assumed to be constant (∼4 and 13.5 ms, respectively). We suggest that the organization of the IHC ribbon synapses not only enables sustained release of neurotransmitter but also imposes temporal regularity on the release process, particularly when operating at high rates.

  8. Needle-free jet injection of intact phospholipid vesicles across the skin: a feasibility study.

    PubMed

    Schlich, Michele; Lai, Francesco; Murgia, Sergio; Valenti, Donatella; Fadda, Anna Maria; Sinico, Chiara

    2016-08-01

    Needle-free liquid jet injectors are devices developed for the delivery of pharmaceutical solutions through the skin. In this paper, we investigated for the first time the ability of these devices to deliver intact lipid vesicles. Diclofenac sodium loaded phospholipid vesicles of two types, namely liposomes and transfersomes, were prepared and fully characterized. The lipid vesicles were delivered through a skin specimen using a jet injector and the collected samples were analyzed to assess vesicle structural integrity, drug retention and release kinetics after the injection. In this regard, data concerning size, size distribution, surface charge of vesicles and bilayer integrity and thickness, before and after the injections, were measured by dynamic light scattering experiments, cryo-electron microscopy, and X-ray scattering techniques. Finally, the effect of vesicle fast jet injection through the skin on drug release kinetics was checked by in vitro experiments. The retention of the morphological, physico-chemical, and technological features after injection, proved the integrity of vesicles after skin crossing as a high-speed liquid jet. The delivery of undamaged vesicular carriers beneath the skin is of utmost importance to create a controlled release drug depot in the hypoderm, which may be beneficial for several localized therapies. Overall results reported in this paper may broaden the range of application of liquid jet injectors to lipid vesicle based formulations thus combining beneficial performance of painless devices with those of liposomal drug delivery systems.

  9. Histones Cause Aggregation and Fusion of Lipid Vesicles Containing Phosphatidylinositol-4-Phosphate

    PubMed Central

    Lete, Marta G.; Sot, Jesus; Gil, David; Valle, Mikel; Medina, Milagros; Goñi, Felix M.; Alonso, Alicia

    2015-01-01

    In a previous article, we demonstrated that histones (H1 or histone octamers) interact with negatively charged bilayers and induce extensive aggregation of vesicles containing phosphatidylinositol-4-phosphate (PIP) and, to a lesser extent, vesicles containing phosphatidylinositol (PI). Here, we found that vesicles containing PIP, but not those containing PI, can undergo fusion induced by histones. Fusion was demonstrated through the observation of intervesicular mixing of total lipids and inner monolayer lipids, and by ultrastructural and confocal microscopy studies. Moreover, in both PI- and PIP-containing vesicles, histones caused permeabilization and release of vesicular aqueous contents, but the leakage mechanism was different (all-or-none for PI and graded release for PIP vesicles). These results indicate that histones could play a role in the remodeling of the nuclear envelope that takes place during the mitotic cycle. PMID:25692591

  10. Histones cause aggregation and fusion of lipid vesicles containing phosphatidylinositol-4-phosphate.

    PubMed

    Lete, Marta G; Sot, Jesus; Gil, David; Valle, Mikel; Medina, Milagros; Goñi, Felix M; Alonso, Alicia

    2015-02-17

    In a previous article, we demonstrated that histones (H1 or histone octamers) interact with negatively charged bilayers and induce extensive aggregation of vesicles containing phosphatidylinositol-4-phosphate (PIP) and, to a lesser extent, vesicles containing phosphatidylinositol (PI). Here, we found that vesicles containing PIP, but not those containing PI, can undergo fusion induced by histones. Fusion was demonstrated through the observation of intervesicular mixing of total lipids and inner monolayer lipids, and by ultrastructural and confocal microscopy studies. Moreover, in both PI- and PIP-containing vesicles, histones caused permeabilization and release of vesicular aqueous contents, but the leakage mechanism was different (all-or-none for PI and graded release for PIP vesicles). These results indicate that histones could play a role in the remodeling of the nuclear envelope that takes place during the mitotic cycle.

  11. Adhesion of phospholipid vesicles to Chinese hamster fibroblasts: Role of cell surface proteins

    PubMed Central

    Pagano, RE; Takeichi, M

    1977-01-01

    The adhesion of artificially generated lipid membrane vesicles to Chinese hamster V79 fibroblasts in suspension was used as a model system for studying membrane interactions. Below their gel-liquid crystalline phase transition temperature, vesicles comprised of dipalmitoyl lecithin (DPL) or dimyristoyl lecithin (DML) absorbed to the surfaces of EDTA- dissociated cells. These adherent vesicles could not be removed by repeated washings of the treated cells but could be released into the medium by treatment with trypsin. EM autoradiographic studies of cells treated with[(3)H]DML or [(3)H]DPL vesicles showed that most of the radioactive lipids were confined to the cell periphery. Scanning electron microscopy and fluorescence microscopy further confirmed the presence of adherent vesicles at the cell surface. Adhesion of DML or DPL vesicles to EDTA-dissociated cells modified the lactoperoxidase-catalyzed iodination pattern of the cell surface proteins; the inhibition of labeling of two proteins with an approximately 60,000- dalton mol wt was particularly evident. Incubation of cells wit h (3)H-lipid vesicles followed by sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis showed that some of the (3)H-lipid migrated preferentially with these approximately 60,000-mol wt proteins. Studies of the temperature dependence of vesicle uptake and subsequent release by trypsin showed that DML or DPL vesicle adhesion to EDTA- dissociated cells increased with decreasing temperatures. In contrast, cells trypsinized before incubation with vesicles showed practically no temperature dependence of vesicle uptake. These results suggest two pathways for adhesion of lipid vesicles to the cell surface-a temperature-sensitive one involving cell surface proteins, and a temperature-independent one. These findings are discussed in terms of current models for cell-cell interactions. PMID:407233

  12. pH-responsive jello: gelatin gels containing fatty acid vesicles.

    PubMed

    Dowling, Matthew B; Lee, Jae-Ho; Raghavan, Srinivasa R

    2009-08-04

    We describe a new way to impart pH-responsive properties to gels of biopolymers such as gelatin. This approach involves the embedding of pH-sensitive nanosized vesicles within the gel. The vesicles employed here are those of sodium oleate (NaOA), a fatty-acid-based amphiphile with a single C18 tail. In aqueous solution, NaOA undergoes a transition from vesicles at a pH approximately 8 to micelles at a pH higher than approximately 10. Here, we combine NaOA and gelatin at pH 8.3 to create a vesicle-loaded gel and then bring the gel in contact with a pH 10 buffer solution. As the buffer diffuses into the gel, the vesicles within the gel get transformed into micelles. Accordingly, a vesicle-micelle front moves through the gel, and this can be visually identified by the difference in turbidity between the two regions. Vesicle disruption can also be done in a spatially selective manner to create micelle-rich domains within a vesicle-loaded gel. A possible application of the above approach is in the area of pH-dependent controlled release. A vesicle-to-micelle transition releases hydrophilic solutes encapsulated within the vesicles into the bulk gel, and in turn these solutes can rapidly diffuse out of the gel into the external bath. Experiments with calcein dye confirm this concept and show that we can indeed use the pH in the bath to tune the release rate of solutes from vesicle-loaded gels.

  13. 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.

  14. Extracellular Vesicles as New Players in Cellular Senescence

    PubMed Central

    Urbanelli, Lorena; Buratta, Sandra; Sagini, Krizia; Tancini, Brunella; Emiliani, Carla

    2016-01-01

    Cell senescence is associated with the secretion of many factors, the so-called “senescence-associated secretory phenotype”, which may alter tissue microenvironment, stimulating the organism to clean up senescent cells and replace them with newly divided ones. Therefore, although no longer dividing, these cells are still metabolically active and influence the surrounding tissue. Much attention has been recently focused not only on soluble factors released by senescent cells, but also on extracellular vesicles as conveyors of senescence signals outside the cell. Here, we give an overview of the role of extracellular vesicles in biological processes and signaling pathways related to senescence and aging. PMID:27571072

  15. Urinary extracellular vesicles and the kidney: biomarkers and beyond.

    PubMed

    Salih, Mahdi; Zietse, Robert; Hoorn, Ewout J

    2014-06-01

    Extracellular vesicles have been isolated in various body fluids, including urine. The cargo of urinary extracellular vesicles (uEVs) is composed of proteins and nucleic acids reflecting the physiological and possibly pathophysiological state of cells lining the nephron. Because urine is a noninvasive and readily available biofluid, the discovery of uEVs has opened a new field of biomarker research. Their potential use as diagnostic, prognostic, or therapeutic biomarkers for various kidney diseases, including glomerulonephritis, acute kidney injury, tubular disorders, and polycystic kidney disease, is currently being explored. Some challenges, however, remain. These challenges include the need to standardize isolation methods, normalization between samples, and validation of candidate biomarkers. Also, the development of a high-throughput platform to isolate and analyze uEVs, for example, an enzyme-linked immunosorbent assay, is desirable. Here, we review recent studies on uEVs dealing with kidney physiology and pathophysiology. Furthermore, we discuss new and exciting developments regarding vesicles, including their role in cell-to-cell communication and the possibility of using vesicles as a therapy for kidney disorders.

  16. Calcium regulates vesicle replenishment at the cone ribbon synapse.

    PubMed

    Babai, Norbert; Bartoletti, Theodore M; Thoreson, Wallace B

    2010-11-24

    Cones release glutamate-filled vesicles continuously in darkness, and changing illumination modulates this release. Because sustained release in darkness is governed by vesicle replenishment rates, we analyzed how cone membrane potential regulates replenishment. Synaptic release from cones was measured by recording postsynaptic currents in Ambystoma tigrinum horizontal or OFF bipolar cells evoked by depolarization of simultaneously voltage-clamped cones. We measured replenishment after attaining a steady state between vesicle release and replenishment using trains of test pulses. Increasing Ca(2+) currents (I(Ca)) by changing the test step from -30 to -10 mV increased replenishment. Lengthening -30 mV test pulses to match the Ca(2+) influx during 25 ms test pulses to -10 mV produced similar replenishment rates. Reducing Ca(2+) driving force by using test steps to +30 mV slowed replenishment. Using UV flashes to reverse inhibition of I(Ca) by nifedipine accelerated replenishment. Increasing [Ca(2+)](i) by flash photolysis of caged Ca(2+) also accelerated replenishment. Replenishment, but not the initial burst of release, was enhanced by using an intracellular Ca(2+) buffer of 0.5 mm EGTA rather than 5 mm EGTA, and diminished by 1 mm BAPTA. This suggests that although release and replenishment exhibited similar Ca(2+) dependencies, release sites are <200 nm from Ca(2+) channels but replenishment sites are >200 nm away. Membrane potential thus regulates replenishment by controlling Ca(2+) influx, principally by effects on replenishment mechanisms but also by altering releasable pool size. This in turn provides a mechanism for converting changes in light intensity into changes in sustained release at the cone ribbon synapse.

  17. 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

  18. Altered mechanisms underlying the abnormal glutamate release in amyotrophic lateral sclerosis at a pre-symptomatic stage of the disease.

    PubMed

    Bonifacino, Tiziana; Musazzi, Laura; Milanese, Marco; Seguini, Mara; Marte, Antonella; Gallia, Elena; Cattaneo, Luca; Onofri, Franco; Popoli, Maurizio; Bonanno, Giambattista

    2016-11-01

    Abnormal Glu release occurs in the spinal cord of SOD1(G93A) mice, a transgenic animal model for human ALS. Here we studied the mechanisms underlying Glu release in spinal cord nerve terminals of SOD1(G93A) mice at a pre-symptomatic disease stage (30days) and found that the basal release of Glu was more elevated in SOD1(G93A) with respect to SOD1 mice, and that the surplus of release relies on synaptic vesicle exocytosis. Exposure to high KCl or ionomycin provoked Ca(2+)-dependent Glu release that was likewise augmented in SOD1(G93A) mice. Equally, the Ca(2+)-independent hypertonic sucrose-induced Glu release was abnormally elevated in SOD1(G93A) mice. Also in this case, the surplus of Glu release was exocytotic in nature. We could determine elevated cytosolic Ca(2+) levels, increased phosphorylation of Synapsin-I, which was causally related to the abnormal Glu release measured in spinal cord synaptosomes of pre-symptomatic SOD1(G93A) mice, and increased phosphorylation of glycogen synthase kinase-3 at the inhibitory sites, an event that favours SNARE protein assembly. Western blot experiments revealed an increased number of SNARE protein complexes at the nerve terminal membrane, with no changes of the three SNARE proteins and increased expression of synaptotagmin-1 and β-Actin, but not of an array of other release-related presynaptic proteins. These results indicate that the abnormal exocytotic Glu release in spinal cord of pre-symptomatic SOD1(G93A) mice is mainly based on the increased size of the readily releasable pool of vesicles and release facilitation, supported by plastic changes of specific presynaptic mechanisms.

  19. Extracellular vesicles shuffling intercellular messages: for good or for bad.

    PubMed

    Lo Cicero, Alessandra; Stahl, Philip D; Raposo, Graça

    2015-08-01

    The release of extracellular vesicles (EVs) is a highly conserved process exploited by diverse organisms as a mode of intercellular communication. Vesicles of sizes ranging from 30 to 1000nm, or even larger, are generated by blebbing of the plasma membrane (microvesicles) or formed in multivesicular endosomes (MVEs) to be secreted by exocytosis as exosomes. Exosomes, microvesicles and other EVs contain membrane and cytosolic components that include proteins, lipids and RNAs, a composition that differs related to their site of biogenesis. Several mechanisms are involved in vesicle formation at the plasma membrane or in endosomes, which is reflected in their heterogeneity, size and composition. EVs have significant promise for therapeutics and diagnostics and for understanding physiological and pathological processes all of which have boosted research to find modulators of their composition, secretion and targeting.

  20. Synaptojanin is recruited by endophilin to promote synaptic vesicle uncoating.

    PubMed

    Verstreken, Patrik; Koh, Tong-Wey; Schulze, Karen L; Zhai, R Grace; Hiesinger, P Robin; Zhou, Yi; Mehta, Sunil Q; Cao, Yu; Roos, Jack; Bellen, Hugo J

    2003-11-13

    We describe the isolation and characterization of Drosophila synaptojanin (synj) mutants. synj encodes a phosphatidylinositol phosphatase involved in clathrin-mediated endocytosis. We show that Synj is specifically localized to presynaptic terminals and is associated with synaptic vesicles. The electrophysiological and ultrastructural defects observed in synj mutants are strikingly similar to those found in endophilin mutants, and Synj and Endo colocalize and interact biochemically. Moreover, synj; endo double mutant synaptic terminals exhibit properties that are very similar to terminals of each single mutant, and overexpression of Endophilin can partially rescue the functional defects in partial loss-of-function synj mutants. Interestingly, Synj is mislocalized and destabilized at synapses devoid of Endophilin, suggesting that Endophilin recruits and stabilizes Synj on newly formed vesicles to promote vesicle uncoating. Our data also provide further evidence that kiss-and-run is able to maintain neurotransmitter release when synapses are not extensively challenged.

  1. Bassoon Speeds Vesicle Reloading at a Central Excitatory Synapse

    PubMed Central

    Hallermann, Stefan; Fejtova, Anna; Schmidt, Hartmut; Weyhersmüller, Annika; Silver, R. Angus; Gundelfinger, Eckart D.; Eilers, Jens

    2010-01-01

    Summary Sustained rate-coded signals encode many types of sensory modalities. Some sensory synapses possess specialized ribbon structures, which tether vesicles, to enable high-frequency signaling. However, central synapses lack these structures, yet some can maintain signaling over a wide bandwidth. To analyze the underlying molecular mechanisms, we investigated the function of the active zone core component Bassoon in cerebellar mossy fiber to granule cell synapses. We show that short-term synaptic depression is enhanced in Bassoon knockout mice during sustained high-frequency trains but basal synaptic transmission is unaffected. Fluctuation and quantal analysis as well as quantification with constrained short-term plasticity models revealed that the vesicle reloading rate was halved in the absence of Bassoon. Thus, our data show that the cytomatrix protein Bassoon speeds the reloading of vesicles to release sites at a central excitatory synapse. PMID:21092860

  2. Emerging Roles of Extracellular Vesicles in the Nervous System

    PubMed Central

    Bali, Jitin; Barr, Maureen M.; Court, Felipe A.; Krämer-Albers, Eva-Maria; Picou, Frederic; Raposo, Graça; van der Vos, Kristan E.; van Niel, Guillaume; Wang, Juan

    2014-01-01

    Information exchange executed by extracellular vesicles, including exosomes, is a newly described form of intercellular communication important in the development and physiology of neural systems. These vesicles can be released from cells, are packed with information including signaling proteins and both coding and regulatory RNAs, and can be taken up by target cells, thereby facilitating the transfer of multilevel information. Recent studies demonstrate their critical role in physiological processes, including nerve regeneration, synaptic function, and behavior. These vesicles also have a sinister role in the propagation of toxic amyloid proteins in neurodegenerative conditions, including prion diseases and Alzheimer's and Parkinson's diseases, in inducing neuroinflammation by exchange of information between the neurons and glia, as well as in aiding tumor progression in the brain by subversion of normal cells. This article provides a summary of topics covered in a symposium and is not meant to be a comprehensive review of the subject. PMID:25392515

  3. Dynamic flow-through approach to evaluate readily bioaccessible antioxidants in solid food samples.

    PubMed

    Maia, Miguel A; Soares, Tânia R P; Mota, Ana I P; Rosende, María; Magalhães, Luís M; Miró, Manuel; Segundo, Marcela A

    2017-05-01

    Release of bioactive compounds from food matrices is regarded as the first step towards their human bioavailability. The objective of this work was the implementation of an affordable and robust flow-through device for expedient dynamic leaching experiments aiming at the assessment of readily bioaccessible antioxidant compounds in solid food commodities. A simple configuration is proposed using commercially available devices containing regenerated cellulose filters placed in polypropylene holders to entrap the solid sample, featuring a disposable, single use extraction chamber. The kinetic extraction profile of fast leachable antioxidants from different food matrices was evaluated using the ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)) assay, fitting a first-order reaction model for readily bioaccessible compounds (R>0.9). The leaching rate constant values associated to the fast leachable antioxidant compounds were 0.060-0.446min(-1) and 0.105-0.210min(-1) for water and ethanol/water (1:1, v/v) applied as extractants, respectively. Furthermore, no statistically significant differences were found between the estimated values of bioaccessible antioxidant compounds by the kinetic model and the values attained using conventional batch-wise extraction methodology, ranging from 3.37 to 60.3 µmol of Trolox ((±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) per g of sample. Extension of the method using U. S. Pharmacopeia surrogate biological media (stomach (pH 1.2) and intestinal (pH 7.5) fluids without enzymes) to NIST-1570a spinach leaves provided gastrointestinal compartment-dependent kinetic leaching rates (0.120 and 0.198min(-1), respectively) and total antioxidant content (45.5 and 52.5µmol of Trolox per g of sample, respectively).

  4. 16 CFR 1101.13 - Public ability to ascertain readily identity of manufacturer or private labeler.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 16 Commercial Practices 2 2014-01-01 2014-01-01 false Public ability to ascertain readily identity...)(1) § 1101.13 Public ability to ascertain readily identity of manufacturer or private labeler. The... readily ascertain from the information itself the identity of the manufacturer or private labeler of...

  5. 16 CFR 1101.13 - Public ability to ascertain readily identity of manufacturer or private labeler.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 16 Commercial Practices 2 2011-01-01 2011-01-01 false Public ability to ascertain readily identity...)(1) § 1101.13 Public ability to ascertain readily identity of manufacturer or private labeler. The... readily ascertain from the information itself the identity of the manufacturer or private labeler of...

  6. 16 CFR 1101.13 - Public ability to ascertain readily identity of manufacturer or private labeler.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 16 Commercial Practices 2 2013-01-01 2013-01-01 false Public ability to ascertain readily identity...)(1) § 1101.13 Public ability to ascertain readily identity of manufacturer or private labeler. The... readily ascertain from the information itself the identity of the manufacturer or private labeler of...

  7. 16 CFR 1101.13 - Public ability to ascertain readily identity of manufacturer or private labeler.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 16 Commercial Practices 2 2012-01-01 2012-01-01 false Public ability to ascertain readily identity...)(1) § 1101.13 Public ability to ascertain readily identity of manufacturer or private labeler. The... readily ascertain from the information itself the identity of the manufacturer or private labeler of...

  8. 17 CFR 37.300 - Core Principle 3-Swaps not readily susceptible to manipulation.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... readily susceptible to manipulation. 37.300 Section 37.300 Commodity and Securities Exchanges COMMODITY FUTURES TRADING COMMISSION SWAP EXECUTION FACILITIES Swaps Not Readily Susceptible to Manipulation § 37.300 Core Principle 3—Swaps not readily susceptible to manipulation. The swap execution facility...

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

    PubMed Central

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

    2010-01-01

    The assembly of SNARE complexes between syntaxin, SNAP-25 and synaptobrevin is required to prime synaptic vesicles for fusion. Since Munc18 and tomosyn compete for syntaxin interactions, the interplay between these proteins is predicted to be important in regulating synaptic transmission. We explored this possibility, by examining genetic interactions between C. elegans unc-18(Munc18), unc-64(syntaxin) and tom-1(tomosyn). We have previously demonstrated that unc-18 mutants have reduced synaptic transmission, whereas tom-1 mutants exhibit enhanced release. Here we show that the unc-18 mutant release defect is associated with loss of two morphologically distinct vesicle pools; those tethered within 25 nm of the plasma membrane and those docked with the plasma membrane. In contrast, priming defective unc-13 mutants accumulate tethered vesicles, while docked vesicles are greatly reduced, indicating tethering is UNC-18-dependent and occurs in the absence of priming. C. elegans unc-64 mutants phenocopy unc-18 mutants, losing both tethered and docked vesicles, whereas overexpression of open syntaxin preferentially increases vesicle docking, suggesting UNC-18/closed syntaxin interactions are responsible for vesicle tethering. Given the competition between vertebrate tomosyn and Munc18, for syntaxin binding, we hypothesized that C. elegans TOM-1 may inhibit both UNC-18-dependent vesicle targeting steps. Consistent with this hypothesis, tom-1 mutants exhibit enhanced UNC-18 plasma membrane localization and a concomitant increase in both tethered and docked synaptic vesicles. Furthermore, in tom-1;unc-18 double mutants the docked, primed vesicle pool is preferentially rescued relative to unc-18 single mutants. Together these data provide evidence for the differential regulation of two vesicle targeting steps by UNC-18 and TOM-1 through competitive interactions with syntaxin. PMID:21423527

  10. Synaptic vesicles in rat hippocampal boutons recycle to different pools in a use-dependent fashion

    PubMed Central

    Vanden Berghe, Pieter; Klingauf, Jürgen

    2006-01-01

    Efficient vesicle membrane recycling at presynaptic terminals is pivotal for preventing depletion and maintaining high firing rates in neuronal networks. We used a new approach, based on the combination of spectrally different optical probes, to investigate how stimulation determines the fate of synaptic vesicles after endocytosis. We found that in the small central synapses of rat hippocampal neurones low frequency stimulation (40 action potentials at 2 Hz) targets vesicles preferentially to vesicle pools that were kinetically faster. Vesicles taken up during endocytosis triggered by high frequency stimulation (400 action potentials, 20 Hz) were also placed in the back of the release queue. We performed a spatial analysis of the recycled vesicles in living hippocampal boutons using two spectrally different FM-dyes (FM1-43 and FM5-95). By using these consecutively, vesicles endocytosed by either stimulation protocol were labelled with a different colour. This revealed that the kinetic arrangement was also reflected in the spatial organization of vesicles within the bouton. Next, we identified the postsynaptic site of the active zone by transfecting the neurones with postsynaptic density protein PSD-95-CFP. The data from these triple colour experiments suggest that retrieval after low frequency stimulation keeps vesicles in a more confined region closer to the active zone as identified by PSD-95-CFP expression at the postsynaptic site. PMID:16439431

  11. Tweek, an evolutionary conserved proteinis required for synaptic vesicle recycling

    PubMed Central

    Verstreken, Patrik; Ohyama, Tomoko; Haueter, Claire; Habets, Ron L.P.; Lin, Yong Q.; Swan, Laura E.; Ly, Cindy V.; Venken, Koen J. T.; De Camilli, Pietro; Bellen, Hugo J.

    2009-01-01

    Synaptic vesicle endocytosis is critical to maintain synaptic communication during intense stimulation. Here we describe Tweek, a conserved protein that is required for synaptic vesicle recycling. tweek mutants show reduced FM 1–43 uptake, cannot maintain release during intense stimulation and harbor larger than normal synaptic vesicles, implicating it in vesicle recycling at the synapse. Interestingly, the levels of a fluorescent PI(4,5)P2 reporter are reduced at tweek mutant synapses and the probe is aberrantly localized during stimulation. In addition, various endocytic adaptors known to bind PI(4,5)P2 are mislocalized and the defects in FM 1–43 dye uptake and adaptor localization are partially suppressed by removing one copy of the phosphoinositide-phosphatase synaptojanin, suggesting a role for Tweek in maintaining proper phosphoinositide levels at synapses. Our data implicate Tweek in regulating synaptic vesicle recycling via an action mediated at least in part by the regulation of PI(4,5)P2 levels or availability at the synapse. PMID:19640479

  12. Extracellular Vesicles in Alzheimer’s Disease: Friends or Foes? Focus on Aβ-Vesicle Interaction

    PubMed Central

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

    2015-01-01

    The intercellular transfer of amyloid-β (Aβ) and tau proteins has received increasing attention in Alzheimer’s disease (AD). Among other transfer modes, Aβ and tau dissemination has been suggested to occur through release of Extracellular Vesicles (EVs), which may facilitate delivery of pathogenic proteins over large distances. Recent evidence indicates that EVs carry on their surface, specific molecules which bind to extracellular Aβ, opening the possibility that EVs may also influence Aβ assembly and synaptotoxicity. In this review we focus on studies which investigated the impact of EVs in Aβ-mediated neurodegeneration and showed either detrimental or protective role for EVs in the pathology. PMID:25741766

  13. Preparation and Evaluation of Nano-vesicles of Brimonidine Tartrate as an Ocular Drug Delivery System

    PubMed Central

    Prabhu, P; Nitish, Kumar R; Koland, M; Harish, NM; Vijayanarayan, K; Dhondge, G; Charyulu, RN

    2010-01-01

    The objective of the present investigation was to design a vesicular formulation of brimonidine tartrate and evaluate its ability to reduce the dosing frequency and improve the therapeutic efficacy of the drug. Nano-vesicles of brimonidine tartrate were prepared by film hydration method. The prepared vesicles were evaluated for photomicroscopic characteristics, entrapment efficiency, in vitro, and ex-in vitro drug release and in vivo intraocular pressure (IOP) lowering activity. The methods employed for preparation of vesicles produced nano vesicles of acceptable shape and size. The in vitro, and ex-in vitro drug release studies showed that there was slow and prolonged release of the drug, which followed zero-order kinetics. The IOP-lowering activity of nano vesicles was determined and compared with that of pure drug solution and showed that the IOP-lowering action of nano-vesicles sustained for a longer period of time. Stability studies revealed that the vesicle formulations were stable at the temperature range of 2-8°C, with no change in shape and drug content. The results of the study indicate that it is possible to develop a safe and physiologically effective topical formulation that is also convenient for patients. PMID:21264093

  14. MicroRNA exocytosis by large dense-core vesicle fusion

    PubMed Central

    Gümürdü, Alican; Yildiz, Ramazan; Eren, Erden; Karakülah, Gökhan; Ünver, Turgay; GENÇ, Şermin; Park, Yongsoo

    2017-01-01

    Neurotransmitters and peptide hormones are secreted into outside the cell by a vesicle fusion process. Although non-coding RNA (ncRNA) that include microRNA (miRNA) regulates gene expression inside the cell where they are transcribed, extracellular miRNA has been recently discovered outside the cells, proposing that miRNA might be released to participate in cell-to-cell communication. Despite its importance of extracellular miRNA, the molecular mechanisms by which miRNA can be stored in vesicles and released by vesicle fusion remain enigmatic. Using next-generation sequencing, vesicle purification techniques, and synthetic neurotransmission, we observe that large dense-core vesicles (LDCVs) contain a variety of miRNAs including miR-375. Furthermore, miRNA exocytosis is mediated by the SNARE complex and accelerated by Ca2+. Our results suggest that miRNA can be a novel neuromodulator that can be stored in vesicles and released by vesicle fusion together with classical neurotransmitters. PMID:28358390

  15. Preparation and Evaluation of Nano-vesicles of Brimonidine Tartrate as an Ocular Drug Delivery System.

    PubMed

    Prabhu, P; Nitish, Kumar R; Koland, M; Harish, Nm; Vijayanarayan, K; Dhondge, G; Charyulu, Rn

    2010-10-01

    The objective of the present investigation was to design a vesicular formulation of brimonidine tartrate and evaluate its ability to reduce the dosing frequency and improve the therapeutic efficacy of the drug. Nano-vesicles of brimonidine tartrate were prepared by film hydration method. The prepared vesicles were evaluated for photomicroscopic characteristics, entrapment efficiency, in vitro, and ex-in vitro drug release and in vivo intraocular pressure (IOP) lowering activity. The methods employed for preparation of vesicles produced nano vesicles of acceptable shape and size. The in vitro, and ex-in vitro drug release studies showed that there was slow and prolonged release of the drug, which followed zero-order kinetics. The IOP-lowering activity of nano vesicles was determined and compared with that of pure drug solution and showed that the IOP-lowering action of nano-vesicles sustained for a longer period of time. Stability studies revealed that the vesicle formulations were stable at the temperature range of 2-8°C, with no change in shape and drug content. The results of the study indicate that it is possible to develop a safe and physiologically effective topical formulation that is also convenient for patients.

  16. Dysregulations of Synaptic Vesicle Trafficking in Schizophrenia.

    PubMed

    Egbujo, Chijioke N; Sinclair, Duncan; Hahn, Chang-Gyu

    2016-08-01

    Schizophrenia is a serious psychiatric illness which is experienced by about 1 % of individuals worldwide and has a debilitating impact on perception, cognition, and social function. Over the years, several models/hypotheses have been developed which link schizophrenia to dysregulations of the dopamine, glutamate, and serotonin receptor pathways. An important segment of these pathways that have been extensively studied for the pathophysiology of schizophrenia is the presynaptic neurotransmitter release mechanism. This set of molecular events is an evolutionarily well-conserved process that involves vesicle recruitment, docking, membrane fusion, and recycling, leading to efficient neurotransmitter delivery at the synapse. Accumulated evidence indicate dysregulation of this mechanism impacting postsynaptic signal transduction via different neurotransmitters in key brain regions implicated in schizophrenia. In recent years, after ground-breaking work that elucidated the operations of this mechanism, research efforts have focused on the alterations in the messenger RNA (mRNA) and protein expression of presynaptic neurotransmitter release molecules in schizophrenia and other neuropsychiatric conditions. In this review article, we present recent evidence from schizophrenia human postmortem studies that key proteins involved in the presynaptic release mechanism are dysregulated in the disorder. We also discuss the potential impact of dysfunctional presynaptic neurotransmitter release on the various neurotransmitter systems implicated in schizophrenia.

  17. 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.

  18. 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.

  19. The origin, function, and diagnostic potential of RNA within extracellular vesicles present in human biological fluids.

    PubMed

    Taylor, Douglas D; Gercel-Taylor, Cicek

    2013-01-01

    We have previously demonstrated that tumor cells release membranous structures into their extracellular environment, which are termed exosomes, microvesicles or extracellular vesicles depending on specific characteristics, including size, composition and biogenesis pathway. These cell-derived vesicles can exhibit an array of proteins, lipids and nucleic acids derived from the originating tumor. This review focuses of the transcriptome (RNA) of these extracellular vesicles. Based on current data, these vesicular components play essential roles as conveyers of intercellular communication and mediators of many of the pathological conditions associated with cancer development, progression and therapeutic failures. These extracellular vesicles express components responsible for angiogenesis promotion, stromal remodeling, signal pathway activation through growth factor/receptor transfer, chemoresistance, and genetic exchange. These tumor-derived extracellular vesicles not only to represent a central mediator of the tumor microenvironment, but their presence in the peripheral circulation may serve as a surrogate for tumor biopsies, enabling real-time diagnosis and disease monitoring.

  20. Additive effects on the energy barrier for synaptic vesicle fusion cause supralinear effects on the vesicle fusion rate

    PubMed Central

    Schotten, Sebastiaan; Meijer, Marieke; Walter, Alexander Matthias; Huson, Vincent; Mamer, Lauren; Kalogreades, Lawrence; ter Veer, Mirelle; Ruiter, Marvin; Brose, Nils; Rosenmund, Christian

    2015-01-01

    The energy required to fuse synaptic vesicles with the plasma membrane (‘activation energy’) is considered a major determinant in synaptic efficacy. From reaction rate theory, we predict that a class of modulations exists, which utilize linear modulation of the energy barrier for fusion to achieve supralinear effects on the fusion rate. To test this prediction experimentally, we developed a method to assess the number of releasable vesicles, rate constants for vesicle priming, unpriming, and fusion, and the activation energy for fusion by fitting a vesicle state model to synaptic responses induced by hypertonic solutions. We show that complexinI/II deficiency or phorbol ester stimulation indeed affects responses to hypertonic solution in a supralinear manner. An additive vs multiplicative relationship between activation energy and fusion rate provides a novel explanation for previously observed non-linear effects of genetic/pharmacological perturbations on synaptic transmission and a novel interpretation of the cooperative nature of Ca2+-dependent release. DOI: http://dx.doi.org/10.7554/eLife.05531.001 PMID:25871846

  1. Extracellular vesicles are rapidly purified from human plasma by PRotein Organic Solvent PRecipitation (PROSPR).

    PubMed

    Gallart-Palau, Xavier; Serra, Aida; Wong, Andrew See Weng; Sandin, Sara; Lai, Mitchell K P; Chen, Christopher P; Kon, Oi Lian; Sze, Siu Kwan

    2015-09-30

    Extracellular vesicles (EVs) such as exosomes and microvesicles mediate intercellular communication and regulate a diverse range of crucial biological processes. Host cells that are damaged, infected or transformed release biomarker-containing EVs into the peripheral circulation, where they can be readily accessed for use in diagnostic or prognostic testing. However, current methods of EV isolation from blood plasma are complex and often require relatively large sample volumes, hence are inefficient for widespread use in clinical settings. Here, we report a novel and inexpensive method of rapidly isolating EVs from small volumes of human blood plasma by PRotein Organic Solvent PRecipitation (PROSPR). PROSPR encompasses a rapid three-step protocol to remove soluble proteins from plasma via precipitation in cold acetone, leaving the lipid-encapsulated EVs behind in suspension. This generates higher purity EVs that can then be obtained from filtration or classical ultracentrifugation methods. We foresee that PROSPR-based purification of EVs will significantly accelerate the discovery of new disease biomarkers and the characterization of EVs with potential for clinical applications.

  2. 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.

  3. 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

  4. Presynaptic Ca2+ influx and vesicle exocytosis at the mouse endbulb of Held: a comparison of two auditory nerve terminals

    PubMed Central

    Lin, Kun-Han; Oleskevich, Sharon; Taschenberger, Holger

    2011-01-01

    Abstract The functional properties of mammalian presynaptic nerve endings remain elusive since most terminals of the central nervous system are not accessible to direct electrophysiological recordings. In this study, direct recordings were performed for the first time at endbulb of Held terminals to characterize passive membrane properties, voltage-gated Ca2+ channels (VGCCs) and Ca2+-dependent exocytosis. Endbulb of Held terminals arise from endings of auditory nerve fibres contacting spherical bushy cells (SBCs) in the anterior ventral cochlear nucleus (AVCN). These terminals had a high mean input resistance (1.1 GΩ) and a small mean capacitance (4.3 pF). Presynaptic VGCCs were predominantly of the P/Q type (86%) and expressed at a high density with an estimated average number of 6400 channels per terminal. Presynaptic Ca2+ currents (ICa(V)) activated and deactivated rapidly. Simulations of action potential (AP)-driven gating of VGCCs suggests that endbulb APs trigger brief Ca2+ influx with a mean half-width of 240 μs and a peak amplitude of 0.45 nA which results from the opening of approximately 2600 channels. Unlike Ca2+ currents at the calyx of Held, ICa(V) of endbulb terminals showed no inactivation during trains of AP-like presynaptic depolarizations. Endbulb terminals are endowed with a large readily releasable vesicle pool (1064 vesicles) of which only a small fraction (<10%) is consumed during a single AP-like stimulus. Fast presynaptic APs together with rapidly gating VGCCs will generate brief intracellular Ca2+ transients that favour highly synchronous transmitter release. Collectively these characteristics ensure sustained and precise transmission of timing information from auditory stimuli at the endbulb→SBC synapse. PMID:21746778

  5. Inhibiting Extracellular Vesicle Release from Human Cardiosphere Derived Cells with Lentiviral Knockdown of nSMase2 Differentially Effects Proliferation and Apoptosis in Cardiomyocytes, Fibroblasts and Endothelial Cells In Vitro

    PubMed Central

    Young, Rebeccah F.; Ashraf, Hashmat; Canty, John M.

    2016-01-01

    Numerous studies have shown a beneficial effect of cardiosphere-derived cell (CDC) therapy on regeneration of injured myocardium. Paracrine signaling by CDC secreted exosomes may contribute to improved cardiac function. However, it has not yet been demonstrated by a genetic approach that exosome release contributes to the therapeutic effect of transplanted CDCs. By employing a lentiviral knockdown (KD) strategy against neutral spingomyelinase 2 (nSMase2), a crucial gene in exosome secretion, we have defined the role of physiologically secreted human CDC-derived exosomes on cardiac fibroblast, endothelial cell and primary cardiomyocyte proliferation, cell death, migration and angiogenesis using a series of in vitro coculture assays. We found that secretion of hCDC-derived exosomes was effectively inhibited by nSMase2 lentiviral KD and shRNAi expression was stable and constitutive. hCDC exosome release contributed to the angiogenic and pro-migratory effects of hCDCs on HUVECs, decreased proliferation of fibroblasts, and decreased apoptosis of cardiomyocytes. These in vitro reactions support a role for exosome secretion as a paracrine mechanism of stem cell-mediated cardiac repair in vivo. Importantly, we have established a novel tool to test constitutive inhibition of exosome secretion in stem cell populations in animal models of cardiac disease. PMID:27806113

  6. The discovery of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex and the molecular regulation of synaptic vesicle transmitter release: the 2010 Kavli Prize in neuroscience.

    PubMed

    Hussain, S; Davanger, S

    2011-09-08

    Brain function depends on a crucial feature: The ability of individual neurons to share packets of information, known as quantal transmission. Given the sheer number of tasks the brain has to deal with, this information sharing must be extremely rapid. Synapses are specialized points of contact between neurons, where fast transmission takes place. Though the basic elements and functions of the synapse had been established since the 1950s, the molecular basis for regulation of fast synaptic transmitter release was not known 20 years ago. However, around 1990, crucial discoveries were made by Richard Scheller, James Rothman, and Thomas Südhof, leading a few years later to the formulation of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) hypothesis and a new understanding of the molecular events controlling vesicular release of transmitter in synapses. The 2010 Kavli Prize in neuroscience was awarded to these three researchers, "for their work to reveal the precise molecular basis of the transfer of signals between nerve cells in the brain."

  7. Presynaptic D1 heteroreceptors and mGlu autoreceptors act at individual cortical release sites to modify glutamate release.

    PubMed

    Hikima, Takuya; Garcia-Munoz, Marianela; Arbuthnott, Gordon William

    2016-05-15

    The aim of this work was to study release of glutamic acid (GLU) from one-axon terminal or bouton at-a-time using cortical neurons grown in vitro to study the effect of presynaptic auto- and heteroreceptor stimulation. Neurons were infected with release reporters SypHx2 or iGluSnFR at 7 or 3 days-in-vitro (DIV) respectively. At 13-15 DIV single synaptic boutons were identified from images obtained from a confocal scanning microscope before and after field electrical stimulation. We further stimulated release by raising intracellular levels of cAMP with forskolin (10µM). Forskolin-mediated effects were dependent on protein kinase A (PKA) and did not result from an increase in endocytosis, but rather from an increase in the size of the vesicle readily releasable pool. Once iGluSnFR was confirmed as more sensitive than SypHx2, it was used to study the participation of presynaptic auto- and heteroreceptors on GLU release. Although most receptor agonizts (carbamylcholine, nicotine, dopamine D2, BDNF) did not affect electrically stimulated GLU release, a significant increase was observed in the presence of metabotropic D1/D5 heteroreceptor agonist (SKF38393 10µM) that was reversed by PKA inhibitors. Interestingly, stimulation of group II metabotropic mGLU2/3 autoreceptors (LY379268 50nM) induced a decrease in GLU release that was reversed by the specific mGLU2/3 receptor antagonist (LY341495 1µM) and also by PKA inhibitors (KT5720 200nM and PKI14-22 400nM). These changes in release probability at individual release sites suggest another level of control of the distribution of transmitter substances in cortical tissue.

  8. 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

  9. 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

  10. 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

  11. Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses.

    PubMed

    Granseth, Björn; Odermatt, Benjamin; Royle, Stephen J; Lagnado, Leon

    2006-09-21

    The maintenance of synaptic transmission requires that vesicles be recycled after releasing neurotransmitter. Several modes of retrieval have been proposed to operate at small synaptic terminals of central neurons, including a fast "kiss-and-run" mechanism that releases neurotransmitter through a fusion pore. Using an improved fluorescent reporter comprising pHluorin fused to synaptophysin, we find that only a slow mode of endocytosis (tau = 15 s) operates at hippocampal synapses when vesicle fusion is triggered by a single nerve impulse or short burst. This retrieval mechanism is blocked by overexpression of the C-terminal fragment of AP180 or by knockdown of clathrin using RNAi, and it is associated with the movement of clathrin and vesicle proteins out of the synapse. These results indicate that clathrin-mediated endocytosis is the major, if not exclusive, mechanism of vesicle retrieval after physiological stimuli.

  12. CAPS-1 and CAPS-2 are essential synaptic vesicle priming proteins.

    PubMed

    Jockusch, Wolf J; Speidel, Dina; Sigler, Albrecht; Sørensen, Jakob B; Varoqueaux, Frederique; Rhee, Jeong-Seop; Brose, Nils

    2007-11-16

    Before transmitter-filled synaptic vesicles can fuse with the plasma membrane upon stimulation they have to be primed to fusion competence. The regulation of this priming process controls the strength and plasticity of synaptic transmission between neurons, which in turn determines many complex brain functions. We show that CAPS-1 and CAPS-2 are essential components of the synaptic vesicle priming machinery. CAPS-deficient neurons contain no or very few fusion competent synaptic vesicles, which causes a selective impairment of fast phasic transmitter release. Increases in the intracellular Ca(2+) levels can transiently revert this defect. Our findings demonstrate that CAPS proteins generate and maintain a highly fusion competent synaptic vesicle pool that supports phasic Ca(2+) triggered release of transmitters.

  13. 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

  14. Synapsin knockdown is associated with decreased neurite outgrowth, functional synaptogenesis impairment, and fast high-frequency neurotransmitter release.

    PubMed

    Brenes, Oscar; Giachello, Carlo Natale Giuseppe; Corradi, Anna Margherita; Ghirardi, Mirella; Montarolo, Pier Giorgio

    2015-10-01

    Synapsins (Syns) are an evolutionarily conserved family of synaptic vesicle-associated proteins related to fine tuning of synaptic transmission. Studies with mammals have partially clarified the different roles of Syns; however, the presence of different genes and isoforms and the development of compensatory mechanisms hinder accurate data interpretation. Here, we use a simple in vitro monosynaptic Helix neuron connection, reproducing an in vivo physiological connection as a reliable experimental model to investigate the effects of Syn knockdown. Cells overexpressing an antisense construct against Helix Syn showed a time-dependent decrease of Syn immunostaining, confirming protein loss. At the morphological level, Syn-silenced cells showed a reduction in neurite linear outgrowth and branching and in the size and number of synaptic varicosities. Functionally, Syn-silenced cells presented a reduced ability to form synaptic connections; however, functional chemical synapses showed similar basal excitatory postsynaptic potentials and similar short-term plasticity paradigms. In addition, Syn-silenced cells presented faster neurotransmitter release and decreased postsynaptic response toward the end of long tetanic presynaptic stimulations, probably related to an impairment of the synaptic vesicle trafficking resulting from a different vesicle handling, with an increased readily releasable pool and a compromised reserve pool.

  15. 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.

  16. 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.

  17. 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

  18. Regulation of neurotransmitter release kinetics by NSF.

    PubMed

    Schweizer, F E; Dresbach, T; DeBello, W M; O'Connor, V; Augustine, G J; Betz, H

    1998-02-20

    NSF (N-ethylmaleimide-sensitive factor) is an adenosine triphosphatase (ATPase) that contributes to a protein complex essential for membrane fusion. The synaptic function of this protein was investigated by injecting, into the giant presynaptic terminal of squid, peptides that inhibit the ATPase activity of NSF stimulated by the soluble NSF attachment protein (SNAP). These peptides reduced the amount and slowed the kinetics of neurotransmitter release as a result of actions that required vesicle turnover and occurred at a step subsequent to vesicle docking. These results define NSF as an essential participant in synaptic vesicle exocytosis that regulates the kinetics of neurotransmitter release and, thereby, the integrative properties of synapses.

  19. 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

  20. Single-vesicle architecture of synaptobrevin2 in astrocytes

    PubMed Central

    Singh, Priyanka; Jorgačevski, Jernej; Kreft, Marko; Grubišić, Vladimir; Stout, Randy F.; Potokar, Maja; Parpura, Vladimir; Zorec, Robert

    2015-01-01

    Exocytic transmitter release is regulated by the SNARE complex, which contains a vesicular protein, synaptobrevin2 (Sb2). However, Sb2 vesicular arrangement is unclear. Here we use super-resolution fluorescence microscopy to study the prevalence and distribution of endogenous and exogenous Sb2 in single vesicles of astrocytes, the most abundant glial cells in the brain. We tag Sb2 protein at C- and N termini with a pair of fluorophores, which allows us to determine the Sb2 length and geometry. To estimate total number of Sb2 proteins per vesicle and the quantity necessary for the formation of fusion pores, we treat cells with ATP to stimulate Ca2+-dependent exocytosis, increase intracellular alkalinity to enhance the fluorescence presentation of yellow-shifted pHluorin (YpH), appended to the vesicle lumen domain of Sb2, and perform photobleaching of YpH fluorophores. Fluorescence intensity analysis reveals that the total number of endogenous Sb2 units or molecules per vesicle is ≤25. PMID:24807050

  1. Key proteins involved in insulin vesicle exocytosis and secretion

    PubMed Central

    Xiong, Qian-Yin; Yu, Cui; Zhang, Yao; Ling, Liefeng; Wang, Lizhuo; Gao, Jia-Lin

    2017-01-01

    In vivo insulin secretion is predominantly affected by blood glucose concentration, blood concentration of amino acids, gastrointestinal hormones and free nerve functional status, in addition to other factors. Insulin is one of the most important hormones in the body, and its secretion is precisely controlled by nutrients, neurotransmitters and hormones. The insulin exocytosis process is similar to the neurotransmitter release mechanism. There are various types of proteins and lipids that participate in the insulin secretory vesicle fusion process, such as soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, Ras-related proteins and vacuolar-type H+-ATPase (V-ATPase). Notably, the SNARE protein is the molecular basis of exocytotic activity. In the current review, the role of the vesicle membrane proteins (synaptobrevins, vesicle associated membrane proteins and target membrane proteins) and auxiliary proteins (Rab proteins and Munc-18 proteins) in vesicle fusion activity were summarized. A summary of these key proteins involved in insulin granule secretion will facilitate understanding of the pathogenesis of diabetes. PMID:28357064

  2. Single-vesicle architecture of synaptobrevin2 in astrocytes.

    PubMed

    Singh, Priyanka; Jorgačevski, Jernej; Kreft, Marko; Grubišić, Vladimir; Stout, Randy F; Potokar, Maja; Parpura, Vladimir; Zorec, Robert

    2014-05-07

    Exocytic transmitter release is regulated by the SNARE complex, which contains a vesicular protein, synaptobrevin2 (Sb2). However, Sb2 vesicular arrangement is unclear. Here we use super-resolution fluorescence microscopy to study the prevalence and distribution of endogenous and exogenous Sb2 in single vesicles of astrocytes, the most abundant glial cells in the brain. We tag Sb2 protein at C- and N termini with a pair of fluorophores, which allows us to determine the Sb2 length and geometry. To estimate total number of Sb2 proteins per vesicle and the quantity necessary for the formation of fusion pores, we treat cells with ATP to stimulate Ca2+-dependent exocytosis, increase intracellular alkalinity to enhance the fluorescence presentation of yellow-shifted pHluorin (YpH), appended to the vesicle lumen domain of Sb2, and perform photobleaching of YpH fluorophores. Fluorescence intensity analysis reveals that the total number of endogenous Sb2 units or molecules per vesicle is ≤25.

  3. 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

  4. Hydrogen ions control synaptic vesicle ion channel activity in Torpedo electromotor neurones.

    PubMed

    Ahdut-Hacohen, Ronit; Duridanova, Dessislava; Meiri, Halina; Rahamimoff, Rami

    2004-04-15

    During exocytosis the synaptic vesicle fuses with the surface membrane and undergoes a pH jump. When the synaptic vesicle is inside the presynaptic nerve terminal its internal pH is about 5.5 and after fusion, the inside of the vesicle comes in contact with the extracellular medium with a pH of about 7.25. We examined the effect of such pH jump on the opening of the non-specific ion channel in the synaptic vesicle membrane, in the context of the post-fusion hypothesis of transmitter release control. The vesicles were isolated from Torpedo ocellata electromotor neurones. The pH dependence of the opening of the non-specific ion channel was examined using the fused vesicle-attached configuration of the patch clamp technique. The rate of opening depends on both pH and voltage. Increasing the pH from 5.5 to 7.25 activated dramatically the non-specific ion channel of the vesicle membrane. The single channel conductance did not change significantly with the alteration in the pH, and neither did the mean channel open time. These results support the hypothesis that during partial fusion of the vesicle with the surface membrane, ion channels in the vesicle membrane open, admit ions and thus help in the ion exchange process mechanism, leading to the release of the transmitter from the intravesicular ion exchange matrix. This process may have also a pathophysiological significance in conditions of altered pH.

  5. How the stimulus defines the dynamics of vesicle pool recruitment, fusion mode, and vesicle recycling in neuroendocrine cells.

    PubMed

    Cárdenas, Ana María; Marengo, Fernando D

    2016-06-01

    The pattern of stimulation defines important characteristics of the secretory process in neurons and neuroendocrine cells, including the pool of secretory vesicles being recruited, the type and amount of transmitters released, the mode of membrane retrieval, and the mechanisms associated with vesicle replenishment. This review analyzes the mechanisms that regulate these processes in chromaffin cells, as well as in other neuroendocrine and neuronal models. A common factor in these mechanisms is the spatial and temporal distribution of the Ca(2+) signal generated during cell stimulation. For instance, neurosecretory cells and neurons have pools of vesicles with different locations with respect to Ca(2+) channels, and those pools are therefore differentially recruited following different patterns of stimulation. In this regard, a brief stimulus will induce the exocytosis of a small pool of vesicles that is highly coupled to voltage-dependent Ca(2+) channels, whereas longer or more intense stimulation will provoke a global Ca(2+) increase, promoting exocytosis irrespective of vesicle location. The pattern of stimulation, and therefore the characteristics of the Ca(2+) signal generated by the stimulus also influence the mode of exocytosis and the type of endocytosis. Indeed, low-frequency stimulation favors kiss-and-run exocytosis and clathrin-independent fast endocytosis, whereas higher frequencies promote full fusion and clathrin-dependent endocytosis. This latter type of endocytosis is accelerated at high-frequency stimulation. Synaptotagmins, calcineurin, dynamin, complexin, and actin remodeling, appear to be involved in the mechanisms that determine the response of these processes to Ca(2+) . In chromaffin cells, a brief stimulus induces the exocytosis of a small pool of vesicles that is highly coupled to voltage-dependent Ca(2+) channels (A), whereas longer or high-frequency stimulation provokes a global Ca(2+) increase, promoting exocytosis irrespective of

  6. Lipid interaction of Pseudomonas aeruginosa exotoxin A. Acid-triggered permeabilization and aggregation of lipid vesicles.

    PubMed Central

    Menestrina, G; Pederzolli, C; Forti, S; Gambale, F

    1991-01-01

    We have investigated the interaction of Pseudomonas exotoxin A with small unilamellar vesicles comprised of different phospholipids as a function of pH, toxin, and lipid concentration. We have found that this toxin induces vesicle permeabilization, as measured by the release of a fluorescent dye. Permeabilization is due to the formation of ion-conductive channels which we have directly observed in planar lipid bilayers. The toxin also produces vesicle aggregation, as indicated by an increase of the turbidity. Aggregation and permeabilization have completely different time course and extent upon toxin dose and lipid composition, thus suggesting that they are two independent events. Both time constants decrease by lowering the pH of the bulk phase or by introducing a negative lipid into the vesicles. Our results indicate that at least three steps are involved in the interaction of Pseudomonas exotoxin A with lipid vesicles. After protonation of one charged group the toxin becomes competent to bind to the surface of the vesicles. Binding is probably initiated by an electrostatic interaction because it is absolutely dependent on the presence of acidic phospholipids. Binding is a prerequisite for the subsequent insertion of the toxin into the lipid bilayer, with a special preference for phosphatidylglycerol-containing membranes, to form ionic channels. At high toxin and vesicle concentrations, bound toxin may also induce aggregation of the vesicles, particularly when phosphatidic acid is present in the lipid mixture. A quenching of the intrinsic tryptophan fluorescence of the protein, which is induced by lowering the pH of the solution, becomes more drastic in the presence of lipid vesicles. However, this further quenching takes so long that it cannot be a prerequisite to either vesicle permeabilization or aggregation. Pseudomonas exotoxin A shares many of these properties with other bacterial toxins like diphtheria and tetanus toxin. Images FIGURE 7 FIGURE 8 FIGURE 12

  7. Procoagulant and immunogenic properties of melanoma exosomes, microvesicles and apoptotic vesicles

    PubMed Central

    Muhsin-Sharafaldine, Morad-Rémy; Saunderson, Sarah C.; Dunn, Amy C.; Faed, James M.; Kleffmann, Torsten; McLellan, Alexander D.

    2016-01-01

    Extracellular vesicles (EV) are lipid particles released from eukaryotic cells into the extracellular fluid. Depending on the cell type or mechanism of release, vesicles vary in form and function and exert distinct functions in coagulation and immunity. Tumor cells may constitutively shed vesicles known as exosomes or microvesicles (MV). Alternatively, apoptosis induces the release of apoptotic blebs or vesicles (ApoV) from the plasma membrane. EV have been implicated in thrombotic events (the second highest cause of death in cancer patients) and tumor vesicles contribute to the anti-cancer immune response. In this study, we utilized the well characterized B16 melanoma model to determine the molecular composition and procoagulant and immunogenic potential of exosomes, MV and ApoV. Distinct patterns of surface and cytoplasmic molecules (tetraspanins, integrins, heat shock proteins and histones) were expressed between the vesicle types. Moreover, in vitro coagulation assays revealed that membrane-derived vesicles, namely MV and ApoV, were more procoagulant than exosomes–with tissue factor and phosphatidylserine critical for procoagulant activity. Mice immunized with antigen-pulsed ApoV and challenged with B16 tumors were protected out to 60 days, while lower protection rates were afforded by MV and exosomes. Together the results demonstrate distinct phenotypic and functional differences between vesicle types, with important procoagulant and immunogenic functions emerging for membrane-derived MV and ApoV versus endosome-derived exosomes. This study highlights the potential of EV to contribute to the prothrombotic state, as well as to anti-cancer immunity. PMID:27462921

  8. 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.

  9. Extracellular Vesicles and Autophagy in Osteoarthritis

    PubMed Central

    Guo, Weimin; Chen, Mingxue; Huang, Jingxiang; Yuan, Zhiguo; Zhang, Yu; Wang, Mingjie; Li, Penghao; Wang, Aiyuan; Wang, Yu; Sui, Xiang; Zhang, Li; Xu, Wenjing; Lu, Shibi

    2016-01-01

    Osteoarthritis (OA) is a type of chronic joint disease that is characterized by the degeneration and loss of articular cartilage and hyperplasia of the synovium and subchondral bone. There is reasonable knowledge about articular cartilage physiology, biochemistry, and chondrocyte metabolism. However, the etiology and pathogenesis of OA remain unclear and need urgent clarification to guide the early diagnosis and treatment of OA. Extracellular vesicles (EVs) are small membrane-linking particles that are released from cells. In recent decades, several special biological properties have been found in EV, especially in terms of cartilage. Autophagy plays a critical role in the regulation of cellular homeostasis. Likewise, more and more research has gradually focused on the effect of autophagy on chondrocyte proliferation and function in OA. The synthesis and release of EV are closely associated with autophagy. At the same time, both EV and autophagy play a role in OA development. Based on the mechanism of EV and autophagy in OA development, EV may be beneficial in the early diagnosis of OA; on the other hand, the combination of EV and autophagy-related regulatory drugs may provide insight into possible OA therapeutic strategies. PMID:28078284

  10. 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.

  11. Time-coded neurotransmitter release at excitatory and inhibitory synapses

    PubMed Central

    Rodrigues, Serafim; Desroches, Mathieu; Krupa, Martin; Cortes, Jesus M.; Sejnowski, Terrence J.; Ali, Afia B.

    2016-01-01

    Communication between neurons at chemical synapses is regulated by hundreds of different proteins that control the release of neurotransmitter that is packaged in vesicles, transported to an active zone, and released when an input spike occurs. Neurotransmitter can also be released asynchronously, that is, after a delay following the spike, or spontaneously in the absence of a stimulus. The mechanisms underlying asynchronous and spontaneous neurotransmitter release remain elusive. Here, we describe a model of the exocytotic cycle of vesicles at excitatory and inhibitory synapses that accounts for all modes of vesicle release as well as short-term synaptic plasticity (STSP). For asynchronous release, the model predicts a delayed inertial protein unbinding associated with the SNARE complex assembly immediately after vesicle priming. Experiments are proposed to test the model’s molecular predictions for differential exocytosis. The simplicity of the model will also facilitate large-scale simulations of neural circuits. PMID:26858411

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

    PubMed Central

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

    2016-01-01

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

  13. Multiple roles of calcium ions in the regulation of neurotransmitter release.

    PubMed

    Neher, Erwin; Sakaba, Takeshi

    2008-09-25

    The intracellular calcium concentration ([Ca(2+)]) has important roles in the triggering of neurotransmitter release and the regulation of short-term plasticity (STP). Transmitter release is initiated by quite high concentrations within microdomains, while short-term facilitation is strongly influenced by the global buildup of "residual calcium." A global rise in [Ca(2+)] also accelerates the recruitment of release-ready vesicles, thereby controlling the degree of short-term depression (STD) during sustained activity, as well as the recovery of the vesicle pool in periods of rest. We survey data that lead us to propose two distinct roles of [Ca(2+)] in vesicle recruitment: one accelerating "molecular priming" (vesicle docking and the buildup of a release machinery), the other promoting the tight coupling between releasable vesicles and Ca(2+) channels. Such coupling is essential for rendering vesicles sensitive to short [Ca(2+)] transients, generated during action potentials.

  14. 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.

  15. 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

  16. Proteomic analysis of Trypanosoma cruzi secretome: characterization of two populations of extracellular vesicles and soluble proteins.

    PubMed

    Bayer-Santos, Ethel; Aguilar-Bonavides, Clemente; Rodrigues, Silas Pessini; Cordero, Esteban Maurício; Marques, Alexandre Ferreira; Varela-Ramirez, Armando; Choi, Hyungwon; Yoshida, Nobuko; da Silveira, José Franco; Almeida, Igor C

    2013-02-01

    Microorganisms use specialized systems to export virulence factors into host cells. Secretion of effector proteins into the extracellular environment has been described in Trypanosoma cruzi; however, a comprehensive proteomic analysis of the secretome and the secretion mechanisms involved remain elusive. Here, we present evidence that T. cruzi releases proteins associated with vesicles that are formed by at least two different mechanisms. Transmission electron microscopy showed larger vesicles budding from the plasma membrane of noninfective epimastigotes and infective metacyclic trypomastigotes, as well as smaller vesicles within the flagellar pocket of both forms. Parasite conditioned culture supernatant was fractionated and characterized by morphological, immunochemical, and proteomic analyses. Three fractions were obtained by differential ultracentrifugation: the first enriched in larger vesicles resembling ectosomes, the second enriched in smaller vesicles resembling exosomes, and a third fraction enriched in soluble proteins not associated with extracellular vesicles. Label-free quantitative proteomic analysis revealed a rich collection of proteins involved in metabolism, signaling, nucleic acid binding, and parasite survival and virulence. These findings support the notion that T. cruzi uses different secretion pathways to excrete/secrete proteins. Moreover, our results suggest that metacyclic forms may use extracellular vesicles to deliver cargo into host cells.

  17. Purification of a vesicle-vacuole fraction functionally linked to aflatoxin synthesis in Aspergillus parasiticus

    PubMed Central

    CHANDA, Anindya; ROZE, Ludmila; PASTOR, Alicia; FRAME, Melinda; LINZ, John E.

    2009-01-01

    Current studies in our laboratory demonstrate a functional link between vesicles, vacuoles and aflatoxin biosynthesis in the filamentous fungus, Aspergillus parasiticus. Under aflatoxin inducing conditions in liquid yeast-extract sucrose medium, A. parasiticus undergoes a shift from vacuole biogenesis to accumulation of an enhanced number of vesicles which exhibit significant heterogeneity in size and density. As a first step in conducting a detailed analysis of the role of these organelles in aflatoxin synthesis, we developed a novel method to purify the vesicle and vacuole fraction using protoplasts prepared from cells harvested during aflatoxin synthesis. The method includes the following steps: 1] preparation of protoplasts from mycelia grown for 36h under aflatoxin inducing conditions; 2] release of vesicles and vacuoles from purified protoplasts in the presence of Triton X-100; and 3] fractionation of the vesicles and vacuoles using a “one-step high density cushion”. The vesicle-vacuole fraction showed a 35 fold enrichment in alpha-mannosidase activity (vacuole marker) and non-detectable succinate dehydrogenase and lactate dehydrogenase activities (mitochondrial and cytoplasmic markers, respectively). Confocal laser scanning microscopy with the vacuole dyes MDY-64 and CMAC demonstrated that the fraction contained pure vesicles and vacuoles and was devoid of membranous debris. Transmission electron microscopy (TEM) confirmed that no mitochondria or unbroken protoplasts contaminated the purified fraction. The purified organelles exhibited significant size heterogeneity with a range of sizes similar to that observed in whole cells and protoplasts. PMID:19358865

  18. Extracellular vesicles: structure, function, and potential clinical uses in renal diseases

    PubMed Central

    Borges, F.T.; Reis, L.A.; Schor, N.

    2013-01-01

    Interest in the role of extracellular vesicles in various diseases including cancer has been increasing. Extracellular vesicles include microvesicles, exosomes, apoptotic bodies, and argosomes, and are classified by size, content, synthesis, and function. Currently, the best characterized are exosomes and microvesicles. Exosomes are small vesicles (40-100 nm) involved in intercellular communication regardless of the distance between them. They are found in various biological fluids such as plasma, serum, and breast milk, and are formed from multivesicular bodies through the inward budding of the endosome membrane. Microvesicles are 100-1000 nm vesicles released from the cell by the outward budding of the plasma membrane. The therapeutic potential of extracellular vesicles is very broad, with applications including a route of drug delivery and as biomarkers for diagnosis. Extracellular vesicles extracted from stem cells may be used for treatment of many diseases including kidney diseases. This review highlights mechanisms of synthesis and function, and the potential uses of well-characterized extracellular vesicles, mainly exosomes, with a special focus on renal functions and diseases. PMID:24141609

  19. Giant unilamellar vesicles formed by hybrid films of agarose and lipids display altered mechanical properties.

    PubMed

    Lira, Rafael B; Dimova, Rumiana; Riske, Karin A

    2014-10-07

    Giant unilamellar vesicles (GUVs) are presumably the current most popular biomimetic membrane model. Preparation of GUVs in physiological conditions using the classical electroformation method is challenging. To circumvent these difficulties, a new method was recently reported, by which GUVs spontaneously swell from hybrid films of agarose and lipids. However, agarose is left encapsulated in the vesicles in different amounts. In this work, we thoroughly characterize the mechanical properties of these agarose-GUVs in response to electric pulses, which induce vesicle deformation and can lead to membrane poration. We show that the relaxation dynamics of deformed vesicles, both in the presence and absence of poration, is significantly slowed down for agarose-GUVs when compared to agarose-free GUVs. In the presence of poration, agarose polymers prevent complete pore closure and lead to high membrane permeability. A fraction of the vesicles were found to encapsulate agarose in the form of a gel-like meshwork. These vesicles rupture and open up after electroporation and the meshwork is expelled through a macropore. When the agarose-GUVs are heated above the melting temperature of agarose for 2 h before use, vesicle response is (partially) recovered due to substantial release of encapsulated agarose during temperature treatment. Our findings reveal potential artifactual behavior of agarose-GUVs in processes involving morphological changes in the membrane as well as poration.

  20. 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.

  1. 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.

  2. 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

  3. The EARP Complex and Its Interactor EIPR-1 Are Required for Cargo Sorting to Dense-Core Vesicles

    PubMed Central

    Topalidou, Irini; Cattin-Ortolá, Jérôme; MacCoss, Michael J.

    2016-01-01

    The dense-core vesicle is a secretory organelle that mediates the regulated release of peptide hormones, growth factors, and biogenic amines. Dense-core vesicles originate from the trans-Golgi of neurons and neuroendocrine cells, but it is unclear how this specialized organelle is formed and acquires its specific cargos. To identify proteins that act in dense-core vesicle biogenesis, we performed a forward genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We previously reported the identification of two conserved proteins that interact with the small GTPase RAB-2 to control normal dense-core vesicle cargo-sorting. Here we identify several additional conserved factors important for dense-core vesicle cargo sorting: the WD40 domain protein EIPR-1 and the endosome-associated recycling protein (EARP) complex. By assaying behavior and the trafficking of dense-core vesicle cargos, we show that mutants that lack EIPR-1 or EARP have defects in dense-core vesicle cargo-sorting similar to those of mutants in the RAB-2 pathway. Genetic epistasis data indicate that RAB-2, EIPR-1 and EARP function in a common pathway. In addition, using a proteomic approach in rat insulinoma cells, we show that EIPR-1 physically interacts with the EARP complex. Our data suggest that EIPR-1 is a new interactor of the EARP complex and that dense-core vesicle cargo sorting depends on the EARP-dependent trafficking of cargo through an endosomal sorting compartment. PMID:27191843

  4. 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.

  5. 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

  6. Autophagy-associated dengue vesicles promote viral transmission avoiding antibody neutralization

    PubMed Central

    Wu, Yan-Wei; Mettling, Clément; Wu, Shang-Rung; Yu, Chia-Yi; Perng, Guey-Chuen; Lin, Yee-Shin; Lin, Yea-Lih

    2016-01-01

    One of the major defense mechanisms against virus spread in vivo is the blocking of viral infectibility by neutralizing antibodies. We describe here the identification of infectious autophagy-associated dengue vesicles released from infected cells. These vesicles contain viral proteins E, NS1, prM/M, and viral RNA, as well as host lipid droplets and LC3-II, an autophagy marker. The viral RNA can be protected within the autophagic organelles since anti-dengue neutralizing antibodies do not have an effect on the vesicle-mediated transmission that is able to initiate a new round of infection in target cells. Importantly, such infectious vesicles were also detected in a patient serum. Our study suggests that autophagy machinery plays a new role in dengue virus transmission. This discovery explains the inefficiency of neutralizing antibody upon dengue infection as a potential immune evasion mechanism in vivo. PMID:27558165

  7. Therapeutic application of extracellular vesicles in acute and chronic renal injury.

    PubMed

    Rovira, Jordi; Diekmann, Fritz; Campistol, Josep M; Ramírez-Bajo, María José

    2016-07-23

    A new cell-to-cell communication system was discovered in the 1990s, which involves the release of vesicles into the extracellular space. These vesicles shuttle bioactive particles, including proteins, mRNA, miRNA, metabolites, etc. This particular communication has been conserved throughout evolution, which explains why most cell types are capable of producing vesicles. Extracellular vesicles (EVs) are involved in the regulation of different physiological processes, as well as in the development and progression of several diseases. EVs have been widely studied over recent years, especially those produced by embryonic and adult stem cells, blood cells, immune system and nervous system cells, as well as tumour cells. EV analysis from bodily fluids has been used as a diagnostic tool for cancer and recently for different renal diseases. However, this review analyses the importance of EVs generated by stem cells, their function and possible clinical application in renal diseases and kidney transplantation.

  8. Unique Lipid Chemistry of Synaptic Vesicle and Synaptosome Membrane Revealed Using Mass Spectrometry.

    PubMed

    Lewis, Kenneth T; Maddipati, Krishna R; Naik, Akshata R; Jena, Bhanu P

    2017-03-02

    Synaptic vesicles measuring 30-50 nm in diameter containing neurotransmitters either completely collapse at the presynaptic membrane or dock and transiently fuse at the base of specialized 15 nm cup-shaped lipoprotein structures called porosomes at the presynaptic membrane of synaptosomes to release neurotransmitters. Recent study reports the unique composition of major lipids associated with neuronal porosomes. Given that lipids greatly influence the association and functions of membrane proteins, differences in lipid composition of synaptic vesicle and the synaptosome membrane was hypothesized. To test this hypothesis, the lipidome of isolated synaptosome, synaptosome membrane, and synaptic vesicle preparation were determined by using mass spectrometry in the current study. Results from the study demonstrate the enriched presence of triacyl glycerols and sphingomyelins in synaptic vesicles, as opposed to the enriched presence of phospholipids in the synaptosome membrane fraction, reflecting on the tight regulation of nerve cells in compartmentalization of membrane lipids at the nerve terminal.

  9. Rationally Separating the Corona and Membrane Functions of Polymer Vesicles for Enhanced T₂ MRI and Drug Delivery.

    PubMed

    Qin, Jingya; Liu, Qiuming; Zhang, Junxue; Chen, Jing; Chen, Shuai; Zhao, Yao; Du, Jianzhong

    2015-07-01

    It is an important challenge to in situ grow ultrafine super-paramagnetic iron oxide nanoparticles (SPIONs) in drug carriers such as polymer vesicles (also called polymersomes) while keeping their biodegradability for enhanced T2-weighted magnetic resonance imaging (MRI) and drug delivery. Herein, we present a new strategy by rationally separating the corona and membrane functions of polymer vesicles to solve the above problem. We designed a poly(ethylene oxide)-block-poly(ε-caprolactone)-block-poly(acrylic acid) (PEO43-b-PCL98-b-PAA25) triblock copolymer and self-assembled it into polymer vesicle. The PAA chains in the vesicle coronas are responsible for the in situ nanoprecipitation of ultrafine SPIONs, while the vesicle membrane composed of PCL is biodegradable. The SPIONs-decorated vesicle is water-dispersible, biocompatible, and slightly cytotoxic to normal human cells. Dynamic light scattering, transmission electron microscopy, energy disperse spectroscopy, and vibrating sample magnetometer revealed the formation of ultrafine super-paramagnetic Fe3O4 nanoparticles (1.9 ± 0.3 nm) in the coronas of polymer vesicles. Furthermore, the CCK-8 assay revealed low cytotoxicity of vesicles against normal L02 liver cells without and with Fe3O4 nanoparticles. The in vitro and in vivo MRI experiments confirmed the enhanced T2-weighted MRI sensitivity and excellent metastasis in mice. The loading and release experiments of an anticancer drug, doxorubicin hydrochloride (DOX·HCl), indicated that the Fe3O4-decorated magnetic vesicles have potential applications as a nanocarrier for anticancer drug delivery. Moreover, the polymer vesicle is degradable in the presence of enzyme such as Pseudomonas lipases, and the ultrafine Fe3O4 nanoparticles in the vesicle coronas are confirmed to be degradable under weakly acidic conditions. Overall, this decoration-in-vesicle-coronas strategy provides us with a new insight for preparing water-dispersible ultrafine super-paramagnetic Fe3O

  10. 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.

  11. 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 ...

  12. Quantitative analysis of synaptic vesicle pool replenishment in cultured cerebellar granule neurons using FM dyes.

    PubMed

    Cheung, Giselle; Cousin, Michael A

    2011-11-11

    After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis(1,2). The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation(1,2). It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli. FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals(3-8). They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane(9). Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal(10), the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified. Here, we present a protocol that has been modified to

  13. Self-assembly in aqueous solution of wheel-shaped Mo154 oxide clusters into vesicles.

    PubMed

    Liu, Tianbo; Diemann, Ekkehard; Li, Huilin; Dress, Andreas W M; Müller, Achim

    2003-11-06

    Surfactants and membrane lipids readily assemble into complex structures such as micelles, liposomes or hollow vesicles owing to their amphiphilic character-the fact that part of their structure is attracted to polar environments while another part is attracted to non-polar environments. The self-assembly of complex structures also occurs in polyoxometallate chemistry, as exemplified by the molybdenum blue solutions known for centuries. But while the presence of nanometre-sized metal oxide aggregates in these solutions has long been recognized, unravelling the composition and formation process of these aggregates proved difficult. Recent work has indicated that discrete, wheel-shaped mixed-valence polyoxomolybdate clusters of the type [Mo154] (refs 2-4) assemble into well-defined nanometre-sized aggregates, including spherical structures. Here we report light-scattering data and transmission electron microscopy images of hollow spherical structures with an average, almost monodisperse radius of about 45 nm and composed of approximately 1,165 [Mo154] wheel-shaped clusters. The clusters appear to lie flat and homogeneously distributed on the vesicle surface. Unlike conventional lipid vesicles, the structures we observe are not stabilized by hydrophobic interactions. Instead, we believe the polyoxomolybdate-based vesicles form owing to a subtle interplay between short-range van der Waals attraction and long-range electrostatic repulsion, with important further stabilization arising from hydrogen bonding involving water molecules encapsulated between the wheel-shaped clusters and in the vesicles' interior.

  14. Matrix-dependent local retention of secretory vesicle cargo in cortical neurons.

    PubMed

    de Wit, Joris; Toonen, Ruud F; Verhage, Matthijs

    2009-01-07

    Neurons secrete many diffusible signals from synaptic and other secretory vesicles. We characterized secretion of guidance cues, neuropeptides, neurotrophins, and proteases from single secretory vesicles using pHluorin-tagged cargo in cortical neurons. Stimulation triggered transient and persistent fusion events. Transient events represented full release followed by cargo diffusion or incomplete release followed by vesicle retrieval, as previously observed in neuroendocrine cells. Unexpectedly, we also observed that certain cargo, such as Semaphorin 3A (Sema3A), was delivered at the cell surface as stable deposits. Stable deposits and transient events were observed for single cargo and both were SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) and calcium dependent. The ratio between stable and transient events did not depend on cargo size, subcellular localization (synaptic vs extrasynaptic secretion), or the presence of the extracellular matrix. Instead, the ratio is cargo specific and depends on an interaction with the vesicle matrix through a basic domain in the cargo protein. Inhibition of this interaction through deletion of the basic domain in Sema3A abolished stable deposits and rendered all events transient. Strikingly, cargo favoring transient release was stably deposited after corelease with cargo favoring stable deposit. These data argue against cargo diffusion after exocytosis as a general principle. Instead, the vesicle matrix retains secreted signals, probably for focal signaling at the cell surface.

  15. Formation of controllable hydrophilic/hydrophobic drug delivery systems by electrospinning of vesicles.

    PubMed

    Li, Wei; Luo, Tian; Yang, Yanjuan; Tan, Xiuniang; Liu, Lifei

    2015-05-12

    Novel multifunctional poly(ethylene oxide) (PEO) nanofibrous membrane, which contains vesicles constructed by mixed surfactant cetyltrimethylammonium bromide (CTAB)/sodium dodecylbenzenesulfonate (SDBS), has been designed as dual drug-delivery system and fabricated via the electrospinning process. 5-FU and paeonolum, which are hydrophilic and hydrophobic anticancer model drugs, can be dissolved in vesicle solution's bond water and lipid bilayer membranes, respectively. The physicochemical properties of the electrospun nanofibrous membrane were systematically studied using scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared (FTIR), and X-ray diffraction (XRD). Drug release behaviors of the electrospun nanofibrous membrane fabricated with different molar ratio of CTAB/SDBS vesicle solution were investigated. The result showed that the releasing amount of hydrophilic drug presented an ascending release manner, while the hydrophobic one showed a descending release behavior with increasing of the molar ratio of CTAB/SDBS. Moreover, the release amount of drugs from drug delivery system can be controlled by the molar ratio of CTAB/SDBS in the vesicle solution easily and conveniently. The distinct properties can be utilized to encapsulate environmental demanding and quantificational materials.

  16. 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.

  17. Amyloid Precursor Protein Is Trafficked and Secreted via Synaptic Vesicles

    PubMed Central

    Riedel, Dietmar; Hua, Yunfeng; Hüve, Jana; Wilhelm, Benjamin G.; Klingauf, Jürgen

    2011-01-01

    A large body of evidence has implicated amyloid precursor protein (APP) and its proteolytic derivatives as key players in the physiological context of neuronal synaptogenesis and synapse maintenance, as well as in the pathology of Alzheimer's Disease (AD). Although APP processing and release are known to occur in response to neuronal stimulation, the exact mechanism by which APP reaches the neuronal surface is unclear. We now demonstrate that a small but relevant number of synaptic vesicles contain APP, which can be released during neuronal activity, and most likely represent the major exocytic pathway of APP. This novel finding leads us to propose a revised model of presynaptic APP trafficking that reconciles existing knowledge on APP with our present understanding of vesicular release and recycling. PMID:21556148

  18. 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.

  19. 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.

  20. 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.

  1. Outer membrane vesicles as platform vaccine technology

    PubMed Central

    Stork, Michiel; van der Ley, Peter

    2015-01-01

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

  2. 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

  3. Fusion of Endosomes Involved in Synaptic Vesicle Recycling

    PubMed Central

    Holroyd, Claudia; Kistner, Ute; Annaert, Wim; Jahn, Reinhard

    1999-01-01

    Recycling of vesicles of the regulated secretory pathway presumably involves passage through an early endosomal compartment as an intermediate step. To learn more about the involvement of endosomes in the recycling of synaptic and secretory vesicles we studied in vitro fusion of early endosomes derived from pheochromocytoma (PC12) cells. Fusion was not affected by cleavage of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins synaptobrevin and syntaxin 1 that operate at the exocytotic limb of the pathway. Furthermore, fusion was inhibited by the fast Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid but not by the slow Ca2+ chelator EGTA. Endosome fusion was restored by the addition of Ca2+ with an optimum at a free Ca2+ concentration of 0.3 × 10−6 M. Other divalent cations did not substitute for Ca2+. A membrane-permeant EGTA derivative caused inhibition of fusion, which was reversed by addition of Ca2+. We conclude that the fusion of early endosomes participating in the recycling of synaptic and neurosecretory vesicles is mediated by a set of SNAREs distinct from those involved in exocytosis and requires the local release of Ca2+ from the endosomal interior. PMID:10473644

  4. Myeloid Extracellular Vesicles: Messengers from the Demented Brain

    PubMed Central

    Nigro, Annamaria; Colombo, Federico; Casella, Giacomo; Finardi, Annamaria; Verderio, Claudia; Furlan, Roberto

    2016-01-01

    Blood-borne monocyte derived cells play a pivotal, initially unrecognized, role in most central nervous system disorders, including diseases initially classified as purely neurodegenerative (i.e., Alzheimer’s disease, Parkinson’s disease, and ALS). Their trafficking to the brain and spinal cord has been extensively studied in classical neuroinflammatory disorders such as multiple sclerosis. Central nervous system resident myeloid cells, namely microglia and perivascular macrophages, also are in the spotlight of investigations on neurological disorders. Myeloid cells, such as infiltrating macrophages and microglia, have been described as having both protective and destructive features in neurological disorders, thus identification of their functional phenotype during disease evolution would be of paramount importance. Extracellular vesicles, namely exosomes and shed vesicles, are released by virtually any cell type and can be detected and identified in terms of cell origin in biological fluids. They therefore constitute an ideal tool to access information on cells residing in an inaccessible site such as the brain. We will review here available information on extracellular vesicles detection in neurological disorders with special emphasis on neurodegenerative diseases. PMID:26858720

  5. Horizontal Transmission of Cytosolic Sup35 Prions by Extracellular Vesicles

    PubMed Central

    Liu, Shu; Hossinger, André; Hofmann, Julia P.; Denner, Philip

    2016-01-01

    ABSTRACT Prions are infectious protein particles that replicate by templating their aggregated state onto soluble protein of the same type. Originally identified as the causative agent of transmissible spongiform encephalopathies, prions in yeast (Saccharomyces cerevisiae) are epigenetic elements of inheritance that induce phenotypic changes of their host cells. The prototype yeast prion is the translation termination factor Sup35. Prions composed of Sup35 or its modular prion domain NM are heritable and are transmitted vertically to progeny or horizontally during mating. Interestingly, in mammalian cells, protein aggregates derived from yeast Sup35 NM behave as true infectious entities that employ dissemination strategies similar to those of mammalian prions. While transmission is most efficient when cells are in direct contact, we demonstrate here that cytosolic Sup35 NM prions are also released into the extracellular space in association with nanometer-sized membrane vesicles. Importantly, extracellular vesicles are biologically active and are taken up by recipient cells, where they induce self-sustained Sup35 NM protein aggregation. Thus, in mammalian cells, extracellular vesicles can serve as dissemination vehicles for protein-based epigenetic information transfer. PMID:27406566

  6. 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.

  7. 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

  8. 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

  9. Modulation of Synaptic Vesicle Exocytosis in Muscle-Dependent Long-Term Depression at the Amphibian Neuromuscular Junction

    PubMed Central

    Etherington, Sarah J.; Johnstone, Victoria P. A.; Everett, Alan W.

    2014-01-01

    We have labeled recycling synaptic vesicles at the somatic Bufo marinus neuromuscular junction with the styryl dye FM2-10 and provide direct evidence for refractoriness of exocytosis associated with a muscle activity-dependent form of long-term depression (LTD) at this synapse. FM2-10 dye unloading experiments demonstrated that the rate of vesicle exocytosis from the release ready pool (RRP) of vesicles was more than halved in the LTD (induced by 20 min of low frequency stimulation). Recovery from LTD, observed as a partial recovery of nerve-evoked muscle twitch amplitude, was accompanied by partial recovery of the refractoriness of RRP exocytosis. Unexpectedly, paired pulse plasticity, another routinely used indicator of presynaptic forms of synaptic plasticity, was unchanged in the LTD. We conclude that the LTD induces refractoriness of the neuromuscular vesicle release machinery downstream of presynaptic calcium entry. PMID:24489862

  10. A Highly Ca2+-Sensitive Pool of Vesicles Contributes to Linearity at the Rod Photoreceptor Ribbon Synapse

    PubMed Central

    Thoreson, Wallace B.; Rabl, Katalin; Townes-Anderson, Ellen; Heidelberger, Ruth

    2011-01-01

    Summary Studies of the properties of synaptic transmission have been carried out at only a few synapses. We analyzed exocytosis from rod photoreceptors with a combination of physiological and ultrastructural techniques. As at other ribbon synapses, we found that rods exhibited rapid kinetics of release, and the number of vesicles in the releasable pool is comparable to the number of vesicles tethered at ribbon-style active zones. However, unlike other previously studied neurons, we identified a highly Ca2+-sensitive pool of releasable vesicles with a relatively shallow relationship between the rate of exocytosis and [Ca2+]i that is nearly linear over a presumed physiological range of intra-terminal [Ca2+]. The low-order [Ca2+] dependence of release promotes a linear relationship between Ca2+ entry and exocytosis that permits rods to relay information about small changes in illumination with high fidelity at the first synapse in vision. PMID:15157421

  11. Formulation and Characterization of Drug Loaded Nonionic Surfactant Vesicles (Niosomes) for Oral Bioavailability Enhancement

    PubMed Central

    Kamboj, Sunil; Saini, Vipin; Bala, Suman

    2014-01-01

    Nonionic surfactant vesicles (niosomes) were formulated with an aim of enhancing the oral bioavailability of tenofovir disoproxil fumarate (TDF), an anti-HIV drug. Niosomes were formulated by conventional thin film hydration technique with different molar ratios of surfactant, cholesterol, and dicetyl phosphate. The formulated niosomes were found spherical in shape, ranging from 2.95 μm to 10.91 μm in size. Vesicles with 1 : 1 : 0.1 ratios of surfactant : cholesterol : dicetyl phosphate with each grade of span were found to have higher entrapment efficiencies, which were further selected for in vitro and in vivo studies. Vesicles formulated with sorbitan monostearate were found to have maximum drug release (99.091%) at the end of 24 hours and followed zero order release kinetics. The results of in vivo study revealed that the niosomes significantly enhanced the oral bioavailability of TDF in rats after a dose of 95 mg/kg. The average relative bioavailability of niosomes in relation to plane drug solution was found to be 2.58, indicating more than twofold increase in oral bioavailability of TDF. Significant increase in mean residential time (MRT) was also found, reflecting release retarding efficacy of the vesicles. In conclusion, niosomes could be a promising delivery for TDF with improved oral bioavailability and prolonged release profiles. PMID:24672401

  12. Mechanical tension contributes to clustering of neurotransmitter vesicles at presynaptic terminals

    PubMed Central

    Siechen, Scott; Yang, Shengyuan; Chiba, Akira; Saif, Taher

    2009-01-01

    Memory and learning in animals are mediated by neurotransmitters that are released from vesicles clustered at the synapse. As a synapse is used more frequently, its neurotransmission efficiency increases, partly because of increased vesicle clustering in the presynaptic neuron. Vesicle clustering has been believed to result primarily from biochemical signaling processes that require the connectivity of the presynaptic terminal with the cell body, the central nervous system, and the postsynaptic cell. Our in vivo experiments on the embryonic Drosophila nervous system show that vesicle clustering at the neuromuscular presynaptic terminal depends on mechanical tension within the axons. Vesicle clustering vanishes upon severing the axon from the cell body, but is restored when mechanical tension is applied to the severed end of the axon. Clustering increases when intact axons are stretched mechanically by pulling the postsynaptic muscle. Using micro mechanical force sensors, we find that embryonic axons that have formed neuromuscular junctions maintain a rest tension of ≈1 nanonewton. If the rest tension is perturbed mechanically, axons restore the rest tension either by relaxing or by contracting over a period of ≈15 min. Our results suggest that neuromuscular synapses employ mechanical tension as a signal to modulate vesicle accumulation and synaptic plasticity. PMID:19620718

  13. AHNAK enables mammary carcinoma cells to produce extracellular vesicles that increase neighboring fibroblast cell motility

    PubMed Central

    Dzik, Luciana M.; Iglesia, Rebeca P.; Cruz, Mário C.; Zelanis, André; de Siqueira, Adriane S.; Serrano, Solange M.T.; Goldberg, Gary S.; Jaeger, Ruy G.; Freitas, Vanessa M.

    2016-01-01

    Extracellular vesicles play important roles in tumor development. Many components of these structures, including microvesicles and exosomes, have been defined. However, mechanisms by which extracellular vesicles affect tumor progression are not fully understood. Here, we investigated vesicular communication between mammary carcinoma cells and neighboring nontransformed mammary fibroblasts. Nonbiased proteomic analysis found that over 1% of the entire proteome is represented in these vesicles, with the neuroblast differentiation associated protein AHNAK and annexin A2 being the most abundant. In particular, AHNAK was found to be the most prominent component of these vesicles based on peptide number, and appeared necessary for their formation. In addition, we report here that carcinoma cells produce vesicles that promote the migration of recipient fibroblasts. These data suggest that AHNAK enables mammary carcinoma cells to produce and release extracellular vesicles that cause disruption of the stroma by surrounding fibroblasts. This paradigm reveals fundamental mechanisms by which vesicular communication between carcinoma cells and stromal cells can promote cancer progression in the tumor microenvironment. PMID:27374178

  14. Interbilayer-crosslinked multilamellar vesicles as synthetic vaccines for potent humoral and cellular immune responses

    NASA Astrophysics Data System (ADS)

    Moon, James J.; Suh, Heikyung; Bershteyn, Anna; Stephan, Matthias T.; Liu, Haipeng; Huang, Bonnie; Sohail, Mashaal; Luo, Samantha; Ho Um, Soong; Khant, Htet; Goodwin, Jessica T.; Ramos, Jenelyn; Chiu, Wah; Irvine, Darrell J.

    2011-03-01

    Vaccines based on recombinant proteins avoid the toxicity and antivector immunity associated with live vaccine (for example, viral) vectors, but their immunogenicity is poor, particularly for CD8+ T-cell responses. Synthetic particles carrying antigens and adjuvant molecules have been developed to enhance subunit vaccines, but in general these materials have failed to elicit CD8+ T-cell responses comparable to those for live vectors in preclinical animal models. Here, we describe interbilayer-crosslinked multilamellar vesicles formed by crosslinking headgroups of adjacent lipid bilayers within multilamellar vesicles. Interbilayer-crosslinked vesicles stably entrapped protein antigens in the vesicle core and lipid-based immunostimulatory molecules in the vesicle walls under extracellular conditions, but exhibited rapid release in the presence of endolysosomal lipases. We found that these antigen/adjuvant-carrying vesicles form an extremely potent whole-protein vaccine, eliciting endogenous T-cell and antibody responses comparable to those for the strongest vaccine vectors. These materials should enable a range of subunit vaccines and provide new possibilities for therapeutic protein delivery.

  15. Dimensional characterization of extracellular vesicles using atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Sebaihi, N.; De Boeck, B.; Yuana, Y.; Nieuwland, R.; Pétry, J.

    2017-03-01

    Extracellular vesicles (EV) are small biological entities released from cells into body fluids. EV are recognized as mediators in intercellular communication and influence important physiological processes. It has been shown that the concentration and composition of EV in body fluids may differ from healthy subjects to patients suffering from particular disease. So, EV have gained a strong scientific and clinical interest as potential biomarkers for diagnosis and prognosis of disease. Due to their small size, accurate detection and characterization of EV remain challenging. The aim of the presented work is to propose a characterization method of erythrocyte-derived EV using atomic force microscopy (AFM). The vesicles are immobilized on anti-CD235a-modified mica and analyzed by AFM under buffer liquid and dry conditions. EV detected under both conditions show very similar sizes namely ~30 nm high and ~90 nm wide. The size of these vesicles remains stable over drying time as long as 7 d at room temperature. Since the detected vesicles are not spherical, EV are characterized by their height and diameter, and not only by the height as is usually done for spherical nanoparticles. In order to obtain an accurate measurement of EV diameters, the geometry of the AFM tip was evaluated to account for the lateral broadening artifact inherent to AFM measurements. To do so, spherical polystyrene (PS) nanobeads and EV were concomitantly deposited on the same mica substrate and simultaneously measured by AFM under dry conditions. By applying this procedure, direct calibration of the AFM tip could be performed together with EV characterization under identical experimental conditions minimizing external sources of uncertainty on the shape and size of the tip, thus allowing standardization of EV measurement.

  16. 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.

  17. The Art Recipe Book, Volume One: 60 Non-toxic Art Materials from Readily Available Materials.

    ERIC Educational Resources Information Center

    Janeczko, Donna

    This collection of recipes is intended for art teachers to provide low-cost, non-toxic materials for classroom use. The materials needed are readily available and can be purchased in quantity to help the budget conscious teacher. Recipes included are for modeling materials, edible modeling materials, paints and inks, adhesives, fixatives, and…

  18. 16 CFR 1101.13 - Public ability to ascertain readily identity of manufacturer or private labeler.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 16 Commercial Practices 2 2010-01-01 2010-01-01 false Public ability to ascertain readily identity of manufacturer or private labeler. 1101.13 Section 1101.13 Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION CONSUMER PRODUCT SAFETY ACT REGULATIONS INFORMATION DISCLOSURE UNDER SECTION 6(b) OF THE CONSUMER PRODUCT SAFETY ACT...

  19. 33 CFR 76.01-1 - Sale of equipment not readily procurable.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... SECURITY AIDS TO NAVIGATION SALE AND TRANSFER OF AIDS TO NAVIGATION EQUIPMENT Sale of Equipment § 76.01-1 Sale of equipment not readily procurable. The Commandant is authorized to sell aids to navigation... to maintain private aids to navigation to mark wrecks, piers, or other obstructions;...

  20. 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.

  1. 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.

  2. 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.

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

    PubMed Central

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

    2013-01-01

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

  4. Supramolecular Vesicles Coassembled from Disulfide-Linked Benzimidazolium Amphiphiles and Carboxylate-Substituted Pillar[6]arenes that Are Responsive to Five Stimuli.

    PubMed

    Jiang, Long; Huang, Xuan; Chen, Dong; Yan, Hua; Li, Xueyuan; Du, Xuezhong

    2017-03-01

    Novel supramolecular vesicles based on host-guest systems were coassembled from carboxylate-substituted pillar[6]arene (CPA[6]) and disulfide-linked benzimidazolium amphiphiles, and the microstructures of the CPA-based supramolecular vesicles were clearly elaborated. The supramolecular vesicles showed controlled drug release in response to five stimuli, with glutathione, pH, CO2 , Zn(2+) ions, and hexanediamine, leading to cleavage of the disulfide bonds, protonation of the carboxylate groups, metal chelation, and competitive binding. This is the first case of a smart pillararene-based supramolecular vesicle being integrated with five stimuli-responsive functions to meet the diverse requirements of controlled drug release. Importantly, each of the five stimuli is closely related to microenvironments of tumors and diseases of the human body. The smart stimuli-responsive supramolecular vesicles have promising applications in drug therapy of tumors and relevant diseases.

  5. 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

  6. 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

  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 from infected cells: potential for direct pathogenesis

    PubMed Central

    Schwab, Angela; Meyering, Shabana S.; Lepene, Ben; Iordanskiy, Sergey; van Hoek, Monique L.; Hakami, Ramin M.; Kashanchi, Fatah

    2015-01-01

    Infections that result in natural or manmade spread of lethal biological agents are a concern and require national and focused preparedness. In this manuscript, as part of an early diagnostics and pathogen treatment strategy, we have focused on extracellular vesicles (EVs) that arise following infections. Although the field of biodefense does not currently have a rich resource in EVs literature, none the less, similar pathogens belonging to the more classical emerging and non-emerging diseases have been studied in their EV/exosomal contents and function. These exosomes are formed in late endosomes and released from the cell membrane in almost every cell type in vivo. These vesicles contain proteins, RNA, and lipids from the cells they originate from and function in development, signal transduction, cell survival, and transfer of infectious material. The current review focuses on how different forms of infection exploit the exosomal pathway and how exosomes can be exploited artificially to treat infection and disease and potentially also be used as a source of vaccine. Virally-infected cells can secrete viral as well as cellular proteins and RNA in exosomes, allowing viruses to cause latent infection and spread of miRNA to nearby cells prior to a subsequent infection. In addition to virally-infected host cells, bacteria, protozoa, and fungi can all release small vesicles that contain pathogen-associated molecular patterns, regulating the neighboring uninfected cells. Examples of exosomes from both virally and bacterially infected cells point toward a re-programming network of pathways in the recipient cells. Finally, many of these exosomes contain cytokines and miRNAs that in turn can effect gene expression in the recipient cells through the classical toll-like receptor and NFκB pathway. Therefore, although exosomes do not replicate as an independent entity, they however facilitate movement of infectious material through tissues and may be the cause of many

  9. 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).

  10. Autophagy modulates articular cartilage vesicle formation in primary articular chondrocytes.

    PubMed

    Rosenthal, Ann K; Gohr, Claudia M; Mitton-Fitzgerald, Elizabeth; Grewal, Rupinder; Ninomiya, James; Coyne, Carolyn B; Jackson, William T

    2015-05-22

    Chondrocyte-derived extracellular organelles known as articular cartilage vesicles (ACVs) participate in non-classical protein secretion, intercellular communication, and pathologic calcification. Factors affecting ACV formation and release remain poorly characterized; although in some cell types, the generation of extracellular vesicles is associated with up-regulation of autophagy. We sought to determine the role of autophagy in ACV production by primary articular chondrocytes. Using an innovative dynamic model with a light scatter nanoparticle counting apparatus, we determined the effects of autophagy modulators on ACV number and content in conditioned medium from normal adult porcine and human osteoarthritic chondrocytes. Healthy articular chondrocytes release ACVs into conditioned medium and show significant levels of ongoing autophagy. Rapamycin, which promotes autophagy, increased ACV numbers in a dose- and time-dependent manner associated with increased levels of autophagy markers and autophagosome formation. These effects were suppressed by pharmacologic autophagy inhibitors and short interfering RNA for ATG5. Caspase-3 inhibition and a Rho/ROCK inhibitor prevented rapamycin-induced increases in ACV number. Osteoarthritic chondrocytes, which are deficient in autophagy, did not increase ACV number in response to rapamycin. SMER28, which induces autophagy via an mTOR-independent mechanism, also increased ACV number. ACVs induced under all conditions had similar ecto-enzyme specific activities and types of RNA, and all ACVs contained LC3, an autophagosome-resident protein. These findings identify autophagy as a critical participant in ACV formation, and augment our understanding of ACVs in cartilage disease and repair.

  11. The small GTPase Cdc42 modulates the number of exocytosis-competent dense-core vesicles in PC12 cells

    SciTech Connect

    Sato, Mai; Kitaguchi, Tetsuya; Ikematsu, Kazuya; Kakeyama, Masaki; Murata, Masayuki; Sato, Ken; Tsuboi, Takashi

    2012-04-06

    Highlights: Black-Right-Pointing-Pointer Regulation of exocytosis by Rho GTPase Cdc42. Black-Right-Pointing-Pointer Cdc42 increases the number of fusion events from newly recruited vesicles. Black-Right-Pointing-Pointer Cdc42 increases the number of exocytosis-competent dense-core vesicles. -- Abstract: Although the small GTPase Rho family Cdc42 has been shown to facilitate exocytosis through increasing the amount of hormones released, the precise mechanisms regulating the quantity of hormones released on exocytosis are not well understood. Here we show by live cell imaging analysis under TIRF microscope and immunocytochemical analysis under confocal microscope that Cdc42 modulated the number of fusion events and the number of dense-core vesicles produced in the cells. Overexpression of a wild-type or constitutively-active form of Cdc42 strongly facilitated high-KCl-induced exocytosis from the newly recruited plasma membrane vesicles in PC12 cells. By contrast, a dominant-negative form of Cdc42 inhibited exocytosis from both the newly recruited and previously docked plasma membrane vesicles. The number of intracellular dense-core vesicles was increased by the overexpression of both a wild-type and constitutively-active form of Cdc42. Consistently, activation of Cdc42 by overexpression of Tuba, a Golgi-associated guanine nucleotide exchange factor for Cdc42 increased the number of intracellular dense-core vesicles, whereas inhibition of Cdc42 by overexpression of the Cdc42/Rac interactive binding domain of neuronal Wiskott-Aldrich syndrome protein decreased the number of them. These findings suggest that Cdc42 facilitates exocytosis by modulating both the number of exocytosis-competent dense-core vesicles and the production of dense-core vesicles in PC12 cells.

  12. Extracellular Vesicles: Composition, Biological Relevance, and Methods of Study

    PubMed Central

    Zaborowski, MikoŁaj P.; Balaj, Leonora; Breakefield, Xandra O.; Lai, Charles P.

    2015-01-01

    The release of extracellular vesicles (EVs), including exosomes and microvesicles, is a phenomenon shared by many cell types as a means of communicating with other cells and also potentially removing cell contents. The cargo of EVs includes the proteins, lipids, nucleic acids, and membrane receptors of the cells from which they originate. EVs released into the extracellular space can enter body fluids and potentially reach distant tissues. Once taken up by neighboring and/or distal cells, EVs can transfer functional cargo that may alter the status of recipient cells, thereby contributing to both physiological and pathological processes. In this article, we will focus on EV composition, mechanisms of uptake, and their biological effects on recipient cells. We will also discuss established and recently developed methods used to study EVs, including isolation, quantification, labeling and imaging protocols, as well as RNA analysis. PMID:26955082

  13. Pharmacology of neurotransmitter release: measuring exocytosis.

    PubMed

    Khvotchev, Mikhail; Kavalali, Ege T

    2008-01-01

    Neurotransmission in the nervous system is initiated at presynaptic terminals by fusion of synaptic vesicles with the plasma membrane and subsequent exocytic release of chemical transmitters. Currently, there are multiple methods to detect neurotransmitter release from nerve terminals, each with their own particular advantages and disadvantages. For instance, most commonly employed methods monitor actions of released chemical substances on postsynaptic receptors or artificial substrates such as carbon fibers. These methods are closest to the physiological setting because they have a rapid time resolution and they measure the action of the endogenous neurotransmitters rather than the signals emitted by exogenous probes. However, postsynaptic receptors only indirectly report neurotransmitter release in a form modified by the properties of receptors themselves, which are often nonlinear detectors of released substances. Alternatively, released chemical substances can be detected biochemically, albeit on a time scale slower than electrophysiological methods. In addition, in certain preparations, where presynaptic terminals are accessible to whole cell recording electrodes, fusion of vesicles with the plasma membrane can be monitored using capacitance measurements. In the last decade, in addition to electrophysiological and biochemical methods, several fluorescence imaging modalities have been introduced which report synaptic vesicle fusion, endocytosis, and recycling. These methods either take advantage of styryl dyes that can be loaded into recycling vesicles or exogenous expression of synaptic vesicle proteins tagged with a pH-sensitive GFP variant at regions facing the vesicle lumen. In this chapter, we will provide an overview of these methods with particular emphasis on their relative strengths and weaknesses and discuss the types of information one can obtain from them.

  14. Characteristics of minerals in vesicles produced by human osteoblasts hFOB 1.19 and osteosarcoma Saos-2 cells stimulated for mineralization.

    PubMed

    Strzelecka-Kiliszek, Agnieszka; Bozycki, Lukasz; Mebarek, Saida; Buchet, Rene; Pikula, Slawomir

    2017-03-29

    Bone cells control initial steps of mineralization by producing extracellular matrix (ECM) proteins and releasing vesicles that trigger apatite nucleation. Using transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) we compared the quality of minerals in vesicles produced by two distinct human cell lines: fetal osteoblastic hFOB 1.19 and osteosarcoma Saos-2. Both cell lines, subjected to osteogenic medium with ascorbic acid (AA) and β-glycerophosphate (β-GP), undergo the entire osteoblastic differentiation program from proliferation to mineralization, produce the ECM and spontaneously release vesicles. We observed that Saos-2 cells mineralized better than hFOB 1.19, as probed by Alizarin Red-S (AR-S) staining, tissue nonspecific alkaline phosphatase (TNAP) activity and by analyzing the composition of minerals in vesicles. Vesicles released from Saos-2 cells contained and were surrounded by more minerals than vesicles released from hFOB 1.19. In addition, there were more F and Cl substituted apatites in vesicles from hFOB 1.19 than in those from Saos-2 cells as determined by ion ratios. Saos-2 and h-FOB 1.19 cells revealed distinct mineralization profiles, indicating that the process of mineralization may proceed differently in various types of cells. Our findings suggest that TNAP activity is correlated with the relative proportions of mineral-filled vesicles and mineral-surrounded vesicles. The origin of vesicles and their properties predetermine the onset of mineralization at the cellular level.

  15. A heterogeneous "resting" pool of synaptic vesicles that is dynamically interchanged across boutons in mammalian CNS synapses.

    PubMed

    Fernandez-Alfonso, Tomas; Ryan, Timothy A

    2008-08-01

    Using pHluorin-tagged synaptic vesicle proteins we have examined the partitioning of these probes into recycling and nonrecycling pools at hippocampal nerve terminals in cell culture. Our studies show that for three of the major synaptic vesicle components, vGlut-1, VAMP-2, and Synaptotagmin I, approximately 50-60% of the tagged protein appears in a recycling pool that responds readily to sustained action potential stimulation by mobilizing and fusing with the plasma membrane, while the remainder is targeted to a nonrecycling, acidic compartment. The fraction of recycling and nonrecycling (or resting) pools varied significantly across boutons within an individual axon, from 100% resting (silent) to 100% recycling. Single-bouton bleaching studies show that recycling and resting pools are dynamic and exchange between synaptic boutons. The quantitative parameters that can be extracted with the approaches outlined here should help elucidate the potential functional role of the resting vesicle pool.

  16. 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

  17. 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.

  18. Cytarabine-AOT catanionic vesicle-loaded biodegradable thermosensitive hydrogel as an efficient cytarabine delivery system.

    PubMed

    Liu, Jing; Jiang, Yue; Cui, Yuting; Xu, Chuanshan; Ji, Xiaoqing; Luan, Yuxia

    2014-10-01

    Carrier with high drug loading content is one of the most important issues in drug delivery system. In the present work, an ion-pair amphiphilic molecule composed of anticancer drug cation and surfactant anion is used for straightforward fabricating vesicles for cancer therapy. Anticancer drug (cytarabine hydrochloride) and anionic surfactant (AOT) are selected for the fabrication of ion-pair amphiphilic molecule. One amphiphilic molecule contains one drug cation, thus the drug loading content is 50% (mol/mol) in theory. The in vitro drug release study shows that the release time of cytarabine is about 3 times of the pure cytarabine solution and the permeability of cytarabine has been improved about 160 times tested by parallel artificial membrane permeability assay model. However, the hemolytic toxicity is largely decreased in the studied concentration range. The in vitro cytotoxicity results show that cytarabine-AOT amphiphiles have a much lower IC50 (drug concentration resulting in 50% cell death) value and a higher cell inhibition rate comparing with their respective components, indicating its effective therapy for leukemic cells. To obtain a longer and a convenient drug release system, the prepared vesicles are further incorporated into the thermosensitive PLGA-PEG-PLGA hydrogel to prepare a subcutaneous administration. The in vivo drug release results indicate that cytarabine-AOT vesicle-loaded hydrogel is a good injectable delivery system for controlled release of cytarabine for cancer therapy.

  19. Effects of boldine on mouse diaphragm and sarcoplasmic reticulum vesicles isolated from skeletal muscle.

    PubMed

    Kang, J J; Cheng, Y W

    1998-02-01

    The effects of boldine [(S)-2,9-dihydroxy-1,10-dimethoxyaporphine], a major alkaloid in the leaves and bark of boldo (Peumus boldus Mol.), on skeletal muscle were studied using mouse diaphragm and isolated sarcoplasmic reticulum membrane vesicles. Boldine, at 10-200 microM, has little effect on the muscle-evoked twitches; however, the ryanodine-induced contracture was potentiated dose-dependently. At higher concentrations of 300 microM, boldine by itself induced muscle contracture of two phases, which were caused by the influx of extracellular Ca2+ and induction of Ca2+ release from the internal Ca2+ storage site, the sarcoplasmic reticulum, respectively. When tested with isolated sarcoplasmic reticulum membrane vesicles, boldine dose-dependently induced Ca2+ release from actively loaded sarcoplasmic reticulum vesicles isolated from skeletal muscle of rabbit or rat which was inhibited by ruthenium red, suggesting that the release was through the Ca2+ release channel, also known as the ryanodine receptor. Boldine also dose-dependently increased apparent [3H]-ryanodine binding with the EC50 value of 50 microM. In conclusion, we have shown that boldine could sensitize the ryanodine receptor and induce Ca2+ release from the internal Ca2+ storage site of skeletal muscle.

  20. 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.

  1. 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.

  2. Structure of a micropipette-aspirated vesicle determined from the bending-energy model.

    PubMed

    Chen, Jeff Z Y

    2012-10-01

    The structure of the system consisting of an aspirating pipette and an aspirated vesicle is investigated with fixed total vesicle volume, total vesicle surface area, and aspirated volume fraction, based on the bending-energy model. Through an energetic consideration, the usage of an aspirated volume fraction can be converted to the aspirating pressure for the determination of a phase diagram; the procedure identifies a first-order transition, between a weakly aspirated state and the strongly aspirated state, as the pressure increases. The physical properties of the system are obtained from minimization of the bending energy by an implementation of the simulated annealing Monte Carlo procedure, which searches for a minimum in a multivariable space. An analysis of the hysteresis effects indicates that the experimentally observed aspirating and releasing critical pressures are related to the location of the spinodal points.

  3. 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.

  4. Homologs of the yeast Tvp38 vesicle-associated protein are conserved in chloroplasts and cyanobacteria

    PubMed Central

    Keller, Rebecca; Schneider, Dirk

    2013-01-01

    Vesicle transfer processes in eukaryotes depend on specific proteins, which mediate the selective packing of cargo molecules for subsequent release out of the cells after vesicle fusion to the plasma membrane. The protein Tvp38 is conserved in yeasts and higher eukaryotes and potentially involved in vesicle transfer processes at the Golgi membrane. Members of the so-called “SNARE-associated proteins of the Tvp38-family” have also been identified in prokaryotes and those belong to the DedA protein family. Tvp38/DedA proteins are also conserved in cyanobacteria and chloroplasts. While only a single member of this family appears to be present in chloroplasts, cyanobacterial genomes typically encode multiple homologous proteins. Mainly based on our understanding of the DedA-homologous proteins of Escherichia coli, it appears likely that the function of these proteins in chloroplast and cyanobacteria involves stabilizing and organizing the structure of internal membrane systems. PMID:24312110

  5. Metformin loaded non-ionic surfactant vesicles: optimization of formulation, effect of process variables and characterization

    PubMed Central

    2013-01-01

    Background Metformin an oral hypoglycemic has been widely used as a fist line of treatment of Type II Diabetes but in a very high dose 2–3 times a day and moreover suffers from a number of side effects like lactic acidosis, gastric discomfort, chest pain, allergic reactions being some of them. The present work was conducted with the aim of sustaining the release of metformin so as to decrease its side effects and also reduce its dosing frequency using a novel delivery system niosomes (non-ionic surfactant vesicles). Non-ionic surfactant vesicles of different surfactants were prepared using thin film hydration technique and were investigated for morphology, entrapment, in-vitro release, TEM (transmission electron microscopy) and physical stability. Optimized formulation was further studied for the effect of Surfactant concentration, DCP (Dicetyl phosphate), Surfactant: cholesterol ratio and volume of hydration. The release studies data was subjected to release kinetics models. Results The prepared vesicles were uniform and spherical in size. Optimized formulation MN3 entrapped the drug with 84.50±0.184 efficiency in the vesicles of the size 487.60±2.646 and showed the most sustained release of 73.89±0.126. Also it was resulted that 100 molar concentration of cholesterol and surfactant, Presence of DCP, equimolar ratio of span 60: cholesterol and 15 ml of volume of hydration were found to be optimum for miosome preparation. Conclusions The present work concluded metformin loaded niosomes to be effective in sustaining the drug release leading to decreased side effects and increased patient compliance. PMID:23351604

  6. 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

  7. Visualization of SNARE-Mediated Hemifusion between Giant Unilamellar Vesicles Arrested by Myricetin

    PubMed Central

    Heo, Paul; Park, Joon-Bum; Shin, Yeon-Kyun; Kweon, Dae-Hyuk

    2017-01-01

    Neurotransmitters are released within a millisecond after Ca2+ arrives at an active zone. However, the vesicle fusion pathway underlying this synchronous release is yet to be understood. At the center of controversy is whether hemifusion, in which outer leaflets are merged while inner leaflets are still separated, is an on-pathway or off-pathway product of Ca2+-triggered exocytosis. Using the single vesicle fusion assay, we recently demonstrated that hemifusion is an on-pathway intermediate that immediately proceeds to full fusion upon Ca2+ triggering. It has been shown that the flavonoid myricetin arrests soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE)-mediated vesicle fusion at hemifusion, but that the hemifused vesicles spontaneously convert to full fusion when the myricetin clamp is removed by the enzyme laccase. In the present study, we visualized SNARE-mediated hemifusion between two SNARE-reconstituted giant unilamellar vesicles (GUVs) arrested by myricetin. The large size of the GUVs enabled us to directly image the hemifusion between them. When two merging GUVs were labeled with different fluorescent dyes, GUV pairs showed asymmetric fluorescence intensities depending on the position on the GUV pair consistent with what is expected for hemifusion. The flow of lipids from one vesicle to the other was revealed with fluorescence recovery after photobleaching (FRAP), indicating that the two membranes had hemifused. These results support the hypothesis that hemifusion may be the molecular status that primes Ca2+-triggered millisecond exocytosis. This study represents the first imaging of SNARE-driven hemifusion between GUVs.

  8. Necessity to adapt land use and land cover classification systems to readily accept radar data

    NASA Technical Reports Server (NTRS)

    Drake, B.

    1977-01-01

    A hierarchial, four level, standardized system for classifying land use/land cover primarily from remote-sensor data (USGS system) is described. The USGS system was developed for nonmicrowave imaging sensors such as camera systems and line scanners. The USGS system is not compatible with the land use/land cover classifications at different levels that can be made from radar imagery, and particularly from synthetic-aperture radar (SAR) imagery. The use of radar imagery for classifying land use/land cover at different levels is discussed, and a possible revision of the USGS system to more readily accept land use/land cover classifications from radar imagery is proposed.

  9. Efficient synthesis and reaction kinetics of readily water soluble esters containing sulfonic groups.

    PubMed

    Idzik, Krzysztof R; Nödler, Karsten; Maier, Friedrich; Licha, Tobias

    2014-12-15

    A series of various readily water soluble esters were synthesized by a very efficient procedure. These compounds can be useful as thermosensitive tracers for studying the cooling progress in a low enthalpy georeservoir exploitable by double flash geothermal power plant systems. The kinetics of their hydrolysis was investigated. Acylation of primary alcohols or phenols was carried out by a method based on a single-phase solvent system consisting of ethyl acetate acting as an organic solvent and triethylamine acting as a catalyst. Products were characterized by 1H-NMR, and 13C-NMR.

  10. Myosin light chain kinase accelerates vesicle endocytosis at the calyx of Held synapse.

    PubMed

    Yue, Hai-Yuan; Xu, Jianhua

    2014-01-01

    Neuronal activity triggers endocytosis at synaptic terminals to retrieve efficiently the exocytosed vesicle membrane, ensuring the membrane homeostasis of active zones and the continuous supply of releasable vesicles. The kinetics of endocytosis depends on Ca(2+) and calmodulin which, as a versatile signal pathway, can activate a broad spectrum of downstream targets, including myosin light chain kinase (MLCK). MLCK is known to regulate vesicle trafficking and synaptic transmission, but whether this kinase regulates vesicle endocytosis at synapses remains elusive. We investigated this issue at the rat calyx of Held synapse, where previous studies using whole-cell membrane capacitance measurement have characterized two common forms of Ca(2+)/calmodulin-dependent endocytosis, i.e., slow clathrin-dependent endocytosis and rapid endocytosis. Acute inhibition of MLCK with pharmacological agents was found to slow down the kinetics of both slow and rapid forms of endocytosis at calyces. Similar impairment of endocytosis occurred when blocking myosin II, a motor protein that can be phosphorylated upon MLCK activation. The inhibition of endocytosis was not accompanied by a change in Ca(2+) channel current. Combined inhibition of MLCK and calmodulin did not induce synergistic inhibition of endocytosis. Together, our results suggest that activation of MLCK accelerates both slow and rapid forms of vesicle endocytosis at nerve terminals, likely by functioning downstream of Ca(2+)/calmodulin.

  11. Dual-mode of insulin action controls GLUT4 vesicle exocytosis.

    PubMed

    Xu, Yingke; Rubin, Bradley R; Orme, Charisse M; Karpikov, Alexander; Yu, Chenfei; Bogan, Jonathan S; Toomre, Derek K

    2011-05-16

    Insulin stimulates translocation of GLUT4 storage vesicles (GSVs) to the surface of adipocytes, but precisely where insulin acts is controversial. Here we quantify the size, dynamics, and frequency of single vesicle exocytosis in 3T3-L1 adipocytes. We use a new GSV reporter, VAMP2-pHluorin, and bypass insulin signaling by disrupting the GLUT4-retention protein TUG. Remarkably, in unstimulated TUG-depleted cells, the exocytic rate is similar to that in insulin-stimulated control cells. In TUG-depleted cells, insulin triggers a transient, twofold burst of exocytosis. Surprisingly, insulin promotes fusion pore expansion, blocked by acute perturbation of phospholipase D, which reflects both properties intrinsic to the mobilized vesicles and a novel regulatory site at the fusion pore itself. Prolonged stimulation causes cargo to switch from approximately 60 nm GSVs to larger exocytic vesicles characteristic of endosomes. Our results support a model whereby insulin promotes exocytic flux primarily by releasing an intracellular brake, but also by accelerating plasma membrane fusion and switching vesicle traffic between two distinct circuits.

  12. Short-term synaptic depression and stochastic vesicle dynamics reduce and shape neuronal correlations.

    PubMed

    Rosenbaum, Robert; Rubin, Jonathan E; Doiron, Brent

    2013-01-01

    Correlated neuronal activity is an important feature in many neural codes, a neural correlate of a variety of cognitive states, as well as a signature of several disease states in the nervous system. The cellular and circuit mechanics of neural correlations is a vibrant area of research. Synapses throughout the cortex exhibit a form of short-term depression where increased presynaptic firing rates deplete neurotransmitter vesicles, which transiently reduces synaptic efficacy. The release and recovery of these vesicles are inherently stochastic, and this stochasticity introduces variability into the conductance elicited by depressing synapses. The impact of spiking and subthreshold membrane dynamics on the transfer of neuronal correlations has been studied intensively, but an investigation of the impact of short-term synaptic depression and stochastic vesicle dynamics on correlation transfer is lacking. We find that short-term synaptic depression and stochastic vesicle dynamics can substantially reduce correlations, shape the timescale over which these correlations occur, and alter the dependence of spiking correlations on firing rate. Our results show that short-term depression and stochastic vesicle dynamics need to be taken into account when modeling correlations in neuronal populations.

  13. Extracellular vesicles: Pharmacological modulators of the peripheral and central signals governing obesity.

    PubMed

    Milbank, Edward; Martinez, M Carmen; Andriantsitohaina, Ramaroson

    2016-01-01

    Obesity and its metabolic resultant dysfunctions such as insulin resistance, hyperglycemia, dyslipidemia and hypertension, grouped as the "metabolic syndrome", are chronic inflammatory disorders that represent one of the most severe epidemic health problems. The imbalance between energy intake and expenditure, leading to an excess of body fat and an increase of cardiovascular and diabetes risks, is regulated by the interaction between central nervous system (CNS) and peripheral signals in order to regulate behavior and finally, the metabolism of peripheral organs. At present, pharmacological treatment of obesity comprises actions in both CNS and peripheral organs. In the last decades, the extracellular vesicles have emerged as participants in many pathophysiological regulation processes. Whether used as biomarkers, targets or even tools, extracellular vesicles provided some promising effects in the treatment of a large variety of diseases. Extracellular vesicles are released by cells from the plasma membrane (microvesicles) or from multivesicular bodies (exosomes) and contain lipids, proteins and nucleic acids, such as DNA, protein coding, and non-coding RNAs. Owing to their composition, extracellular vesicles can (i) activate receptors at the target cell and then, the subsequent intracellular pathway associated to the specific receptor; (ii) transfer molecules to the target cells and thereby change their phenotype and (iii) be used as shuttle of drugs and, thus, to carry specific molecules towards specific cells. Herein, we review the impact of extracellular vesicles in modulating the central and peripheral signals governing obesity.

  14. Deciphering dead-end docking of large dense core vesicles in bovine chromaffin cells.

    PubMed

    Hugo, Sandra; Dembla, Ekta; Halimani, Mahantappa; Matti, Ulf; Rettig, Jens; Becherer, Ute

    2013-10-23

    Large dense core vesicle (LDCV) exocytosis in chromaffin cells follows a well characterized process consisting of docking, priming, and fusion. Total internal reflection fluorescence microscopy (TIRFM) studies suggest that some LDCVs, although being able to dock, are resistant to calcium-triggered release. This phenomenon termed dead-end docking has not been investigated until now. We characterized dead-end vesicles using a combination of membrane capacitance measurement and visualization of LDCVs with TIRFM. Stimulation of bovine chromaffin cells for 5 min with 6 μm free intracellular Ca2+ induced strong secretion and a large reduction of the LDCV density at the plasma membrane. Approximately 15% of the LDCVs were visible at the plasma membrane throughout experiments, indicating they were permanently docked dead-end vesicles. Overexpression of Munc18-2 or SNAP-25 reduced the fraction of dead-end vesicles. Conversely, expressing open-syntaxin increased the fraction of dead-end vesicles. These results indicate the existence of the unproductive target soluble N-ethylmaleimide-sensitive factor attachment protein receptor acceptor complex composed of 2:1 syntaxin-SNAP-25 in vivo. More importantly, they define a novel function for this acceptor complex in mediating dead-end docking.

  15. 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.

  16. Quantification of birefringence readily measures the level of muscle damage in zebrafish

    SciTech Connect

    Berger, Joachim; Sztal, Tamar; Currie, Peter D.

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer Report of an unbiased quantification of the birefringence of muscle of fish larvae. Black-Right-Pointing-Pointer Quantification method readily identifies level of overall muscle damage. Black-Right-Pointing-Pointer Compare zebrafish muscle mutants for level of phenotype severity. Black-Right-Pointing-Pointer Proposed tool to survey treatments that aim to ameliorate muscular dystrophy. -- Abstract: Muscular dystrophies are a group of genetic disorders that progressively weaken and degenerate muscle. Many zebrafish models for human muscular dystrophies have been generated and analysed, including dystrophin-deficient zebrafish mutants dmd that model Duchenne Muscular Dystrophy. Under polarised light the zebrafish muscle can be detected as a bright area in an otherwise dark background. This light effect, called birefringence, results from the diffraction of polarised light through the pseudo-crystalline array of the muscle sarcomeres. Muscle damage, as seen in zebrafish models for muscular dystrophies, can readily be detected by a reduction in the birefringence. Therefore, birefringence is a very sensitive indicator of overall muscle integrity within larval zebrafish. Unbiased documentation of the birefringence followed by densitometric measurement enables the quantification of the birefringence of zebrafish larvae. Thereby, the overall level of muscle integrity can be detected, allowing the identification and categorisation of zebrafish muscle mutants. In addition, we propose that the establish protocol can be used to analyse treatments aimed at ameliorating dystrophic zebrafish models.

  17. 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.

  18. A new efficient method for synaptic vesicle quantification reveals differences between medial prefrontal cortex perforated and nonperforated synapses.

    PubMed

    Nava, Nicoletta; Chen, Fenghua; Wegener, Gregers; Popoli, Maurizio; Nyengaard, Jens Randel

    2014-02-01

    Communication between neurons is mediated by the release of neurotransmitter-containing vesicles from presynaptic terminals. Quantitative characterization of synaptic vesicles can be highly valuable for understanding mechanisms underlying synaptic function and plasticity. We performed a quantitative ultrastructural analysis of cortical excitatory synapses by mean of a new, efficient method, as an alternative to three-dimensional (3D) reconstruction. Based on a hierarchical sampling strategy and unequivocal identification of the region of interest, serial sections from excitatory synapses of medial prefrontal cortex (mPFC) of six Sprague-Dawley rats were acquired with a transmission electron microscope. Unbiased estimates of total 3D volume of synaptic terminals were obtained through the Cavalieri estimator, and adequate correction factors for vesicle profile number estimation were applied for final vesicle quantification. Our analysis was based on 79 excitatory synapses, nonperforated (NPSs) and perforated (PSs) subtypes. We found that total number of docked and reserve-pool vesicles in PSs significantly exceeded that in NPSs (by, respectively, 77% and 78%). These differences were found to be related to changes in size between the two subtypes (active zone area by 86%; bouton volume by 105%) rather than to postsynaptic density shape. Positive significant correlations were found between number of docked and reserve-pool vesicles, active zone area and docked vesicles, and bouton volume and reserve pool vesicles. Our method confirmed the large size of mPFC PSs and a linear correlation between presynaptic features of typical hippocampal synapses. Moreover, a greater number of docked vesicles in PSs may promote a high synaptic strength of these synapses.

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

    PubMed Central

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

    2014-01-01

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

  20. Endogenous Leucine-Rich Repeat Kinase 2 Slows Synaptic Vesicle Recycling in Striatal Neurons

    PubMed Central

    Maas, James W. Jr.; Yang, Jing; Edwards, Robert H.

    2017-01-01

    Dominant mutations in leucine-rich repeat kinase 2 (LRRK2) produce the most common inherited form of Parkinson’s disease (PD) but the function of LRRK2 remains poorly understood. The presynaptic role of multiple genes linked to PD including α-synuclein (α-syn) has suggested that LRRK2 may also influence neurotransmitter release, a possibility supported by recent work. However, the use of disease-associated mutants that cause toxicity complicates the analysis. To determine whether LRRK2 normally influences the synaptic vesicle, we have now used a combination of imaging and electrophysiology to study LRRK2 knockout (KO) mice. Surprisingly, we find that in hippocampal (generally excitatory) neurons, the loss of LRRK2 does not affect synaptic vesicle exocytosis, endocytosis or the mobility of α-syn. Double KO (DKO) mice lacking LRRK1 as well as LRRK2 also show no defect in transmitter release by hippocampal neurons. However, in striatal neurons, which express LRRK2 at higher levels, the loss of LRRK2 leads to modest acceleration of synaptic vesicle endocytosis. Thus, endogenous LRRK2 normally slows synaptic vesicle recycling at striatal terminals. PMID:28280464

  1. Adhesion energy can regulate vesicle fusion and stabilize partially fused states.

    PubMed

    Long, Rong; Hui, Chung-Yuen; Jagota, Anand; Bykhovskaia, Maria

    2012-07-07

    Release of neurotransmitters from nerve terminals occurs by fusion of synaptic vesicles with the plasma membrane, and this process is highly regulated. Although major molecular components that control docking and fusion of vesicles to the synaptic membrane have been identified, the detailed mechanics of this process is not yet understood. We have developed a mathematical model that predicts how adhesion forces imposed by docking and fusion molecular machinery would affect the fusion process. We have computed the membrane stress that is produced by adhesion-driven vesicle bending and find that it is compressive. Further, our computations of the membrane curvature predict that strong adhesion can create a metastable state with a partially opened pore that would correspond to the 'kiss and run' release mode. Our model predicts that the larger the vesicle size, the more likely the metastable state with a transiently opened pore. These results contribute to understanding the mechanics of the fusion process, including possible clamping of the fusion by increasing molecular adhesion, and a balance between 'kiss and run' and full collapse fusion modes.

  2. Selection of high efficient transdermal lipid vesicle for curcumin skin delivery.

    PubMed

    Zhao, Ying-Zheng; Lu, Cui-Tao; Zhang, Yi; Xiao, Jian; Zhao, Ya-Ping; Tian, Ji-Lai; Xu, Yan-Yan; Feng, Zhi-Guo; Xu, Chong-Yong

    2013-09-15

    Curcumin shows effective anti-inflammatory activities but is seldom used in clinic because of its poor solubility in water and vulnerablity to sunshine ultraviolet effect. Novel lipid vesicles have been developed as carriers for skin delivery. In this paper, lipid vesicles-propylene glycol liposomes (PGL), Ethosomes and traditional liposomes, were prepared as curcumin carriers respectively. Their morphology, particle size and encapsulation efficiency and drug release behavior in vitro were evaluated. Transdermal efficiency and deposition quantity in abdominal skin were also measured with Franz diffusion device. Carrageenan-induced paw edema was established to evaluate the anti-inflammatory effect. From the result, the particle size order of lipid vesicles was: PGL (182.4 ± 89.2 nm)Ethosomes>traditional liposomes. PGL had the best encapsulation efficiency of 92.74 ± 3.44%. From anti-inflammatory experiment, PGL showed the highest and longest inhibition on the development of paw edema, followed by Ethosomes and Traditional liposomes. With the elevated entrapment efficiency, good transdermic ability and sustained-release behavior, PGL may represent an efficient transdermal lipid vesicle for skin delivery.

  3. Ultrasound and pH Dually Responsive Polymer Vesicles for Anticancer Drug Delivery

    NASA Astrophysics Data System (ADS)

    Chen, Wenqin; Du, Jianzhong

    2013-07-01

    Recently, smart polymer vesicles have attracted increasing interest due to their endless potential applications such as tunable delivery vehicles for the treatment of degenerative diseases. However, the evolution of stimuli-responsive vesicles from bench to bedside still seems far away for the limitations of current stimuli forms such as temperature, light, redox, etc. Since ultrasound combined with chemotherapy has been widely used in tumor treatment and the pH in tumor tissues is relatively low, we designed herein a novel polymer vesicle that respond to both physical (ultrasound) and chemical (pH) stimuli based on a PEO-b-P(DEA-stat-TMA) block copolymer, where PEO is short for poly(ethylene oxide), DEA for 2-(diethylamino)ethyl methacrylate and TMA for (2-tetrahydrofuranyloxy)ethyl methacrylate. These dually responsive vesicles show noncytotoxicity below 250 μg/mL and can encapsulate anticancer drugs, exhibiting retarded release profile and controllable release rate when subjected to ultrasound radiation or varying pH in tris buffer at 37°C.

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

    PubMed Central

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

    2016-01-01

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

  5. Endogenous Leucine-Rich Repeat Kinase 2 Slows Synaptic Vesicle Recycling in Striatal Neurons.

    PubMed

    Maas, James W Jr; Yang, Jing; Edwards, Robert H

    2017-01-01

    Dominant mutations in leucine-rich repeat kinase 2 (LRRK2) produce the most common inherited form of Parkinson's disease (PD) but the function of LRRK2 remains poorly understood. The presynaptic role of multiple genes linked to PD including α-synuclein (α-syn) has suggested that LRRK2 may also influence neurotransmitter release, a possibility supported by recent work. However, the use of disease-associated mutants that cause toxicity complicates the analysis. To determine whether LRRK2 normally influences the synaptic vesicle, we have now used a combination of imaging and electrophysiology to study LRRK2 knockout (KO) mice. Surprisingly, we find that in hippocampal (generally excitatory) neurons, the loss of LRRK2 does not affect synaptic vesicle exocytosis, endocytosis or the mobility of α-syn. Double KO (DKO) mice lacking LRRK1 as well as LRRK2 also show no defect in transmitter release by hippocampal neurons. However, in striatal neurons, which express LRRK2 at higher levels, the loss of LRRK2 leads to modest acceleration of synaptic vesicle endocytosis. Thus, endogenous LRRK2 normally slows synaptic vesicle recycling at striatal terminals.

  6. 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.

  7. 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

  8. 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.

  9. Exosomes in the Thymus: Antigen Transfer and Vesicles

    PubMed Central

    Skogberg, Gabriel; Telemo, Esbjörn; Ekwall, Olov

    2015-01-01

    Thymocytes go through several steps of maturation and selection in the thymus in order to form a functional pool of effector T-cells and regulatory T-cells in the periphery. Close interactions between thymocytes, thymic epithelial cells, and dendritic cells are of vital importance for the maturation, selection, and lineage decision of the thymocytes. One important question that is still unanswered is how a relatively small epithelial cell population can present a vast array of self-antigens to the manifold larger population of developing thymocytes in this selection process. Here, we review and discuss the literature concerning antigen transfer from epithelial cells with a focus on exosomes. Exosomes are nano-sized vesicles released from a cell into the extracellular space. These vesicles can carry proteins, microRNAs, and mRNAs between cells and are thus able to participate in intercellular communication. Exosomes have been shown to be produced by thymic epithelial cells and to carry tissue-restricted antigens and MHC molecules, which may enable them to participate in the thymocyte selection process. PMID:26257734

  10. Focus on Extracellular Vesicles: Introducing the Next Small Big Thing.

    PubMed

    Kalra, Hina; Drummen, Gregor P C; Mathivanan, Suresh

    2016-02-06

    Intercellular communication was long thought to be regulated exclusively through direct contact between cells or via release of soluble molecules that transmit the signal by binding to a suitable receptor on the target cell, and/or via uptake into that cell. With the discovery of small secreted vesicular structures that contain complex cargo, both in their lumen and the lipid membrane that surrounds them, a new frontier of signal transduction was discovered. These "extracellular vesicles" (EV) were initially thought to be garbage bags through which the cell ejected its waste. Whilst this is a major function of one type of EV, i.e., apoptotic bodies, many EVs have intricate functions in intercellular communication and compound exchange; although their physiological roles are still ill-defined. Additionally, it is now becoming increasingly clear that EVs mediate disease progression and therefore studying EVs has ignited significant interests among researchers from various fields of life sciences. Consequently, the research effort into the pathogenic roles of EVs is significantly higher even though their protective roles are not well established. The "Focus on extracellular vesicles" series of reviews highlights the current state of the art regarding various topics in EV research, whilst this review serves as an introductory overview of EVs, their biogenesis and molecular composition.

  11. Facile preparation of salivary extracellular vesicles for cancer proteomics

    NASA Astrophysics Data System (ADS)

    Sun, Yan; Xia, Zhijun; Shang, Zhi; Sun, Kaibo; Niu, Xiaomin; Qian, Liqiang; Fan, Liu-Yin; Cao, Cheng-Xi; Xiao, Hua

    2016-04-01

    Extracellular vesicles (EVs) are membrane surrounded structures released by cells, which have been increasingly recognized as mediators of intercellular communication. Recent reports indicate that EVs participate in important biological processes and could serve as potential source for cancer biomarkers. As an attractive EVs source with merit of non-invasiveness, human saliva is a unique medium for clinical diagnostics. Thus, we proposed a facile approach to prepare salivary extracellular vesicles (SEVs). Affinity chromatography column combined with filter system (ACCF) was developed to efficiently remove the high abundant proteins and viscous interferences of saliva. Protein profiling in the SEVs obtained by this strategy was compared with conventional centrifugation method, which demonstrated that about 70% more SEVs proteins could be revealed. To explore its utility for cancer proteomics, we analyzed the proteome of SEVs in lung cancer patients and normal controls. Shotgun proteomic analysis illustrated that 113 and 95 proteins have been identified in cancer group and control group, respectively. Among those 63 proteins that have been consistently discovered only in cancer group, 12 proteins are lung cancer related. Our results demonstrated that SEVs prepared through the developed strategy are valuable samples for proteomics and could serve as a promising liquid biopsy for cancer.

  12. Vesiclepedia: A Compendium for Extracellular Vesicles with Continuous Community Annotation

    PubMed Central

    Kalra, Hina; Simpson, Richard J.; Ji, Hong; Aikawa, Elena; Altevogt, Peter; Askenase, Philip; Bond, Vincent C.; Borràs, Francesc E.; Breakefield, Xandra; Budnik, Vivian; Buzas, Edit; Camussi, Giovanni; Clayton, Aled; Cocucci, Emanuele; Falcon-Perez, Juan M.; Gabrielsson, Susanne; Gho, Yong Song; Gupta, Dwijendra; Harsha, H. C.; Hendrix, An; Hill, Andrew F.; Inal, Jameel M.; Jenster, Guido; Krämer-Albers, Eva-Maria; Lim, Sai Kiang; Llorente, Alicia; Lötvall, Jan; Marcilla, Antonio; Mincheva-Nilsson, Lucia; Nazarenko, Irina; Nieuwland, Rienk; Nolte-'t Hoen, Esther N. M.; Pandey, Akhilesh; Patel, Tushar; Piper, Melissa G.; Pluchino, Stefano; Prasad, T. S. Keshava; Rajendran, Lawrence; Raposo, Graca; Record, Michel; Reid, Gavin E.; Sánchez-Madrid, Francisco; Schiffelers, Raymond M.; Siljander, Pia; Stensballe, Allan; Stoorvogel, Willem; Taylor, Douglas; Thery, Clotilde; Valadi, Hadi; van Balkom, Bas W. M.; Vázquez, Jesús; Vidal, Michel; Wauben, Marca H. M.; Yáñez-Mó, María; Zoeller, Margot; Mathivanan, Suresh

    2012-01-01

    Extracellular vesicles (EVs) are membraneous vesicles released by a variety of cells into their microenvironment. Recent studies have elucidated the role of EVs in intercellular communication, pathogenesis, drug, vaccine and gene-vector delivery, and as possible reservoirs of biomarkers. These findings have generated immense interest, along with an exponential increase in molecular data pertaining to EVs. Here, we describe Vesiclepedia, a manually curated compendium of molecular data (lipid, RNA, and protein) identified in different classes of EVs from more than 300 independent studies published over the past several years. Even though databases are indispensable resources for the scientific community, recent studies have shown that more than 50% of the databases are not regularly updated. In addition, more than 20% of the database links are inactive. To prevent such database and link decay, we have initiated a continuous community annotation project with the active involvement of EV researchers. The EV research community can set a gold standard in data sharing with Vesiclepedia, which could evolve as a primary resource for the field. PMID:23271954

  13. Extracellular Vesicles as Therapeutic Tools in Cardiovascular Diseases

    PubMed Central

    Fleury, Audrey; Martinez, Maria Carmen; Le Lay, Soazig

    2014-01-01

    Extracellular vesicles (EVs), including microvesicles (MVs) and exosomes, are small vesicles secreted from a wide variety of cells. Whereas MVs are particles released by the outward budding of the plasma membrane, exosomes are derived from endocytic compartments. Secretion of EVs can be enhanced by specific stimuli, and increased plasma circulating levels of EVs have been correlated with pathophysiological situations. MVs, already present in the blood of healthy individuals, are considerably elevated in several cardiovascular diseases associated with inflammation, suggesting that they can mediate deleterious effects such as endothelial dysfunction or thrombosis. Nonetheless, very recent studies also demonstrate that MVs may act as biological information vectors transferring proteins or genetic material to maintain cell homeostasis, favor cell repair, or even promote angiogenesis. Additionally, exosomes have also been shown to have pro-angiogenic and cardio-protective properties. These beneficial effects, therefore, reveal the potential therapeutical use of EVs in the field of cardiovascular medicine and regenerative therapy. In this review, we will provide an update of cellular processes modulated by EVs of specific interest in the treatment of cardiovascular pathologies. A special focus will be made on the morphogen sonic hedgehog (Shh) associated with EVs (EVsShh+), which have been shown to mediate many pro-angiogenic effects. In addition to offer a potential source of cardiovascular markers, therapeutical potential of EVs reveal exciting opportunities to deliver specific agents by non-immunogenic means to cardiovascular system. PMID:25136343

  14. Facile preparation of salivary extracellular vesicles for cancer proteomics

    PubMed Central

    Sun, Yan; Xia, Zhijun; Shang, Zhi; Sun, Kaibo; Niu, Xiaomin; Qian, Liqiang; Fan, Liu-Yin; Cao, Cheng-Xi; Xiao, Hua

    2016-01-01

    Extracellular vesicles (EVs) are membrane surrounded structures released by cells, which have been increasingly recognized as mediators of intercellular communication. Recent reports indicate that EVs participate in important biological processes and could serve as potential source for cancer biomarkers. As an attractive EVs source with merit of non-invasiveness, human saliva is a unique medium for clinical diagnostics. Thus, we proposed a facile approach to prepare salivary extracellular vesicles (SEVs). Affinity chromatography column combined with filter system (ACCF) was developed to efficiently remove the high abundant proteins and viscous interferences of saliva. Protein profiling in the SEVs obtained by this strategy was compared with conventional centrifugation method, which demonstrated that about 70% more SEVs proteins could be revealed. To explore its utility for cancer proteomics, we analyzed the proteome of SEVs in lung cancer patients and normal controls. Shotgun proteomic analysis illustrated that 113 and 95 proteins have been identified in cancer group and control group, respectively. Among those 63 proteins that have been consistently discovered only in cancer group, 12 proteins are lung cancer related. Our results demonstrated that SEVs prepared through the developed strategy are valuable samples for proteomics and could serve as a promising liquid biopsy for cancer. PMID:27091080

  15. Extracellular vesicles in diagnosis and therapy of kidney diseases.

    PubMed

    Zhang, Wei; Zhou, Xiangjun; Zhang, Hao; Yao, Qisheng; Liu, Yutao; Dong, Zheng

    2016-11-01

    Extracellular vesicles (EV) are endogenously produced, membrane-bound vesicles that contain various molecules. Depending on their size and origins, EVs are classified into apoptotic bodies, microvesicles, and exosomes. A fundamental function of EVs is to mediate intercellular communication. In kidneys, recent research has begun to suggest a role of EVs, especially exosomes, in cell-cell communication by transferring proteins, mRNAs, and microRNAs to recipient cells as nanovectors. EVs may mediate the cross talk between various cell types within kidneys for the maintenance of tissue homeostasis. They may also mediate the cross talk between kidneys and other organs under physiological and pathological conditions. EVs have been implicated in the pathogenesis of both acute kidney injury and chronic kidney diseases, including renal fibrosis, end-stage renal disease, glomerular diseases, and diabetic nephropathy. The release of EVs with specific molecular contents into urine and plasma may be useful biomarkers for kidney disease. In addition, EVs produced by cultured cells may have therapeutic effects for these diseases. However, the role of EVs in kidney diseases is largely unclear, and the mechanism underlying EV production and secretion remains elusive. In this review, we introduce the basics of EVs and then analyze the present information about the involvement, diagnostic value, and therapeutic potential of EVs in major kidney diseases.

  16. Do sympathetic nerves release noradrenaline in "quanta"?

    PubMed

    Stjärne, L

    2000-07-03

    The discovery of excitatory junction potentials (EJPs) in guinea-pig vas deferens by Burnstock and Holman (1960) showed for the first time that a sympathetic transmitter, now known to be ATP, is secreted in "quanta". As it was assumed at the time that EJPS are triggered by noradrenaline, this discovery led to attempts to use the fractional overflow of noradrenaline from sympathetically innervated tissues to assess, indirectly, the number of noradrenaline molecules in the average "quantum". The basic finding was that each pulse released 1/50000 of the tissue content of noradrenaline, when reuptake was blocked and prejunctional alpha(2)-adrenoceptors were intact. This provided the constraints, two extreme alternatives: (i) each pulse releases 0.2-3% of the content of a vesicle from all varicosities, or (ii) each pulse releases the whole content of a vesicle from 0.2 to 3% of the varicosities. New techniques have made it possible to address questions about the release probability in individual sites, or the "quantal" size, more directly. Results by optical (comparison of the labelling of SV2 and synaptotagmin, proteins in the membrane of transmitter vesicles), electrophysiological (excitatory junction currents, EJCs, at single visualized varicosities) and amperometric (the noradrenaline oxidation current at a carbon fibre electrode) methods reveal that transmitter exocytosis in varicosities is intermittent. The EJC and noradrenaline oxidation current responses (in rat arteries) to a train of single pulses were observed to be similar in intermittency and amplitude fluctuation. This suggests that they are caused by exocytosis of single or very few "quanta" of ATP and noradrenaline, respectively, equal to the contents of single vesicles, from a small population of release sites. These findings support, but do not conclusively prove the validity of the "intermittent" model of noradrenaline release. The question if noradrenaline is always secreted in packets of preset size

  17. Munc18-Bound Syntaxin Readily Forms SNARE Complexes with Synaptobrevin in Native Plasma Membranes

    PubMed Central

    Zilly, Felipe E; Sørensen, Jakob B; Jahn, Reinhard; Lang, Thorsten

    2006-01-01

    Munc18–1, a protein essential for regulated exocytosis in neurons and neuroendocrine cells, belongs to the family of Sec1/Munc18-like (SM) proteins. In vitro, Munc18–1 forms a tight complex with the SNARE syntaxin 1, in which syntaxin is stabilized in a closed conformation. Since closed syntaxin is unable to interact with its partner SNAREs SNAP-25 and synaptobrevin as required for membrane fusion, it has hitherto not been possible to reconcile binding of Munc18–1 to syntaxin 1 with its biological function. We now show that in intact and exocytosis-competent lawns of plasma membrane, Munc18–1 forms a complex with syntaxin that allows formation of SNARE complexes. Munc18–1 associated with membrane-bound syntaxin 1 can be effectively displaced by adding recombinant synaptobrevin but not syntaxin 1 or SNAP-25. Displacement requires the presence of endogenous SNAP-25 since no displacement is observed when chromaffin cell membranes from SNAP-25–deficient mice are used. We conclude that Munc18–1 allows for the formation of a complex between syntaxin and SNAP-25 that serves as an acceptor for vesicle-bound synaptobrevin and that thus represents an intermediate in the pathway towards exocytosis. PMID:17002520

  18. Munc18-bound syntaxin readily forms SNARE complexes with synaptobrevin in native plasma membranes.

    PubMed

    Zilly, Felipe E; Sørensen, Jakob B; Jahn, Reinhard; Lang, Thorsten

    2006-10-01

    Munc18-1, a protein essential for regulated exocytosis in neurons and neuroendocrine cells, belongs to the family of Sec1/Munc18-like (SM) proteins. In vitro, Munc18-1 forms a tight complex with the SNARE syntaxin 1, in which syntaxin is stabilized in a closed conformation. Since closed syntaxin is unable to interact with its partner SNAREs SNAP-25 and synaptobrevin as required for membrane fusion, it has hitherto not been possible to reconcile binding of Munc18-1 to syntaxin 1 with its biological function. We now show that in intact and exocytosis-competent lawns of plasma membrane, Munc18-1 forms a complex with syntaxin that allows formation of SNARE complexes. Munc18-1 associated with membrane-bound syntaxin 1 can be effectively displaced by adding recombinant synaptobrevin but not syntaxin 1 or SNAP-25. Displacement requires the presence of endogenous SNAP-25 since no displacement is observed when chromaffin cell membranes from SNAP-25-deficient mice are used. We conclude that Munc18-1 allows for the formation of a complex between syntaxin and SNAP-25 that serves as an acceptor for vesicle-bound synaptobrevin and that thus represents an intermediate in the pathway towards exocytosis.

  19. On the dynamic behavior of three readily available soft tissue simulants

    NASA Astrophysics Data System (ADS)

    Appleby-Thomas, G. J.; Hazell, P. J.; Wilgeroth, J. M.; Shepherd, C. J.; Wood, D. C.; Roberts, A.

    2011-04-01

    Plate-impact experiments have been employed to investigate the dynamic response of three readily available tissue simulants for ballistic purposes: gelatin, ballistic soap (both subdermal tissue simulants), and lard (adipose layers). All three materials exhibited linear Hugoniot equations-of-state in the US-uP plane. While gelatin behaved hydrodynamically under shock, soap and lard appeared to strengthen under increased loading. Interestingly, the simulants under test appeared to strengthen in a material-independent manner on shock arrival (tentatively attributed to a rearrangement of the amorphous molecular chains under loading). However, material-specific behavior was apparent behind the shock. This behavior appeared to correlate with microstructural complexity, suggesting a steric hindrance effect.

  20. Synthesis of readily recyclable biobased plastics by Diels-Alder reaction.

    PubMed

    Ishida, Kazuki; Yoshie, Naoko

    2008-10-08

    Readily recyclable biobased plastics were designed and synthesized utilizing the thermally reversible DA reaction. Furyl-telechelic poly(butylene succinate) prepolymers (PBSF(2)) were extended with bis- and tris-maleimide linkers (M(2) and M(3)) by the DA reaction in the bulk state to produce linear and network polymers, respectively. The DA reaction was able to proceed at 25-80 degrees C, at which crystalline domains of PBSF(2) were present. In the linear polymer system, the molecular weight in the reaction equilibrium was dependent on the chain length of the prepolymer, but almost independent of the reaction temperature. The cycle of DA and retro-DA reactions was repeatable with no prepolymer deterioration.

  1. Low-cost and readily available tissue carriers for the Boston keratoprosthesis: a review of possibilities.

    PubMed

    Cruzat, Andrea; Tauber, Allyson; Shukla, Anita; Paschalis, Eleftherios I; Pineda, Roberto; Dohlman, Claes H

    2013-01-01

    The Boston keratoprosthesis (B-KPro), currently the most commonly used artificial cornea worldwide, can provide rapid visual rehabilitation for eyes with severe corneal opacities not suitable for standard corneal transplantation. However, the B-KPro presently needs a corneal graft as a tissue carrier. Although corneal allograft tissue is readily available in the United States and other developed countries with established eye banks, the worldwide need vastly exceeds supply. Therefore, a simple, safe, and inexpensive alternative to corneal allografts is desirable for the developing world. We are currently exploring reasonable alternative options such as corneal autografts, xenografts, noncorneal autologous tissues, and laboratory-made tissue constructs, as well as modifications to corneal allografts, such as deep-freezing, glycerol-dehydration, gamma irradiation, and cross-linking. These alternative tissue carriers for the B-KPro are discussed with special regard to safety, practicality, and cost for the developing world.

  2. The non-steroidal anti-inflammatory drug diclofenac is readily biodegradable in agricultural soils.

    PubMed

    Al-Rajab, Abdul Jabbar; Sabourin, Lyne; Lapen, David R; Topp, Edward

    2010-12-01

    Diclofenac, 2-[2-[(2,6-dichlorophenyl)amino]phenyl]acetic acid, is an important non-steroidal anti-inflammatory drug widely used for human and animals to reduce inflammation and pain. Diclofenac could potentially reach agricultural lands through the application of municipal biosolids or wastewater, and in the absence of any environmental fate data, we evaluated its persistence in agricultural soils incubated in the laboratory. (14)C-Diclofenac was rapidly mineralized without a lag when added to soils varying widely in texture (sandy loam, loam, clay loam). Over a range of temperature and moisture conditions extractable (14)C-diclofenac residues decreased with half lives <5days. No extractable transformation products were detectable by HPLC. Diclofenac mineralization in the loam soil was abolished by heat sterilization. Addition of biosolids to sterile or non-sterile soil did not accelerate the dissipation of diclofenac. These findings indicate that diclofenac is readily biodegradable in agricultural soils.

  3. [Dentistry and healthcare legislation 10. The law governing complaints: readily accessible filing procedures].

    PubMed

    van der Ven, J M; Eijkman, M A J; Brands, W G

    2014-03-01

    The law promises patients a readily accessible means of filing complaints. Healthcare providers are therefore required to adopt regulations governing complaints which satisfy a number of conditions. Most dentists choose to adopt the regulations which have been established by their professional organization. In addition to handling complaints, there is also a provision for mediation, which is often used by patients. Mediation appears, then, to be a successful provision. Many complaints have their origin in insufficient knowledge of healthcare legislation and patients' rights legislation. This demonstrates that more attention should be given to these subjects in educational programmes and programmes in continuing education. The present law governing complaints is expected to be replaced this year by a new, more comprehensive law in which considerable attention will be devoted to the quality of care as well as to complaints. It seems likely, however, that the new law governing complaints will damage the effective manner in which patients' complaints are dealt with in dentistry today.

  4. Generation of biologically contained, readily transformable, and genetically manageable mutants of the biotechnologically important Bacillus pumilus.

    PubMed

    Wemhoff, Stephanie; Meinhardt, Friedhelm

    2013-09-01

    Bacillus pumilus mutants were generated by targeted deletion of a set of genes eventually facilitating genetic handling and assuring biological containment. The well-defined and stable mutants do not form functional endospores due to the deletion of yqfD, an essential sporulation gene; they are affected in DNA repair, as ΔuvrBA rendered them UV hypersensitive and, thus, biologically contained; they are deficient for the uracil phosphoribosyl-transferase (Δupp), allowing for 5-fluorouracil-based counterselection facilitating rapid allelic exchanges; and they are readily transformable due to the deletion of the restrictase encoding locus (ΔhsdR) of a type I restriction modification system. Vegetative growth as well as extracellular enzyme production and secretion are in no case affected. The combination of such gene deletions allows for development of B. pumilus strains suited for industrial use and further improvements.

  5. DNA sequences within glioma-derived extracellular vesicles can cross the intact blood-brain barrier and be detected in peripheral blood of patients

    PubMed Central

    Lázaro-Ibáñez, Elisa; Peris-Celda, María; Alonso, Marta M.; Guzmán-De-Villoria, Juan; Fernández-Carballal, Carlos; de Mendivil, Ana Ortiz; García-Duque, Sara; Escobedo-Lucea, Carmen; Prat-Acín, Ricardo; Belda-Iniesta, Cristóbal; Ayuso-Sacido, Angel

    2017-01-01

    Tumor-cell-secreted extracellular vesicles (EVs) can cross the disrupted blood-brain barrier (BBB) into the bloodstream. However, in certain gliomas, the BBB remains intact, which might limit EVs release. To evaluate the ability of tumor-derived EVs to cross the BBB, we used an orthotopic xenotransplant mouse model of human glioma-cancer stem cells featuring an intact BBB. We demonstrated that all types of tumor cells-derived EVs−apoptotic bodies, shedding microvesicles and exosomes−cross the intact BBB and can be detected in the peripheral blood, which provides a minimally invasive method for their detection compared to liquid biopsies obtained from cerebrospinal fluid (CSF). Furthermore, these EVs can be readily distinguished from total murine EVs, since they carry human-specific DNA sequences relevant for GBM biology. In a small cohort of glioma patients, we finally demonstrated that peripheral blood EVs cargo can be successfully used to detect the presence of IDH1G395A, an essential biomarker in the current management of human glioma PMID:27902458

  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. 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.

  8. 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.

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

    NASA Astrophysics Data System (ADS)

    Wang, Xi

    peptide activities. The study of vesicle rupture mechanics and mechanical properties provide a means of understanding triggered release of internal payload from vesicular structures. POPC vesicles were also deposited on graphene; a transparent and highly conductive electrode. A combination method of diffusion bonding and template-stripping was used to prepare metal surfaces for graphene growth without concerns of outgassing, thermal and chemical compatibility. Continuous LBM formed on graphene-single crystal Cu, while tubular features with 120°C patterns formed on graphene-Cu foil, indicating the step edge of Cu below graphene may also guide the assembly of tubular LBM features on graphene.

  10. Plasmonic Vesicles of Amphiphilic Nanocrystals: Optically Active Multifunctional Platform for Cancer Diagnosis and Therapy.

    PubMed

    Song, Jibin; Huang, Peng; Duan, Hongwei; Chen, Xiaoyuan

    2015-09-15

    Vesicular structures with compartmentalized, water-filled cavities, such as liposomes of natural and synthetic amphiphiles, have tremendous potential applications in nanomedicine. When block copolymers self-assemble, the result is polymersomes with tailored structural properties and built-in releasing mechanisms, controlled by stimuli-responsive polymer building blocks. More recently, chemists are becoming interested in multifunctional hybrid vesicles containing inorganic nanocrystals with unique optical, electronic, and magnetic properties. In this Account, we review our recent progress in assembling amphiphilic plasmonic nanostructures to create a new class of multifunctional hybrid vesicles and applying them towards cancer diagnosis and therapy. Localized surface plasmon resonance (LSPR) gives plasmonic nanomaterials a unique set of optical properties that are potentially useful for both biosensing and nanomedicine. For instance, the strong light scattering at their LSPR wavelength opens up the applications of plasmonic nanostructures in single particle plasmonic imaging. Their superior photothermal conversion properties, on the other hand, make them excellent transducers for photothermal ablation and contrast agents for photoacoustic imaging. Of particular note for ultrasensitive detection is that the confined electromagnetic field resulting from excitation of LSPR can give rise to highly efficient surface enhanced Raman scattering (SERS) for molecules in close proximity. We have explored several ways to combine well-defined plasmonic nanocrystals with amphiphilic polymer brushes of diverse chemical functionalities. In multiple systems, we have shown that the polymer grafts impart amphiphilicity-driven self-assembly to the hybrid nanoparticles. This has allowed us to synthesize well-defined vesicles in which we have embedded plasmonic nanocrystals in the shell of collapsed hydrophobic polymers. The hydrophilic brushes extend into external and interior aqueous

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. Extracellular vesicles and viruses: Are they close relatives?

    PubMed Central

    Nolte-‘t Hoen, Esther; Cremer, Tom; Gallo, Robert C.; Margolis, Leonid B.

    2016-01-01

    Extracellular vesicles (EVs) released by various cells are small phospholipid membrane-enclosed entities that can carry miRNA. They are now central to research in many fields of biology because they seem to constitute a new system of cell–cell communication. Physical and chemical characteristics of many EVs, as well as their biogenesis pathways, resemble those of retroviruses. Moreover, EVs generated by virus-infected cells can incorporate viral proteins and fragments of viral RNA, being thus indistinguishable from defective (noninfectious) retroviruses. EVs, depending on the proteins and genetic material incorporated in them, play a significant role in viral infection, both facilitating and suppressing it. Deciphering the mechanisms of EV-cell interactions may facilitate the design of EVs that inhibit viral infection and can be used as vehicles for targeted drug delivery. PMID:27432966

  17. Extracellular vesicles of the blood-brain barrier

    PubMed Central

    András, Ibolya E; Toborek, Michal

    2016-01-01

    Extracellular vesicles (ECV), like exosomes, gained recently a lot of attention as potentially playing a significant role in neurodegenerative diseases, particularly in Aβ pathology. While there are a lot of reports on ECV/exosomes derived from a variety of cell types, there is limited information on ECV/exosomes originated from brain microvascular endothelial cells forming the blood-brain barrier (BBB). In this review, we summarize the literature data on brain endothelial ECV/exosomes and present our own data on BBB-derived ECV and their possible involvement in the brain's Aβ pathology. We propose that ECV/exosome release from brain endothelial cells associated with Aβ affects different cells of the neurovascular unit and may be an important contributor to the Aβ deposition in the central nervous system. PMID:27141419

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

    PubMed Central

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

    2007-01-01

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

  19. 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.

  20. Multisensor on-the-go mapping of readily dispersible clay, particle size and soil organic matter

    NASA Astrophysics Data System (ADS)

    Debaene, Guillaume; Niedźwiecki, Jacek; Papierowska, Ewa

    2016-04-01

    Particle size fractions affect strongly the physical and chemical properties of soil. Readily dispersible clay (RDC) is the part of the clay fraction in soils that is easily or potentially dispersible in water when small amounts of mechanical energy are applied to soil. The amount of RDC in the soil is of significant importance for agriculture and environment because clay dispersion is a cause of poor soil stability in water which in turn contributes to soil erodibility, mud flows, and cementation. To obtain a detailed map of soil texture, many samples are needed. Moreover, RDC determination is time consuming. The use of a mobile visible and near-infrared (VIS-NIR) platform is proposed here to map those soil properties and obtain the first detailed map of RDC at field level. Soil properties prediction was based on calibration model developed with 10 representative samples selected by a fuzzy logic algorithm. Calibration samples were analysed for soil texture (clay, silt and sand), RDC and soil organic carbon (SOC) using conventional wet chemistry analysis. Moreover, the Veris mobile sensor platform is also collecting electrical conductivity (EC) data (deep and shallow), and soil temperature. These auxiliary data were combined with VIS-NIR measurement (data fusion) to improve prediction results. EC maps were also produced to help understanding RDC data. The resulting maps were visually compared with an orthophotography of the field taken at the beginning of the plant growing season. Models were developed with partial least square regression (PLSR) and support vector machine regression (SVMR). There were no significant differences between calibration using PLSR or SVMR. Nevertheless, the best models were obtained with PLSR and standard normal variate (SNV) pretreatment and the fusion with deep EC data (e.g. for RDC and clay content: RMSECV = 0,35% and R2 = 0,71; RMSECV = 0,32% and R2 = 0,73 respectively). The best models were used to predict soil properties from the

  1. The 20S proteasome core, active within apoptotic exosome-like vesicles, induces autoantibody production and accelerates rejection.

    PubMed

    Dieudé, Mélanie; Bell, Christina; Turgeon, Julie; Beillevaire, Deborah; Pomerleau, Luc; Yang, Bing; Hamelin, Katia; Qi, Shijie; Pallet, Nicolas; Béland, Chanel; Dhahri, Wahiba; Cailhier, Jean-François; Rousseau, Matthieu; Duchez, Anne-Claire; Lévesque, Tania; Lau, Arthur; Rondeau, Christiane; Gingras, Diane; Muruve, Danie; Rivard, Alain; Cardinal, Héloise; Perreault, Claude; Desjardins, Michel; Boilard, Éric; Thibault, Pierre; Hébert, Marie-Josée

    2015-12-16

    Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naïve mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)-incompatible aortic graft. The 20S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation.

  2. Blood circulation and tissue biodistribution of lipid--quantum dot (L-QD) hybrid vesicles intravenously administered in mice.

    PubMed

    Al-Jamal, Wafa' T; Al-Jamal, Khuloud T; Cakebread, Andrew; Halket, John M; Kostarelos, Kostas

    2009-09-01

    The present work describes the pharmacokinetics of recently developed liposome-quantum dot (L-QD) hybrid vesicles in nude mice following systemic administration. Hydrophobic QD were incorporated into different bilayer compositions, and the serum stability of such hybrid vesicles was evaluated using turbidity and carboxyfluorescein release measurements. L-QD hybrid blood profile and organ biodistribution were also determined by elemental (cadmium) analysis. Following intravenous administration, different tissue biodistribution profiles and tissue affinities were observed depending on the L-QD lipid bilayer characteristics. Immediate blood clearance was observed with cationic (DOTAP/DOPE/Chol) hybrid with rapid lung accumulation, while incorporation of PEG at the surface of zwitterionic vesicles dramatically prolonged their blood circulation half-life after systemic administration. Overall, the L-QD hybrid vesicle system is considered a viable platform that allows QD delivery to different tissues through facile modulation of the hybrid vesicle characteristics. In addition, L-QD offers many opportunities for the development of combinatory therapeutic and imaging (theranostic) modalities by incorporating both drug molecules and QD within the different compartments of a single vesicle.

  3. Extracellular vesicle in vivo biodistribution is determined by cell source, route of administration and targeting.

    PubMed

    Wiklander, Oscar P B; Nordin, Joel Z; O'Loughlin, Aisling; Gustafsson, Ylva; Corso, Giulia; Mäger, Imre; Vader, Pieter; Lee, Yi; Sork, Helena; Seow, Yiqi; Heldring, Nina; Alvarez-Erviti, Lydia; Smith, C I Edvard; Le Blanc, Katarina; Macchiarini, Paolo; Jungebluth, Philipp; Wood, Matthew J A; Andaloussi, Samir El

    2015-01-01

    Extracellular vesicles (EVs) have emerged as important mediators of intercellular communication in a diverse range of biological processes. For future therapeutic applications and for EV biology research in general, understanding the in vivo fate of EVs is of utmost importance. Here we studied biodistribution of EVs in mice after systemic delivery. EVs were isolated from 3 different mouse cell sources, including dendritic cells (DCs) derived from bone marrow, and labelled with a near-infrared lipophilic dye. Xenotransplantation of EVs was further carried out for cross-species comparison. The reliability of the labelling technique was confirmed by sucrose gradient fractionation, organ perfusion and further supported by immunohistochemical staining using CD63-EGFP probed vesicles. While vesicles accumulated mainly in liver, spleen, gastrointestinal tract and lungs, differences related to EV cell origin were detected. EVs accumulated in the tumour tissue of tumour-bearing mice and, after introduction of the rabies virus glycoprotein-targeting moiety, they were found more readily in acetylcholine-receptor-rich organs. In addition, the route of administration and the dose of injected EVs influenced the biodistribution pattern. This is the first extensive biodistribution investigation of EVs comparing the impact of several different variables, the results of which have implications for the design and feasibility of therapeutic studies using EVs.

  4. Extracellular vesicle in vivo biodistribution is determined by cell source, route of administration and targeting

    PubMed Central

    Wiklander, Oscar P. B.; Nordin, Joel Z.; O’Loughlin, Aisling; Gustafsson, Ylva; Corso, Giulia; Mäger, Imre; Vader, Pieter; Lee, Yi; Sork, Helena; Seow, Yiqi; Heldring, Nina; Alvarez-Erviti, Lydia; Smith, CI Edvard; Le Blanc, Katarina; Macchiarini, Paolo; Jungebluth, Philipp; Wood, Matthew J. A.; Andaloussi, Samir EL

    2015-01-01

    Extracellular vesicles (EVs) have emerged as important mediators of intercellular communication in a diverse range of biological processes. For future therapeutic applications and for EV biology research in general, understanding the in vivo fate of EVs is of utmost importance. Here we studied biodistribution of EVs in mice after systemic delivery. EVs were isolated from 3 different mouse cell sources, including dendritic cells (DCs) derived from bone marrow, and labelled with a near-infrared lipophilic dye. Xenotransplantation of EVs was further carried out for cross-species comparison. The reliability of the labelling technique was confirmed by sucrose gradient fractionation, organ perfusion and further supported by immunohistochemical staining using CD63-EGFP probed vesicles. While vesicles accumulated mainly in liver, spleen, gastrointestinal tract and lungs, differences related to EV cell origin were detected. EVs accumulated in the tumour tissue of tumour-bearing mice and, after introduction of the rabies virus glycoprotein-targeting moiety, they were found more readily in acetylcholine-receptor-rich organs. In addition, the route of administration and the dose of injected EVs influenced the biodistribution pattern. This is the first extensive biodistribution investigation of EVs comparing the impact of several different variables, the results of which have implications for the design and feasibility of therapeutic studies using EVs. PMID:25899407

  5. Oscillatory phase separation in giant lipid vesicles induced by transmembrane osmotic differentials

    PubMed Central

    Oglęcka, Kamila; Rangamani, Padmini; Liedberg, Bo; Kraut, Rachel S; Parikh, Atul N

    2014-01-01

    Giant lipid vesicles are closed compartments consisting of semi-permeable shells, which isolate femto- to pico-liter quantities of aqueous core from the bulk. Although water permeates readily across vesicular walls, passive permeation of solutes is hindered. In this study, we show that, when subject to a hypotonic bath, giant vesicles consisting of phase separating lipid mixtures undergo osmotic relaxation exhibiting damped oscillations in phase behavior, which is synchronized with swell–burst lytic cycles: in the swelled state, osmotic pressure and elevated membrane tension due to the influx of water promote domain formation. During bursting, solute leakage through transient pores relaxes the pressure and tension, replacing the domain texture by a uniform one. This isothermal phase transition—resulting from a well-coordinated sequence of mechanochemical events—suggests a complex emergent behavior allowing synthetic vesicles produced from simple components, namely, water, osmolytes, and lipids to sense and regulate their micro-environment. DOI: http://dx.doi.org/10.7554/eLife.03695.001 PMID:25318069

  6. The unconventional secretion of stress-inducible protein 1 by a heterogeneous population of extracellular vesicles.

    PubMed

    Hajj, Glaucia N M; Arantes, Camila P; Dias, Marcos Vinicios Salles; Roffé, Martín; Costa-Silva, Bruno; Lopes, Marilene H; Porto-Carreiro, Isabel; Rabachini, Tatiana; Lima, Flávia R; Beraldo, Flávio H; Prado, Marco A M; Prado, Marco M A; Linden, Rafael; Martins, Vilma R

    2013-09-01

    The co-chaperone stress-inducible protein 1 (STI1) is released by astrocytes, and has important neurotrophic properties upon binding to prion protein (PrP(C)). However, STI1 lacks a signal peptide and pharmacological approaches pointed that it does not follow a classical secretion mechanism. Ultracentrifugation, size exclusion chromatography, electron microscopy, vesicle labeling, and particle tracking analysis were used to identify three major types of extracellular vesicles (EVs) released from astrocytes with sizes ranging from 20-50, 100-200, and 300-400 nm. These EVs carry STI1 and present many exosomal markers, even though only a subpopulation had the typical exosomal morphology. The only protein, from those evaluated here, present exclusively in vesicles that have exosomal morphology was PrP(C). STI1 partially co-localized with Rab5 and Rab7 in endosomal compartments, and a dominant-negative for vacuolar protein sorting 4A (VPS4A), required for formation of multivesicular bodies (MVBs), impaired EV and STI1 release. Flow cytometry and PK digestion demonstrated that STI1 localized to the outer leaflet of EVs, and its association with EVs greatly increased STI1 activity upon PrP(C)-dependent neuronal signaling. These results indicate that astrocytes secrete a diverse population of EVs derived from MVBs that contain STI1 and suggest that the interaction between EVs and neuronal surface components enhances STI1-PrP(C) signaling.

  7. Superpriming of synaptic vesicles as a common basis for intersynapse variability and modulation of synaptic strength

    PubMed Central

    Taschenberger, Holger; Woehler, Andrew; Neher, Erwin

    2016-01-01

    Glutamatergic synapses show large variations in strength and short-term plasticity (STP). We show here that synapses displaying an increased strength either after posttetanic potentiation (PTP) or through activation of the phospholipase-C–diacylglycerol pathway share characteristic properties with intrinsically strong synapses, such as (i) pronounced short-term depression (STD) during high-frequency stimulation; (ii) a conversion of that STD into a sequence of facilitation followed by STD after a few conditioning stimuli at low frequency; (iii) an equalizing effect of such conditioning stimulation, which reduces differences among synapses and abolishes potentiation; and (iv) a requirement of long periods of rest for reconstitution of the original STP pattern. These phenomena are quantitatively described by assuming that a small fraction of “superprimed” synaptic vesicles are in a state of elevated release probability (p ∼ 0.5). This fraction is variable in size among synapses (typically about 30%), but increases after application of phorbol ester or during PTP. The majority of vesicles, released during repetitive stimulation, have low release probability (p ∼ 0.1), are relatively uniform in number across synapses, and are rapidly recruited. In contrast, superprimed vesicles need several seconds to be regenerated. They mediate enhanced synaptic strength at the onset of burst-like activity, the impact of which is subject to modulation by slow modulatory transmitter systems. PMID:27432975

  8. SUMOylation of synapsin Ia maintains synaptic vesicle availability and is reduced in an autism mutation

    PubMed Central

    Tang, Leo T. -H.; Craig, Tim J.; Henley, Jeremy M.

    2015-01-01

    Synapsins are key components of the presynaptic neurotransmitter release machinery. Their main role is to cluster synaptic vesicles (SVs) to each other and anchor them to the actin cytoskeleton to establish the reserve vesicle pool, and then release them in response to appropriate membrane depolarization. Here we demonstrate that SUMOylation of synapsin Ia (SynIa) at K687 is necessary for SynIa function. Replacement of endogenous SynIa with a non-SUMOylatable mutant decreases the size of the releasable vesicle pool and impairs stimulated SV exocytosis. SUMOylation enhances SynIa association with SVs to promote the efficient reclustering of SynIa following neuronal stimulation and maintain its presynaptic localization. The A548T mutation in SynIa is strongly associated with autism and epilepsy and we show that it leads to defective SynIa SUMOylation. These results identify SUMOylation as a fundamental regulator of SynIa function and reveal a novel link between reduced SUMOylation of SynIa and neurological disorders. PMID:26173895

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. Producing Long-term Series of Whole-Stream Metabolism Using Readily Available Data

    NASA Astrophysics Data System (ADS)

    Pai, H.; Villamizar, S. R.; Harmon, T. C.

    2015-12-01

    Continuous water quality and river discharge data that are readily available through government websites may be used to produce valuable information about key processes within a river ecosystem. In this work, we describe in detail the steps for acquisition and processing of river flow, dissolved oxygen, temperature, and specific conductance data that, combined with atmospheric data and physical properties of the river reach of interest, allow for the production of a long-term series of whole stream metabolism estimates, an important piece of information for the purposes of understanding the structure and function of river ecosystems. The restoration reach of the San Joaquin River in California (USA) has been intensively instrumented since 2010 and serves as an ideal case for testing this tool. The set of scripts, written in R code, can be used immediately for any other river in California for which the key parameters (river flow, dissolved oxygen, temperature, and specific conductivity) are available, and can be modified by the new users to fit their particular site conditions.

  17. Model of Tryptophan Metabolism, Readily Scalable Using Tissue-specific Gene Expression Data*

    PubMed Central

    Stavrum, Anne-Kristin; Heiland, Ines; Schuster, Stefan; Puntervoll, Pål; Ziegler, Mathias

    2013-01-01

    Tryptophan is utilized in various metabolic routes including protein synthesis, serotonin, and melatonin synthesis and the kynurenine pathway. Perturbations in these pathways have been associated with neurodegenerative diseases and cancer. Here we present a comprehensive kinetic model of the complex network of human tryptophan metabolism based upon existing kinetic data for all enzymatic conversions and transporters. By integrating tissue-specific expression data, modeling tryptophan metabolism in liver and brain returned intermediate metabolite concentrations in the physiological range. Sensitivity and metabolic control analyses identified expected key enzymes to govern fluxes in the branches of the network. Combining tissue-specific models revealed a considerable impact of the kynurenine pathway in liver on the concentrations of neuroactive derivatives in the brain. Moreover, using expression data from a cancer study predicted metabolite changes that resembled the experimental observations. We conclude that the combination of the kinetic model with expression data represents a powerful diagnostic tool to predict alterations in tryptophan metabolism. The model is readily scalable to include more tissues, thereby enabling assessment of organismal tryptophan metabolism in health and disease. PMID:24129579

  18. Is the lower atmosphere a readily accessible reservoir of culturable, antimicrobial compound-producing Actinomycetales?

    PubMed Central

    Weber, Carolyn F.; Werth, Jason T.

    2015-01-01

    Recent metagenomic studies have revealed that microbial diversity in the atmosphere rivals that of surface environments. This indicates that the atmosphere may be worth bioprospecting in for novel microorganisms, especially those selected for by harsh atmospheric conditions. This is interesting in light of the antibiotic resistance crisis and renewed interests in bioprospecting for members of the Actinomycetales, which harbor novel secondary metabolite-producing pathways and produce spores that make them well suited for atmospheric travel. The latter leads to the hypothesis that the atmosphere may be a promising environment in which to search for novel Actinomycetales. Although ubiquitous in soils, where bioprospecting efforts for Actinomycetales have been and are largely still focused, we present novel data indicating that culturable members of this taxonomic order are 3–5.6 times more abundant in air samples collected at 1.5, 4.5, 7.5, and 18 m above the ground, than in the underlying soil. These results support the hypothesis that mining the vast and readily accessible lower atmosphere for novel Actinomycetales in the search for undescribed secondary metabolites could prove fruitful. PMID:26300868

  19. Field and Lava Flow Experiment Analysis of Vesicle Deformation as a Means of Determining Ancient Flow Direction

    NASA Astrophysics Data System (ADS)

    McColl, B.; Teasdale, R.

    2006-12-01

    The goal of this work is to test whether flow direction of ancient lavas can be determined from orientations of preserved vesicles. We have attempted to correlate field observations with lab experiments as a means of understanding the development of deformed vesicles. This work focuses on vesicles deformed parallel to the lava flow direction. On a fieldtrip, we observed deformed vesicles in basaltic lava flows at cinder cones in the Coso Volcanic Field. Other basalt flows with similarly deformed vesicles are also documented in the Lovejoy Basalt (Chico, CA) and in flows at Lava Beds National Monument, Medicine Lake Volcanic Field. We believe that the vesicles were deformed during lava flow emplacement and cooling. Analog flow experiments used materials with Newtonian behavior (honey, syrup) but Bingham fluid behavior is more similar to natural lavas so gelatin was also attempted. Experiments started with the analog fluids on a horizontal surface. Air was then injected into the fluids with a hypodermic needle and then the surface was inclined to approximately 4-5 degrees. The deformation of the bubbles in the analog fluids was recorded with digital photos taken from above the flows. In some cases, bubbles rose to the surface of the flow and were not deformed parallel to the flow direction. In other cases, bubbles were deformed and we recorded a bulbous end and elongate tail parallel to the flow direction. In all cases the bulbous end of deformed vesicles are directed down stream and a tail stretches behind. Honey best preserved vesicle deformation. Bubbles in syrup rose to the surface too quickly to document (even when syrup was chilled). Air injected into gelatin caused shear, releasing the air without forming bubbles. Future work will address analog material issues by using wax or polyethylene glycol (PEG). These materials are likely to better represent rheologies of basalt lavas during flow emplacement.

  20. Disruption of the presynaptic cytomatrix protein bassoon degrades ribbon anchorage, multiquantal release, and sound encoding at the hair cell afferent synapse.

    PubMed

    Jing, Zhizi; Rutherford, Mark A; Takago, Hideki; Frank, Thomas; Fejtova, Anna; Khimich, Darina; Moser, Tobias; Strenzke, Nicola

    2013-03-06

    Inner hair cells (IHCs) of the cochlea use ribbon synapses to transmit auditory information faithfully to spiral ganglion neurons (SGNs). In the present study, we used genetic disruption of the presynaptic scaffold protein bassoon in mice to manipulate the morphology and function of the IHC synapse. Although partial-deletion mutants lacking functional bassoon (Bsn(ΔEx4/5)) had a near-complete loss of ribbons from the synapses (up to 88% ribbonless synapses), gene-trap mutants (Bsn(gt)) showed weak residual expression of bassoon and 56% ribbonless synapses, whereas the remaining 44% had a loosely anchored ribbon. Patch-clamp recordings and synaptic CaV1.3 immunolabeling indicated a larger number of Ca(2+) channels for Bsn(gt) IHCs compared with Bsn(ΔEx4/5) IHCs and for Bsn(gt) ribbon-occupied versus Bsn(gt) ribbonless synapses. An intermediate phenotype of Bsn(gt) IHCs was also found by membrane capacitance measurements for sustained exocytosis, but not for the size of the readily releasable vesicle pool. The frequency and amplitude of EPSCs were reduced in Bsn(ΔEx4/5) mouse SGNs, whereas their postsynaptic AMPA receptor clusters were largely unaltered. Sound coding in SGN, assessed by recordings of single auditory nerve fibers and their population responses in vivo, was similarly affected in Bsn(gt) and Bsn(ΔEx4/5) mice. Both genotypes showed impaired sound onset coding and reduced evoked and spontaneous spike rates. In summary, reduced bassoon expression or complete lack of full-length bassoon impaired sound encoding to a similar extent, which is consistent with the comparable reduction of the readily releasable vesicle pool. This suggests that the remaining loosely anchored ribbons in Bsn(gt) IHCs were functionally inadequate or that ribbon independent mechanisms dominated the coding deficit.

  1. 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

  2. Erk1/2 inhibit synaptic vesicle exocytosis through L type calcium channels

    PubMed Central

    Subramanian, Jaichandar; Morozov, Alexei

    2011-01-01

    L type calcium channels play only a minor role in basal neurotransmitter release in brain neurons, but contribute significantly after induction of plasticity. Very little is known about mechanisms that enable L type calcium channel participation in neurotransmitter release. Here, using mouse primary cortical neurons, we found that inhibition of Erk1/2 enhanced synaptic vesicle exocytosis by increasing calcium influx through L type calcium channels. Furthermore, inhibition of Erk1/2 increased the surface fraction of these channels. These findings indicate a novel inhibitory effect of Erk1/2 on synaptic transmission through L type calcium channels. PMID:21430174

  3. High-throughput homogeneous immunoassay readily identifies monoclonal antibody to serovariant clostridial neurotoxins.

    PubMed

    Corbett, Cindi R; Elias, Mohammad D; Simpson, Lance L; Yuan, Xin-Yong; Cassan, Robyn R; Ballegeer, Erin; Kabani, Amin; Plummer, Francis A; Berry, Jody D

    2007-12-01

    High-throughput screening can create the potential ability to screen large numbers of monoclonal antibodies (mAb) in a short time period. A major bottleneck in the hybridoma method for mAb development has historically been the inability to sift through large numbers of hybridoma culture supernatants to identify clones secreting mAbs of the desired specificity. Herein, we develop a homogeneous fluorometric microvolume assay technology (FMAT) and compare it to conventional ELISA screening techniques for monoclonal antibody against soluble protein toxin fragments of the Clostridium botulinum types A, B and E neurotoxin (BoNT) proteins. In total 8,744 hybridoma clones were screened to identify 29 stable hybridomas to neurotoxin binding domain; six of these would have been missed by ELISA alone. Screening of hybridoma supernatants on days 1 and 4 following cloning from semi-solid HAT agarose reveals that FMAT provides a reliable method for screening hybridoma clones to purified protein toxins. The homogeneous FMAT utilizes far less reagent (antigen and hybridoma supernatant) allowing for simultaneous screening against multiple serovariant antigens early in the hybridoma cloning cycle. This reduces costs for reagents and labour by lowering numbers of clones being maintained with undesired specificity. Furthermore, this assay easily accommodates replicate screening which facilitates identification of cross-reactivity to neurotoxin serotypes, thus readily identifying mAb to serovariant antigens. These findings have broad application in accelerating mAb development to serovariant cell-surface or bead bound targets without arraying devices. In summary, FMAT provides a reliable method for the screening of mAbs against C. botulinum neurotoxins.

  4. Virtual screening using combinatorial cyclic peptide libraries reveals protein interfaces readily targetable by cyclic peptides.

    PubMed

    Duffy, Fergal J; O'Donovan, Darragh; Devocelle, Marc; Moran, Niamh; O'Connell, David J; Shields, Denis C

    2015-03-23

    Protein-protein and protein-peptide interactions are responsible for the vast majority of biological functions in vivo, but targeting these interactions with small molecules has historically been difficult. What is required are efficient combined computational and experimental screening methods to choose among a number of potential protein interfaces worthy of targeting lead macrocyclic compounds for further investigation. To achieve this, we have generated combinatorial 3D virtual libraries of short disulfide-bonded peptides and compared them to pharmacophore models of important protein-protein and protein-peptide structures, including short linear motifs (SLiMs), protein-binding peptides, and turn structures at protein-protein interfaces, built from 3D models available in the Protein Data Bank. We prepared a total of 372 reference pharmacophores, which were matched against 108,659 multiconformer cyclic peptides. After normalization to exclude nonspecific cyclic peptides, the top hits notably are enriched for mimetics of turn structures, including a turn at the interaction surface of human α thrombin, and also feature several protein-binding peptides. The top cyclic peptide hits also cover the critical "hot spot" interaction sites predicted from the interaction crystal structure. We have validated our method by testing cyclic peptides predicted to inhibit thrombin, a key protein in the blood coagulation pathway of important therapeutic interest, identifying a cyclic peptide inhibitor with lead-like activity. We conclude that protein interfaces most readily targetable by cyclic peptides and related macrocyclic drugs may be identified computationally among a set of candidate interfaces, accelerating the choice of interfaces against which lead compounds may be screened.

  5. Invasive Bighead and Silver Carps Form Different Sized Shoals that Readily Intermix

    PubMed Central

    Ghosal, Ratna; Xiong, Peter X.; Sorensen, Peter W.

    2016-01-01

    Two species of congeneric filter-feeding microphagous carps from Asia, the bighead and the silver carp, were recently introduced to North America and have become highly invasive. These species of carp have similar food habits but the silver carp has the unique habit of jumping when disturbed. Both species have complex but poorly understood social behaviors and while both are thought to aggregate (form groups) and shoal (form tight social groups), this possibility has not yet been examined in these species. The present study examined the grouping tendencies of these species in the laboratory and the effects of fish density and species identity on it. Using nearest neighbor distance (NND) as a metric, we showed that both juvenile bighead and juvenile silver carp grouped (aggregate) strongly (P<0.05) but to different extents, and that fish density had no effect (P>0.05) on this behavior. Within aggregations, bighead carp tended to form a single large shoal while silver carp formed shoals of 2–3 individuals. Further, when tested as mixed-species groups, bighead and silver carp readily shoaled with each other but not with the common carp, which is from Eurasia and a member of another feeding guild. Due to their similar feeding strategies, we speculate that the bighead and silver carp tend to aggregate and shoal to facilitate both their foraging efforts and to avoid predation, while the differences in the size of the shoals they form may seemingly reflect their different anti-predation strategies. These complex shoaling behaviors likely influence Asian carp distribution in rivers, and thus how they might be sampled and managed. PMID:27276024

  6. Most of the Dominant Members of Amphibian Skin Bacterial Communities Can Be Readily Cultured

    PubMed Central

    Becker, Matthew H.; Hughey, Myra C.; Swartwout, Meredith C.; Jensen, Roderick V.; Belden, Lisa K.

    2015-01-01

    Currently, it is estimated that only 0.001% to 15% of bacteria in any given system can be cultured by use of commonly used techniques and media, yet culturing is critically important for investigations of bacterial function. Despite this situation, few studies have attempted to link culture-dependent and culture-independent data for a single system to better understand which members of the microbial community are readily cultured. In amphibians, some cutaneous bacterial symbionts can inhibit establishment and growth of the fungal pathogen Batrachochytrium dendrobatidis, and thus there is great interest in using these symbionts as probiotics for the conservation of amphibians threatened by B. dendrobatidis. The present study examined the portion of the culture-independent bacterial community (based on Illumina amplicon sequencing of the 16S rRNA gene) that was cultured with R2A low-nutrient agar and whether the cultured bacteria represented rare or dominant members of the community in the following four amphibian species: bullfrogs (Lithobates catesbeianus), eastern newts (Notophthalmus viridescens), spring peepers (Pseudacris crucifer), and American toads (Anaxyrus americanus). To determine which percentage of the community was cultured, we clustered Illumina sequences at 97% similarity, using the culture sequences as a reference database. For each amphibian species, we cultured, on average, 0.59% to 1.12% of each individual's bacterial community. However, the average percentage of bacteria that were culturable for each amphibian species was higher, with averages ranging from 2.81% to 7.47%. Furthermore, most of the dominant operational taxonomic units (OTUs), families, and phyla were represented in our cultures. These results open up new research avenues for understanding the functional roles of these dominant bacteria in host health. PMID:26162880

  7. 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.

  8. Structural basis of synaptic vesicle assembly promoted by α-synuclein

    PubMed Central

    Fusco, Giuliana; Pape, Tillmann; Stephens, Amberley D.; Mahou, Pierre; Costa, Ana Rita; Kaminski, Clemens F.; Kaminski Schierle, Gabriele S.; Vendruscolo, Michele; Veglia, Gianluigi; Dobson, Christopher M.; De Simone, Alfonso

    2016-01-01

    α-synuclein (αS) is an intrinsically disordered protein whose fibrillar aggregates are the major constituents of Lewy bodies in Parkinson's disease. Although the specific function of αS is still unclear, a general consensus is forming that it has a key role in regulating the process of neurotransmitter release, which is associated with the mediation of synaptic vesicle interactions and assembly. Here we report the analysis of wild-type αS and two mutational variants linked to familial Parkinson's disease to describe the structural basis of a molecular mechanism enabling αS to induce the clustering of synaptic vesicles. We provide support for this ‘double-anchor' mechanism by rationally designing and experimentally testing a further mutational variant of αS engineered to promote stronger interactions between synaptic vesicles. Our results characterize the nature of the active conformations of αS that mediate the clustering of synaptic vesicles, and indicate their relevance in both functional and pathological contexts. PMID:27640673

  9. Endothelial Cell-Surface Gp60 Activates Vesicle Formation and Trafficking via Gi-Coupled Src Kinase Signaling Pathway

    PubMed Central

    Minshall, Richard D.; Tiruppathi, Chinnaswamy; Vogel, Stephen M.; Niles, Walter D.; Gilchrist, Annette; Hamm, Heidi E.; Malik, Asrar B.

    2000-01-01

    We tested the hypothesis that the albumin-docking protein gp60, which is localized in caveolae, couples to the heterotrimeric GTP binding protein Gi, and thereby activates plasmalemmal vesicle formation and the directed migration of vesicles in endothelial cells (ECs). We used the water-soluble styryl pyridinium dye N-(3-triethylaminopropyl)-4-(p-dibutylaminostyryl) pyridinium dibromide (FM 1-43) to quantify vesicle trafficking by confocal and digital fluorescence microscopy. FM 1-43 and fluorescently labeled anti-gp60 antibody (Ab) were colocalized in endocytic vesicles within 5 min of gp60 activation. Vesicles migrated to the basolateral surface where they released FM 1-43, the fluid phase styryl probe. FM 1-43 fluorescence disappeared from the basolateral EC surface without the loss of anti-gp60 Ab fluorescence. Activation of cell-surface gp60 by cross-linking (using anti-gp60 Ab and secondary Ab) in EC grown on microporous filters increased transendothelial 125I-albumin permeability without altering liquid permeability (hydraulic conductivity), thus, indicating the dissociation of hydraulic conductivity from the albumin permeability pathway. The findings that the sterol-binding agent, filipin, prevented gp60-activated vesicle formation and that caveolin-1 and gp60 were colocalized in vesicles suggest the caveolar origin of endocytic vesicles. Pertussis toxin pretreatment and expression of the dominant negative construct encoding an 11–amino acid Gαi carboxyl-terminal peptide inhibited endothelial 125I-albumin endocytosis and vesicle formation induced by gp60 activation. Expression of dominant negative Src (dn-Src) and overexpression of wild-type caveolin-1 also prevented gp60-activated endocytosis. Caveolin-1 overexpression resulted in the sequestration of Gαi with the caveolin-1, whereas dn-Src inhibited Gαi binding to caveolin-1. Thus, vesicle formation induced by gp60 and migration of vesicles to the basolateral membrane requires the interaction of gp60

  10. Protein-protein interactions and protein modules in the control of neurotransmitter release.

    PubMed Central

    Benfenati, F; Onofri, F; Giovedí, S

    1999-01-01

    Information transfer among neurons is operated by neurotransmitters stored in synaptic vesicles and released to the extracellular space by an efficient process of regulated exocytosis. Synaptic vesicles are organized into two distinct functional pools, a large reserve pool in which vesicles are restrained by the actin-based cytoskeleton, and a quantitatively smaller releasable pool in which vesicles approach the presynaptic membrane and eventually fuse with it on stimulation. Both synaptic vesicle trafficking and neurotransmitter release depend on a precise sequence of events that include release from the reserve pool, targeting to the active zone, docking, priming, fusion and endocytotic retrieval of synaptic vesicles. These steps are mediated by a series of specific interactions among cytoskeletal, synaptic vesicle, presynaptic membrane and cytosolic proteins that, by acting in concert, promote the spatial and temporal regulation of the exocytotic machinery. The majority of these interactions are mediated by specific protein modules and domains that are found in many proteins and are involved in numerous intracellular processes. In this paper, the possible physiological role of these multiple protein-protein interactions is analysed, with ensuing updating and clarification of the present molecular model of the process of neurotransmitter release. PMID:10212473

  11. 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

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

    PubMed Central

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

    2014-01-01

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

  13. 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

  14. 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

  15. Role of calcium and vesicle-docking proteins in remobilising dormant neuromuscular junctions in desert frogs.

    PubMed

    Lavidis, Nickolas A; Hudson, Nicholas J; Choy, Peng T; Lehnert, Sigrid A; Franklin, Craig E

    2008-01-01

    Despite prolonged immobility the desert frog, Cyclorana alboguttata, suffers little impairment in muscle function. To determine compensatory mechanisms at neuromuscular junctions, transmitter release was examined along primary terminals in C. alboguttata iliofibularis muscle. Using extracellular recording we found the amplitudes of evoked endplate currents were significantly smaller in dormant frogs. In active frogs we identified two negatively sloping proximal-distal gradients of transmitter frequency and quantal content; a shallow proximal-distal gradient with low probability of transmitter release (<0.2) and a second much steeper proximal-distal gradient for quantal content with high probability release sites (>0.6). During aestivation, only a shallow gradient was identified. The high probability release sites in control frogs were inhibited during aestivation by a mechanism that could be reversed by (1) increasing the extracellular calcium concentration, and (2) increasing the frequency of stimulation. This suggests that transmitter vesicles are available during aestivation but not released. We quantified expression of messenger RNA transcripts coding for the transmitter vesicle-docking proteins synaptotagmin 1, syntaxin 1B and UNC-13. All three were rare transcripts maintained at control values during aestivation. Neuromuscular remobilisation after dormancy in C. alboguttata is more likely a product of rapidly reversible physiologic mechanisms than reorganisations of the neuromuscular transcriptome.

  16. CD30 on extracellular vesicles from malignant Hodgkin cells supports damaging of CD30 ligand-expressing bystander cells with Brentuximab-Vedotin, in vitro

    PubMed Central

    Hansen, Hinrich P.; Trad, Ahmad; Dams, Maria; Zigrino, Paola; Moss, Marcia; Tator, Maximilian; Schön, Gisela; Grenzi, Patricia C; Bachurski, Daniel; Aquino, Bruno; Dürkop, Horst; Reiners, Katrin S; von Bergwelt-Baildon, Michael; Hallek, Michael; Grötzinger, Joachim; Engert, Andreas; Leme, Adriana F Paes; von Strandmann, Elke Pogge

    2016-01-01

    The goal of targeted immunotherapy in cancer is to damage both malignant and tumor-supporting cells of the microenvironment but spare unaffected tissue. The malignant cells in classical Hodgkin lymphoma (cHL) selectively express CD30. They release this receptor on extracellular vesicles (EVs) for the tumor-supporting communication with CD30 ligand (CD30L)-positive bystander cells. Here, we investigated how CD30-positive EVs influence the efficacy of the CD30 antibody drug conjugate (ADC) Brentuximab Vedotin (SGN-35). The malignant cells and the EVs expressed the active sheddase ADAM10. ADAM10 cleaved and released the CD30 ectodomain (sCD30), causing a gradual depletion of SGN-35 binding sites on EVs and creating a soluble competitor of the ADC therapy. In a 3D semi-solid tumor microenvironment model, the EVs were retained in the matrix whereas sCD30 penetrated readily into the surrounding culture medium. This resulted in a lowered ratio of EV-associated CD30 (CD30EV) to sCD30 in the surrounding medium in comparison to non-embedded cultures. A low percentage of CD30EV was also detected in the plasma of cHL patients, supporting the clinical relevance of the model. The adherence of CD30EV but not sCD30 to CD30−/CD30L+ mast cells and eosinophils allowed the indirect binding of SGN-35. Moreover, SGN-35 damaged CD30-negative cells, provided they were loaded with CD30+ EVs. PMID:27105521

  17. Osmotic behaviour of polyhedral non-ionic surfactant vesicles (niosomes).

    PubMed

    Arunothayanun, P; Uchegbu, I F; Florence, A T

    1999-06-01

    In addition to common spherical non-ionic surfactant vesicles (niosomes), disc-like, tubular, and polyhedral niosomes have also been reported. The permeability and osmotic activity of niosomes are important in determining their use as controlled-release drug-delivery systems. These properties have been compared for polyhedral niosomes prepared by hydrating a mixture of a hexadecyl diglycerol ether (C16G2), a poly(24)oxyethylene cholesteryl ether (Solulan C24), 91:9 or 98:2, and conventional spherical niosomes prepared from the same surfactants but with cholesterol. When subjected to osmotic gradients, polyhedral niosomes, the membranes of which are in the gel phase, swell and shrink less than their spherical counterparts and they are more permeable to the hydrophilic solute 5(6)-carboxyfluorescein. In 2 M NaCl the rate of release of carboxyfluorescein from polyhedral niosomes (both containing 9% Solulan C24) into either a hypotonic (water) or an isotonic medium (2 M NaCl) was low. This contrasted with similarly loaded spherical niosomes and polyhedral niosomes containing 2% Solulan C24, from which release was high in hypotonic media (e.g. water) but less in an isotonic medium (2 M NaCl). For both polyhedral and spherical niosomes encapsulating carboxyfluorescein (pKa = 6.4), release rates were higher at pH 8 than at pH 5. Polyhedral niosomes are thus, in general, less osmotically active than spherical niosomes because of their rigid but highly permeable membranes. The unusual polyhedral membrane impermeability to carboxyfluorescein co-entrapped with salt in hypotonic media is a function of Solulan C24 content, and is possibly a result of salting out of the polyoxyethylene chains; this is, therefore, a property that might be manipulated in the design of a drug-delivery system.

  18. 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.

  19. 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.

  20. 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.

  1. WET MARS: plentiful, readily-available martian water and its implications

    SciTech Connect

    Hyde, R; Ishikawa, M; Nuckolls, J; Whitehead, J; Wood, L

    1999-08-12

    on Martian atmospheric water. All of the oxygen, water and propellants necessary for life-support, extensive exploration and Earth-return can be provided quite readily by the host planet. The most challenging technical problem with respect to human expeditions to Mars is that of escaping from Earth's deep, 11.2 km/s gravity well. Living on Mars, exploring it extensively and returning to Earth each are technically much less difficult, thanks in no small part to the effective ''wetness'' of Mars.

  2. LEAP into the Pfizer Global Virtual Library (PGVL) space: creation of readily synthesizable design ideas automatically.

    PubMed

    Hu, Qiyue; Peng, Zhengwei; Kostrowicki, Jaroslav; Kuki, Atsuo

    2011-01-01

    Pfizer Global Virtual Library (PGVL) of 10(13) readily synthesizable molecules offers a tremendous opportunity for lead optimization and scaffold hopping in drug discovery projects. However, mining into a chemical space of this size presents a challenge for the concomitant design informatics due to the fact that standard molecular similarity searches against a collection of explicit molecules cannot be utilized, since no chemical information system could create and manage more than 10(8) explicit molecules. Nevertheless, by accepting a tolerable level of false negatives in search results, we were able to bypass the need for full 10(13) enumeration and enabled the efficient similarity search and retrieval into this huge chemical space for practical usage by medicinal chemists. In this report, two search methods (LEAP1 and LEAP2) are presented. The first method uses PGVL reaction knowledge to disassemble the incoming search query molecule into a set of reactants and then uses reactant-level similarities into actual available starting materials to focus on a much smaller sub-region of the full virtual library compound space. This sub-region is then explicitly enumerated and searched via a standard similarity method using the original query molecule. The second method uses a fuzzy mapping onto candidate reactions and does not require exact disassembly of the incoming query molecule. Instead Basis Products (or capped reactants) are mapped into the query molecule and the resultant asymmetric similarity scores are used to prioritize the corresponding reactions and reactant sets. All sets of Basis Products are inherently indexed to specific reactions and specific starting materials. This again allows focusing on a much smaller sub-region for explicit enumeration and subsequent standard product-level similarity search. A set of validation studies were conducted. The results have shown that the level of false negatives for the disassembly-based method is acceptable when the

  3. Glucose and lactate are equally effective in energizing activity-dependent synaptic vesicle turnover in purified cortical neurons.

    PubMed

    Morgenthaler, F D; Kraftsik, R; Catsicas, S; Magistretti, P J; Chatton, J-Y

    2006-08-11

    This study examines the role of glucose and lactate as energy substrates to sustain synaptic vesicle cycling. Synaptic vesicle turnover was assessed in a quantitative manner by fluorescence microscopy in primary cultures of mouse cortical neurons. An electrode-equipped perfusion chamber was used to stimulate cells both by electrical field and potassium depolarization during image acquisition. An image analysis procedure was elaborated to select in an unbiased manner synaptic boutons loaded with the fluorescent dye N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM1-43). Whereas a minority of the sites fully released their dye content following electrical stimulation, others needed subsequent K(+) depolarization to achieve full release. This functional heterogeneity was not significantly altered by the nature of metabolic substrates. Repetitive stimulation sequences of FM1-43 uptake and release were then performed in the absence of any metabolic substrate and showed that the number of active sites dramatically decreased after the first cycle of loading/unloading. The presence of 1 mM glucose or lactate was sufficient to sustain synaptic vesicle cycling under these conditions. Moreover, both substrates were equivalent for recovery of function after a phase of decreased metabolic substrate availability. Thus, lactate appears to be equivalent to glucose for sustaining synaptic vesicle turnover in cultured cortical neurons during activity.

  4. 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

  5. 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.

  6. 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

  7. Outer membrane vesicles of Tannerella forsythia: biogenesis, composition, and virulence.

    PubMed

    Friedrich, V; Gruber, C; Nimeth, I; Pabinger, S; Sekot, G; Posch, G; Altmann, F; Messner, P; Andrukhov, O; Schäffer, C

    2015-12-01

    Tannerella forsythia is the only 'red-complex' bacterium covered by an S-layer, which has been shown to affect virulence. Here, outer membrane vesicles (OMVs) enriched with putative glycoproteins are described as a new addition to the virulence repertoire of T. forsythia. Investigations of this bacterium are hampered by its fastidious growth requirements and the recently discovered mismatch of the available genome sequence (92A2 = ATCC BAA-2717) and the widely used T. forsythia strain (ATCC 43037). T. forsythia was grown anaerobically in serum-free medium and biogenesis of OMVs was analyzed by electron and atomic force microscopy. This revealed OMVs with a mean diameter of ~100 nm budding off from the outer membrane while retaining the S-layer. An LC-ESI-TOF/TOF proteomic analysis of OMVs from three independent biological replicates identified 175 proteins. Of these, 14 exhibited a C-terminal outer membrane translocation signal that directs them to the cell/vesicle surface, 61 and 53 were localized to the outer membrane and periplasm, respectively, 22 were predicted to be extracellular, and 39 to originate from the cytoplasm. Eighty proteins contained the Bacteroidales O-glycosylation motif, 18 of which were confirmed as glycoproteins. Release of pro-inflammatory mediators from the human monocytic cell line U937 and periodontal ligament fibroblasts upon stimulation with OMVs followed a concentration-dependent increase that was more pronounced in the presence of soluble CD14 in conditioned media. The inflammatory response was significantly higher than that caused by whole T. forsythia cells. Our study represents the first characterization of T. forsythia OMVs, their proteomic composition and immunogenic potential.

  8. 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.

  9. 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

  10. Acute lymphoblastic leukaemia cells produce large extracellular vesicles containing organelles and an active cytoskeleton

    PubMed Central

    Johnson, Suzanne M.; Dempsey, Clare; Parker, Catriona; Mironov, Aleksandr; Bradley, Helen; Saha, Vaskar

    2017-01-01

    ABSTRACT Extracellular vesicles have been described in non-paracrine cellular interactions in cancer. We report a similar phenomenon in B-cell precursor (BCP) acute lymphoblastic leukaemia (ALL). Using advanced microscopy and high throughput screening, we further characterise a subset of large vesicles (LEVs) identified in cell lines, murine models of human BCP-ALL and clinical samples. Primary ALL blasts and cell lines released heterogeneous anucleate vesicles <6 micron into extracellular fluids. Larger LEVs were enclosed in continuous membranes, contained intact organelles and demonstrated an organised cytoskeleton. An excess of circulating CD19-positive LEVs were observed in diagnostic samples and isolated from mice engrafted with BCP-ALL primary cells. LEVs exhibited dynamic shape change in vitro and were internalised by other leukaemic cell lines leading to phenotypic transformation analogous to the cell of origin. In patient-derived xenografts, LEVs were released by primary ALL cells into extracellular spaces and internalised by murine mesenchymal cells in vivo. Collectively these data highlight the heterogeneity but accessibility of LEVs in clinical samples and their potential to provide a unique insight into the biology of the cell of origin and to their development as novel biomarkers to aid diagnosis and improve therapeutic outcomes. PMID:28386390

  11. Ultrastructural analysis of chlamydial antigen-containing vesicles everting from the Chlamydia trachomatis inclusion.

    PubMed

    Giles, David K; Whittimore, Judy D; LaRue, Richard W; Raulston, Jane E; Wyrick, Priscilla B

    2006-05-01

    Several chlamydial antigens have been detected in the infected epithelial cell cytosol and on the host cell surface prior to their presumed natural release at the end of the 72-96 h developmental cycle. These extra-inclusion antigens are proposed to influence vital host cell functions, antigen trafficking and presentation and, ultimately, contribute to a prolonged inflammatory response. To begin to dissect the mechanisms for escape of these antigens from the chlamydial inclusion, which are enhanced on exposure to antibiotics, polarized endometrial epithelial cells (HEC-1B) were infected with Chlamydia trachomatis serovar E for 36 h or 48 h. Infected cells were then exposed to chemotactic human polymorphonuclear neutrophils not loaded or pre-loaded in vitro with the antibiotic azithromycin. Viewed by electron microscopy, the azithromycin-mediated killing of chlamydiae involved an increase in chlamydial outer membrane blebbing followed by the appearance of the blebs in larger vesicles (i) everting from but still associated with the inclusion as well as (ii) external to the inclusion. Evidence that the vesicles originated from the chlamydial inclusion membrane was shown by immuno-localization of inclusion membrane proteins A, F, and G on the vesicular membranes. Chlamydial heat shock protein 60 (chsp60) copies 2 and 3, but not copy 1, were released from RB and incorporated into the everted inclusion membrane vesicles and delivered to the infected cell surface. These data represent direct evidence for one mechanism of early antigen delivery, albeit membrane-bound, beyond the confines of the chlamydial inclusion.

  12. Fast retrieval and autonomous regulation of single spontaneously recycling synaptic vesicles.

    PubMed

    Leitz, Jeremy; Kavalali, Ege T

    2014-11-21

    Presynaptic terminals release neurotransmitters spontaneously in a manner that can be regulated by Ca(2+). However, the mechanisms underlying this regulation are poorly understood because the inherent stochasticity and low probability of spontaneous fusion events has curtailed their visualization at individual release sites. Here, using pH-sensitive optical probes targeted to synaptic vesicles, we visualized single spontaneous fusion events and found that they are retrieved extremely rapidly with faster re-acidification kinetics than their action potential-evoked counterparts. These fusion events were coupled to postsynaptic NMDA receptor-driven Ca(2+) signals, and at elevated Ca(2+) concentrations there was an increase in the number of vesicles that would undergo fusion. Furthermore, spontaneous vesicle fusion propensity in a synapse was Ca(2+)-dependent but regulated autonomously: independent of evoked fusion probability at the same synapse. Taken together, these results expand classical quantal analysis to incorporate endocytic and exocytic phases of single fusion events and uncover autonomous regulation of spontaneous fusion.

  13. WET MARS: Plentiful, Readily-Available Martian Water and its Implications

    SciTech Connect

    Hyde, R.; Ishikawa, M.; Nuckolls, J.; Whitehead, J.; Wood, L.

    1999-09-14

    exploiting Martian atmospheric water. All of the oxygen, water and propellants necessary for life-support, extensive exploration and Earth-return can be provided readily by the host planet. Crewed exploration of Mars launched from LEO with on1.V 2 Shuttle-loads of equipment and consumables--a commercial total cost-equivalent of {approx}$650 M--thereby becomes feasible. The most challenging current problem with respect to human expeditions to Mars is escape from Earth's deep, 11.2 km/s gravity well, and is largely an economic issue. Living on Mars, exploring it extensively and returning to Earth, each hitherto major technical issues, are actually much less difficult, thanks in no small part to the effective ''wetness'' of Mars. Similar considerations apply to other water-rich locations in the Solar system, e.g. Europa.

  14. From glioblastoma to endothelial cells through extracellular vesicles: messages for angiogenesis.

    PubMed

    Giusti, Ilaria; Delle Monache, Simona; Di Francesco, Marianna; Sanità, Patrizia; D'A