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Sample records for synaptic vesicle formation

  1. Synaptic Vesicle Endocytosis

    PubMed Central

    Saheki, Yasunori; De Camilli, Pietro

    2012-01-01

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

  2. The role of synaptotagmin I C2A calcium-binding domain in synaptic vesicle clustering during synapse formation

    PubMed Central

    Gardzinski, Peter; Lee, David W K; Fei, Guang-He; Hui, Kwokyin; Huang, Guan J; Sun, Hong-Shuo; Feng, Zhong-Ping

    2007-01-01

    Synaptic vesicles aggregate at the presynaptic terminal during synapse formation via mechanisms that are poorly understood. Here we have investigated the role of the putative calcium sensor synaptotagmin I in vesicle aggregation during the formation of soma–soma synapses between identified partner cells using a simple in vitro synapse model in the mollusc Lymnaea stagnalis. Immunocytochemistry, optical imaging and electrophysiological recording techniques were used to monitor synapse formation and vesicle localization. Within 6 h, contact between appropriate synaptic partner cells up-regulated global synaptotagmin I expression, and induced a localized aggregation of synaptotagmin I at the contact site. Cell contacts between non-synaptic partner cells did not affect synaptotagmin I expression. Application of an human immunodeficiency virus type-1 transactivator (HIV-1 TAT)-tagged peptide corresponding to loop 3 of the synaptotagmin I C2A domain prevented synaptic vesicle aggregation and synapse formation. By contrast, a TAT-tagged peptide containing the calcium-binding motif of the C2B domain did not affect synaptic vesicle aggregation or synapse formation. Calcium imaging with Fura-2 demonstrated that TAT–C2 peptides did not alter either basal or evoked intracellular calcium levels. These results demonstrate that contact with an appropriate target cell is necessary to initiate synaptic vesicle aggregation during nascent synapse formation and that the initial aggregation of synaptic vesicles is dependent on loop 3 of the C2A domain of synaptotagmin I. PMID:17317745

  3. Coordinated trafficking of synaptic vesicle and active zone proteins prior to synapse formation.

    PubMed

    Bury, Luke A D; Sabo, Shasta L

    2011-05-10

    The proteins required for synaptic transmission are rapidly assembled at nascent synapses, but the mechanisms through which these proteins are delivered to developing presynaptic terminals are not understood. Prior to synapse formation, active zone proteins and synaptic vesicle proteins are transported along axons in distinct organelles referred to as piccolo-bassoon transport vesicles (PTVs) and synaptic vesicle protein transport vesicles (STVs), respectively. Although both PTVs and STVs are recruited to the same site in the axon, often within minutes of axo-dendritic contact, it is not known whether or how PTV and STV trafficking is coordinated before synapse formation. Here, using time-lapse confocal imaging of the dynamics of PTVs and STVs in the same axon, we show that vesicle trafficking is coordinated through at least two mechanisms. First, a significant proportion of STVs and PTVs are transported together before forming a stable terminal. Second, individual PTVs and STVs share pause sites within the axon. Importantly, for both STVs and PTVs, encountering the other type of vesicle increases their propensity to pause. To determine if PTV-STV interactions are important for pausing, PTV density was reduced in axons by expression of a dominant negative construct corresponding to the syntaxin binding domain of syntabulin, which links PTVs with their KIF5B motor. This reduction in PTVs had a minimal effect on STV pausing and movement, suggesting that an interaction between STVs and PTVs is not responsible for enhancing STV pausing. Our results indicate that trafficking of STVs and PTVs is coordinated even prior to synapse development. This novel coordination of transport and pausing might provide mechanisms through which all of the components of a presynaptic terminal can be rapidly accumulated at sites of synapse formation.

  4. Cholesterol supports the retinoic acid-induced synaptic vesicle formation in differentiating human SH-SY5Y neuroblastoma cells.

    PubMed

    Sarkanen, Jertta-Riina; Nykky, Jonna; Siikanen, Jutta; Selinummi, Jyrki; Ylikomi, Timo; Jalonen, Tuula O

    2007-09-01

    Synaptic vesicle formation, vesicle activation and exo/endocytosis in the pre-synaptic area are central steps in neuronal communication. The formation and localization of synaptic vesicles in human SH-SY5Y neuroblastoma cells, differentiated with 12-o-tetradecanoyl-phorbol-13-acetate, dibutyryl cyclic AMP, all-trans-retinoic acid (RA) and cholesterol, was studied by fluorescence microscopy and immunocytochemical methods. RA alone or together with cholesterol, produced significant neurite extension and formation of cell-to-cell contacts. Synaptic vesicle formation was followed by anti-synaptophysin (SypI) and AM1-43 staining. SypI was only weakly detected, mainly in cell somata, before 7 days in vitro, after which it was found in neurites. Depolarization of the differentiated cells with high potassium solution increased the number of fluorescent puncta, as well as SypI and AM1-43 co-localization. In addition to increase in the number of synaptic vesicles, RA and cholesterol also increased the number and distribution of lysosome-associated membrane protein 2 labeled lysosomes. RA-induced Golgi apparatus fragmentation was partly avoided by co-treatment with cholesterol. The SH-SY5Y neuroblastoma cell line, differentiated by RA and cholesterol and with good viability in culture, is a valuable tool for basic studies of neuronal metabolism, specifically as a model for dopaminergic neurons.

  5. 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

  6. BDNF mobilizes synaptic vesicles and enhances synapse formation by disrupting cadherin-beta-catenin interactions.

    PubMed

    Bamji, Shernaz X; Rico, Beatriz; Kimes, Nikole; Reichardt, Louis F

    2006-07-17

    Neurons of the vertebrate central nervous system have the capacity to modify synapse number, morphology, and efficacy in response to activity. Some of these functions can be attributed to activity-induced synthesis and secretion of the neurotrophin brain-derived neurotrophic factor (BDNF); however, the molecular mechanisms by which BDNF mediates these events are still not well understood. Using time-lapse confocal analysis, we show that BDNF mobilizes synaptic vesicles at existing synapses, resulting in small clusters of synaptic vesicles "splitting" away from synaptic sites. We demonstrate that BDNF's ability to mobilize synaptic vesicle clusters depends on the dissociation of cadherin-beta-catenin adhesion complexes that occurs after tyrosine phosphorylation of beta-catenin. Artificially maintaining cadherin-beta-catenin complexes in the presence of BDNF abolishes the BDNF-mediated enhancement of synaptic vesicle mobility, as well as the longer-term BDNF-mediated increase in synapse number. Together, this data demonstrates that the disruption of cadherin-beta-catenin complexes is an important molecular event through which BDNF increases synapse density in cultured hippocampal neurons.

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. Synaptic Vesicle Pools: An Update

    PubMed Central

    Denker, Annette; Rizzoli, Silvio O.

    2010-01-01

    During the last few decades synaptic vesicles have been assigned to a variety of functional and morphological classes or “pools”. We have argued in the past (Rizzoli and Betz, 2005) that synaptic activity in several preparations is accounted for by the function of three vesicle pools: the readily releasable pool (docked at active zones and ready to go upon stimulation), the recycling pool (scattered throughout the nerve terminals and recycling upon moderate stimulation), and finally the reserve pool (occupying most of the vesicle clusters and only recycling upon strong stimulation). We discuss here the advancements in the vesicle pool field which took place in the ensuing years, focusing on the behavior of different pools under both strong stimulation and physiological activity. Several new findings have enhanced the three-pool model, with, for example, the disparity between recycling and reserve vesicles being underlined by the observation that the former are mobile, while the latter are “fixed”. Finally, a number of altogether new concepts have also evolved such as the current controversy on the identity of the spontaneously recycling vesicle pool. PMID:21423521

  12. Clathrin regenerates synaptic vesicles from endosomes

    PubMed Central

    Watanabe, Shigeki; Trimbuch, Thorsten; Camacho-Pérez, Marcial; Rost, Benjamin R.; Brokowski, Bettina; Söhl-Kielczynski, Berit; Felies, Annegret; Davis, M. Wayne; Rosenmund, Christian; Jorgensen, Erik M.

    2014-01-01

    Summary Ultrafast endocytosis can retrieve a single large endocytic vesicle as fast as 50-100 ms after synaptic vesicle fusion. However, the fate of the large endocytic vesicles is not known. Here we demonstrate that these vesicles transition to a synaptic endosome about one second after stimulation. The endosome is resolved into coated vesicles after 3 seconds, which in turn become small-diameter synaptic vesicles 5-6 seconds after stimulation. We disrupted clathrin function using RNAi and found that clathrin is not required for ultrafast endocytosis but is required to generate synaptic vesicles from the endosome. Ultrafast endocytosis fails when actin polymerization is disrupted, or when neurons are stimulated at room temperature instead of physiological temperature. In the absence of ultrafast endocytosis, synaptic vesicles are retrieved directly from the plasma membrane by clathrin-mediated endocytosis. These results explain in large part discrepancies among published experiments concerning the role of clathrin in synaptic vesicle endocytosis. PMID:25296249

  13. 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

  14. 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

  15. 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

  16. 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

  17. Signaling for Vesicle Mobilization and Synaptic Plasticity

    PubMed Central

    Levitan, Edwin S.

    2008-01-01

    The hypothesis that release of classical neurotransmitters and neuropeptides is facilitated by increasing the mobility of small synaptic vesicles (SSVs) and dense core vesicles (DCVs) could not be tested until the advent of methods for visualizing these secretory vesicles in living nerve terminals. In fact, fluorescence imaging studies have only since 2005 established that activity increases secretory vesicle mobility in motoneuron terminals and chromaffin cells. Mobilization of DCVs and SSVs appears to be due to liberation of hindered vesicles to promote quicker diffusion. However, F-actin and synapsin, which have been featured in mobilization models, are not required for activity-dependent increases in the mobility of DCVs or SSVs. Most recently, the signaling required for sustained mobilization has been identified for Drosophila motoneuron DCVs and shown to increase synaptic transmission. Specifically, presynaptic endoplasmic reticulum ryanodine receptor (RyR)-mediated Ca2+ release activates Ca2+/calmodulin-dependent kinase II (CamKII) to mobilize DCVs and induce post-tetanic potentiation (PTP) of neuropeptide release in the Drosophila neuromuscular junction. The shared signaling for increasing vesicle mobility and PTP links vesicle mobilization and synaptic plasticity. PMID:18446451

  18. 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

  19. Alignment of synaptic vesicle macromolecules with the macromolecules in active zone material that direct vesicle docking.

    PubMed

    Harlow, Mark L; Szule, Joseph A; 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 docking

  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. Involvement of vH+-ATPase in synaptic vesicle swelling

    PubMed Central

    Shin, Leah; Basi, Nirukti; Jeremic, Aleksandar; Lee, Jin-Sook; Cho, Won Jin; Chen, ZhiHui; Abu-Hamdah, Rania; Oupicky, David; Jena, Bhanu P

    2010-01-01

    Secretory vesicle swelling is central to cell secretion, however the underlying mechanism of vesicle swelling, particularly synaptic vesicles, is not completely understood. The Gαi3-PLA2-mediated involvement of water channel AQP-1 in the regulation of secretory vesicle swelling in exocrine pancreas, and the Gαo-mediated AQP-6 involvement in synaptic vesicle swelling in neurons, has previously been reported. Furthermore, the role of vH+-ATPase in neurotransmitter transport into synaptic vesicles, has also been shown. Using nanometer scale precision measurements of isolated synaptic vesicles, the present study reports for the first time, the involvement of vH+-ATPase in GTP-Gαo-mediated synaptic vesicle swelling. Results from this study, demonstrate that the GTP-Gαo-mediated vesicle swelling is vH+-ATPase–dependent, and pH sensitive. Zeta potential measurements of isolated synaptic vesicles further demonstrate, a bafilomycin-sensitive vesicle acidification, following the GTP-Gαo-induced swelling stimulus. Since water channels are bidirectional, and the vH+-ATPase inhibitor bafilomycin decreases both the volume of isolated synaptic vesicles and GTP-mastoparan stimulated swelling, suggests vH+-ATPase to be upstream of AQP-6, in the pathway leading from Gαo-stimulated swelling of synaptic vesicles. Vesicle acidification is therefore a prerequisite for AQP-6 mediated gating of water into synaptic vesicles. PMID:19610106

  2. 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

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

    PubMed

    Cole, Andy A; Chen, Xiaobing; Reese, Thomas S

    2016-03-16

    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. 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. Copyright © 2016 the authors 0270-6474/16/363222-09$15.00/0.

  4. 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

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

    PubMed Central

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

    2015-01-01

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

  6. Epsin1 modulates synaptic vesicle retrieval capacity at CNS synapses

    PubMed Central

    Kyung, Jae Won; Bae, Jae Ryul; Kim, Dae-Hwan; Song, Woo Keun; Kim, Sung Hyun

    2016-01-01

    Synaptic vesicle retrieval is an essential process for continuous maintenance of neural information flow after synaptic transmission. Epsin1, originally identified as an EPS15-interacting protein, is a major component of clathrin-mediated endocytosis. However, the role of Epsin1 in synaptic vesicle endocytosis at CNS synapses remains elusive. Here, we showed significantly altered synaptic vesicle endocytosis in neurons transfected with shRNA targeting Epsin1 during/after neural activity. Endocytosis was effectively restored by introducing shRNA-insensitive Epsin1 into Epsin1-depleted neurons. Domain studies performed on neurons in which domain deletion mutants of Epsin1 were introduced after Epsin1 knockdown revealed that ENTH, CLAP, and NPFs are essential for synaptic vesicle endocytosis, whereas UIMs are not. Strikingly, the efficacy of the rate of synaptic vesicle retrieval (the “endocytic capacity”) was significantly decreased in the absence of Epsin1. Thus, Epsin1 is required for proper synaptic vesicle retrieval and modulates the endocytic capacity of synaptic vesicles. PMID:27557559

  7. Synaptic vesicle chips to assay botulinum neurotoxins

    PubMed Central

    2005-01-01

    BoNTs (botulinum neurotoxins), considered to be the most toxic of all biological substances, inhibit neurotransmission through proteolytic cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins [VAMP (vesicle-associated membrane protein, or synaptobrevin), SNAP-25 (25 kDa synaptosome-associated protein) or syntaxin]. Expansion in the use of BoNTs as therapeutic and cosmetic agents, and the potential threat they constitute as biological weapons, underlines the need for rapid and sensitive in vitro assays. Here, we present new automatized bioassays to detect VAMP cleavage by BoNT/B and F. Western blotting and SPR (surface plasmon resonance) methods revealed that BoNT/B and F totally cleave their substrate on immunoisolated SVs (synaptic vesicles). Real-time monitoring of the immunocapture of native SVs from crude lysates on SPR sensor chips enabled the detection of picogram amounts of different SV proteins. Pre-incubation of a membrane fraction containing SVs with BoNT specifically inhibited capture by anti-VAMP antibodies, and amounts as low as 0.1 pg of BoNT/B were detected. This automated SPR assay is approx. 200 times more sensitive, and 25 times more rapid, than the in vivo BoNT/B test currently used. Moreover, the method can be performed using a few thousand cultured neurons and constitutes a new screening assay for inhibitors. Our data indicate that native VAMP is an optimal substrate for in vitro BoNT assays that can be monitored by SPR. PMID:16011482

  8. 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

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

    PubMed Central

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

    2016-01-01

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

  10. 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

  11. Evidence that vesicles undergo compound fusion on the synaptic ribbon

    PubMed Central

    Matthews, Gary; Sterling, Peter

    2008-01-01

    The ribbon synapse can release a stream of transmitter quanta at very high rates. Although the ribbon tethers numerous vesicles near the presynaptic membrane, most of the tethered vesicles are held at a considerable distance from the plasma membrane. Therefore, it remains unclear how their contents are released. We evoked prolonged bouts of exocytosis from a retinal bipolar cell, fixed within seconds, and then studied the ribbons by electron microscopy. Vesicle density on ribbons was reduced by ~50% compared with cells where exocytosis was blocked with intracellular ATP-γS. Large, irregularly shaped vesicles appeared on the ribbon in cells fixed during repetitive stimulation of exocytosis, and in some cases the large vesicles could be traced in adjacent sections to cisternae open to the medium. The large cisternal structures were attached to the ribbon by filaments similar to those that tether synaptic vesicles to the ribbon, and they occupied the base of the ribbon near the plasma membrane, where normal synaptic vesicles are found in resting cells. We suggest that the cisternae attached to ribbons represent synaptic vesicles that fused by compound exocytosis during strong repetitive stimulation, and thus that vesicles tethered to the ribbon can empty their contents by fusing to other vesicles docked at the presynaptic membrane. Such compound fusion could explain the extremely high release rates and the multivesicular release reported for auditory and visual ribbon synapses. PMID:18495874

  12. Synaptic vesicle membrane proteins interact to form a multimeric complex

    PubMed Central

    1992-01-01

    Potential interactions between membrane components of rat brain synaptic vesicles were analyzed by detergent solubilization followed by size fractionation or immunoprecipitation. The behavior of six synaptic vesicle membrane proteins as well as a plasma membrane protein was monitored by Western blotting. Solubilization of synaptic vesicle membranes in CHAPS resulted in the recovery of a large protein complex that included SV2, p65, p38, vesicle-associated membrane protein, and the vacuolar proton pump. Solubilization in octylglucoside resulted in the preservation of interactions between SV2, p38, and rab3A, while solubilization of synaptic vesicles with Triton X-100 resulted in two predominant interactions, one involving p65 and SV2, and the other involving p38 and vesicle-associated membrane protein. The multicomponent complex preserved with CHAPS solubilization was partially reconstituted following octylglucoside solubilization and subsequent dialysis against CHAPS. Reduction of the CHAPS concentration by gel filtration chromatography resulted in increased recovery of the multicomponent complex. Examination of the large complex isolated from CHAPS-solubilized vesicles by negative stain EM revealed structures with multiple globular domains, some of which were specifically labeled with gold-conjugated antibodies directed against p65 and SV2. The protein interactions defined in this report are likely to underlie aspects of neurotransmitter secretion, membrane traffic, and the spatial organization of vesicles within the nerve terminal. PMID:1730776

  13. [Lipids in the process of synaptic vesicle exo- and endocytosis].

    PubMed

    Zefirov, A L; Petrov, A M

    2010-08-01

    The phenomenon of synaptic transmission is based on the processes of synaptic vesicle exo- and endocytosis carried out with complex protein-dependent mechanisms. The SNARE-complex forming proteins (synaptobrevin, syntaxin, SNAP-25), synaptotagmin, Munc13, Munc18, NSF, alpha-SNAP are involved in exocytosis, while the synaptic vesicle endocytosis is mediated by another protein (clathrin, AP-2, epsin, endophilin, amphiphysin, dynamin, synaptojanin, Hsc70). In recent years, data on critical role of various lipids in exo- and encocytosis are collected. Most interesting results are received about significance of the cholesterol, phosphoinositides, phosphatidic and polynonsaturated fat acids in the exo-endocytosis cycle. Participation of lipid rafts in synaptic vesicle recycling is discussed. In this article, the data of the last years, including the authors' own data about role of some lipids and lipid-modifying enzimes in processes of exo- and endocytosis are presented.

  14. 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

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

    SciTech Connect

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

    1988-07-12

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

  16. Neuronal Depolarization Drives Increased Dopamine Synaptic Vesicle Loading via VGLUT.

    PubMed

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

    2017-08-30

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

  17. Cdk5 and the mystery of synaptic vesicle endocytosis.

    PubMed

    Nguyen, Chan; Bibb, James A

    2003-11-24

    Regulation of endocytosis by protein phosphorylation and dephosphorylation is critical to synaptic vesicle recycling. Two groups have now identified the neuronal kinase Cdk5 (cyclin-dependent kinase 5) as an important regulator of this process. Robinson and coworkers recently demonstrated that Cdk5 is necessary for synaptic vesicle endocytosis (SVE) (Tan et al., 2003), whereas a new report in this issue claims that Cdk5 negatively regulates SVE (Tomizawa et al., 2003). Careful examination of the data reveals a model that helps resolve the apparently contradictory nature of these reports.

  18. Involvement of water channels in synaptic vesicle swelling.

    PubMed

    Jeremic, Aleksandar; Cho, Won Jin; Jena, Bhanu P

    2005-10-01

    Vesicle swelling is critical for secretion; however, the underlying mechanism of synaptic vesicle (SV) swelling is unknown. A G alphai3-phospholipase A2 (PLA2)-mediated involvement of the water channel aquaporin-1 (AQP1) in the regulation of secretory vesicle swelling in the exocrine pancreas has been previously reported. In the present study, the association and involvement of water channels in SV swelling was explored. Results from the study demonstrate that water channels AQP1 and AQP6, and the heterotrimeric Go protein are associated with SVs and participate in their swelling.

  19. The BLOC-1 Subunit Pallidin Facilitates Activity-Dependent Synaptic Vesicle Recycling

    PubMed Central

    Ma, Wenpei; Zhang, Shixing; Paluch, Jeremy; Guo, Wanlin

    2017-01-01

    Abstract Membrane trafficking pathways must be exquisitely coordinated at synaptic terminals to maintain functionality, particularly during conditions of high activity. We have generated null mutations in the Drosophila homolog of pallidin, a central subunit of the biogenesis of lysosome-related organelles complex-1 (BLOC-1), to determine its role in synaptic development and physiology. We find that Pallidin localizes to presynaptic microtubules and cytoskeletal structures, and that the stability of Pallidin protein is highly dependent on the BLOC-1 components Dysbindin and Blos1. We demonstrate that the rapidly recycling vesicle pool is not sustained during high synaptic activity in pallidin mutants, leading to accelerated rundown and slowed recovery. Following intense activity, we observe a loss of early endosomes and a concomitant increase in tubular endosomal structures in synapses without Pallidin. Together, our data reveal that Pallidin subserves a key role in promoting efficient synaptic vesicle recycling and re-formation through early endosomes during sustained activity. PMID:28317021

  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. Overlapping Role of Dynamin Isoforms in Synaptic Vesicle Endocytosis

    PubMed Central

    Raimondi, Andrea; Ferguson, Shawn M.; Lou, Xuelin; Armbruster, Moritz; Paradise, Summer; Giovedi, Silvia; Messa, Mirko; Kono, Nao; Takasaki, Junko; Cappello, Valentina; O’Toole, Eileen; Ryan, Timothy A.; De Camilli, Pietro

    2011-01-01

    The existence of neuron specific endocytic protein isoforms raises questions about their importance for specialized neuronal functions. Dynamin, a GTPase implicated in the fission reaction of endocytosis, is encoded by three genes, two of which, dynamin 1 and 3, are highly expressed in neurons. We show that dynamin 3, thought to play a predominantly postsynaptic role, has a major presynaptic function. While lack of dynamin 3 does not produce an overt phenotype in mice, it worsens the dynamin 1 KO phenotype, leading to perinatal lethality and a more severe defect in activity-dependent synaptic vesicle endocytosis. Thus, dynamin 1 and 3, which together account for the overwhelming majority of brain dynamin, cooperate in supporting optimal rates of synaptic vesicle endocytosis. Persistence of synaptic transmission in their absence indicates that if dynamin plays essential functions in neurons, such functions can be achieved by the very low levels of dynamin 2. PMID:21689597

  2. The transport of neurotransmitters into synaptic vesicles.

    PubMed

    Peter, D; Liu, Y; Brecha, N; Edwards, R H

    1995-01-01

    Using selection in the neurotoxin MPP+, we have isolated a cDNA encoding vesicular amine transport. The transporter protects against MPP+ by sequestering the toxin in vesicles, away from its primary site of action in mitochondria. Unexpectedly, two distinct but highly related genes encode vesicular amine transport in the adrenal gland and the central nervous system. The sequence of both predicts twelve transmembrane domains and weak homology to a class of bacterial antibiotic resistance proteins. The two human genes occur on different chromosomes. In addition, the two transporters show a number of differences in function, including substrate specificity and the interaction with one inhibitor and the amphetamines.

  3. Energy coupling of L-glutamate transport and vacuolar H(+)-ATPase in brain synaptic vesicles.

    PubMed

    Moriyama, Y; Maeda, M; Futai, M

    1990-10-01

    Energy coupling of L-glutamate transport in brain synaptic vesicles has been studied. ATP-dependent acidification of the bovine brain synaptic vesicles was shown to require CI-, to be accelerated by valinomycin and to be abolished by ammonium sulfate, nigericin or CCCP plus valinomycin, and K+. On the other hand, ATP-driven formation of a membrane potential (positive inside) was found to be stimulated by ammonium sulfate, not to be affected by nigericin and to be abolished by CCCP plus valinomycin and K+. Like formation of a membrane potential, ATP-dependent L-[3H]glutamate uptake into vesicles was stimulated by ammonium sulfate, not affected by nigericin and abolished by CCCP plus valinomycin and K+. The L-[3H]glutamate uptake differed in specificity from the transport system in synaptic plasma membranes. Both ATP-dependent H+ pump activity and L-glutamate uptake were inhibited by bafilomycin and cold treatment (common properties of vacuolar H(+)-ATPase). ATP-dependent acidification in the presence of L-glutamate was also observed, suggesting that L-glutamate uptake lowered the membrane potential to drive further entry of H+. These results were consistent with the notion that the vacuolar H(+)-ATPase of synpatic vesicles formed a membrane potential to drive L-glutamate uptake. ATPase activity of the vesicles was not affected by the addition of Cl-, glutamate or nigericin, indicating that an electrochemical H+ gradient had no effect on the ATPase activity.

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

    PubMed Central

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

    2015-01-01

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

  5. 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.

  6. 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.

  7. Adaptor protein complexes 1 and 3 are essential for generation of synaptic vesicles from activity-dependent bulk endosomes.

    PubMed

    Cheung, Giselle; Cousin, Michael A

    2012-04-25

    Activity-dependent bulk endocytosis is the dominant synaptic vesicle retrieval mode during high intensity stimulation in central nerve terminals. A key event in this endocytosis mode is the generation of new vesicles from bulk endosomes, which replenish the reserve vesicle pool. We have identified an essential requirement for both adaptor protein complexes 1 and 3 in this process by employing morphological and optical tracking of bulk endosome-derived synaptic vesicles in rat primary neuronal cultures. We show that brefeldin A inhibits synaptic vesicle generation from bulk endosomes and that both brefeldin A knockdown and shRNA knockdown of either adaptor protein 1 or 3 subunits inhibit reserve pool replenishment from bulk endosomes. Conversely, no plasma membrane function was found for adaptor protein 1 or 3 in either bulk endosome formation or clathrin-mediated endocytosis. Simultaneous knockdown of both adaptor proteins 1 and 3 indicated that they generated the same population of synaptic vesicles. Thus, adaptor protein complexes 1 and 3 play an essential dual role in generation of synaptic vesicles during activity-dependent bulk endocytosis.

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

    PubMed

    Takeda, Kouji; Ueda, Tetsufumi

    2017-01-01

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

  9. DOPAL derived alpha-synuclein oligomers impair synaptic vesicles physiological function

    PubMed Central

    Plotegher, N.; Berti, G.; Ferrari, E.; Tessari, I.; Zanetti, M.; Lunelli, L.; Greggio, E.; Bisaglia, M.; Veronesi, M.; Girotto, S.; Dalla Serra, M.; Perego, C.; Casella, L.; Bubacco, L.

    2017-01-01

    Parkinson’s disease is a neurodegenerative disorder characterized by the death of dopaminergic neurons and by accumulation of alpha-synuclein (aS) aggregates in the surviving neurons. The dopamine catabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) is a highly reactive and toxic molecule that leads to aS oligomerization by covalent modifications to lysine residues. Here we show that DOPAL-induced aS oligomer formation in neurons is associated with damage of synaptic vesicles, and with alterations in the synaptic vesicles pools. To investigate the molecular mechanism that leads to synaptic impairment, we first aimed to characterize the biochemical and biophysical properties of the aS-DOPAL oligomers; heterogeneous ensembles of macromolecules able to permeabilise cholesterol-containing lipid membranes. aS-DOPAL oligomers can induce dopamine leak in an in vitro model of synaptic vesicles and in cellular models. The dopamine released, after conversion to DOPAL in the cytoplasm, could trigger a noxious cycle that further fuels the formation of aS-DOPAL oligomers, inducing neurodegeneration. PMID:28084443

  10. DOPAL derived alpha-synuclein oligomers impair synaptic vesicles physiological function.

    PubMed

    Plotegher, N; Berti, G; Ferrari, E; Tessari, I; Zanetti, M; Lunelli, L; Greggio, E; Bisaglia, M; Veronesi, M; Girotto, S; Dalla Serra, M; Perego, C; Casella, L; Bubacco, L

    2017-01-13

    Parkinson's disease is a neurodegenerative disorder characterized by the death of dopaminergic neurons and by accumulation of alpha-synuclein (aS) aggregates in the surviving neurons. The dopamine catabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) is a highly reactive and toxic molecule that leads to aS oligomerization by covalent modifications to lysine residues. Here we show that DOPAL-induced aS oligomer formation in neurons is associated with damage of synaptic vesicles, and with alterations in the synaptic vesicles pools. To investigate the molecular mechanism that leads to synaptic impairment, we first aimed to characterize the biochemical and biophysical properties of the aS-DOPAL oligomers; heterogeneous ensembles of macromolecules able to permeabilise cholesterol-containing lipid membranes. aS-DOPAL oligomers can induce dopamine leak in an in vitro model of synaptic vesicles and in cellular models. The dopamine released, after conversion to DOPAL in the cytoplasm, could trigger a noxious cycle that further fuels the formation of aS-DOPAL oligomers, inducing neurodegeneration.

  11. 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

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

    PubMed Central

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

    2009-01-01

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

  13. 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

  14. The effects of nerve stimulation and hemicholinium on synaptic vesicles at the mammalian neuromuscular junction

    PubMed Central

    Jones, S. F.; Kwanbunbumpen, Suthiwan

    1970-01-01

    1. Electron micrographs of nerve terminals in rat phrenic nerve—diaphragm preparations have been studied. This has been done before and after prolonged nerve stimulation. The effectiveness of nerve stimulation has been monitored by intracellular micro-electrode recordings from the muscle cells. 2. Characteristic changes in the form and distribution of the nerve terminal mitochondria were noted after nerve stimulation. 3. Synaptic vesicle numbers in the region of nerve terminal less than 1800 Å from the synaptic cleft were significantly greater in tissue taken 2 and 3 min after nerve stimulation, than in unstimulated preparations. 4. The long and short diameters of the synaptic vesicle profiles less than 1800 Å from the synaptic cleft were measured. Analysis of the distribution of the diameters indicated synaptic vesicles to be basically spherical structures. Estimates of synaptic vesicle volume were made from the measurements. Synaptic vesicle volume was significantly reduced in tissue taken 2 and 4 min following nerve stimulation. 5. If hemicholinium, a compound which inhibits acetylcholine synthesis, was present during the period of nerve stimulation, much greater reductions in synaptic vesicle volume occurred. Synaptic vesicle numbers in the region of nerve terminal less than 1800 Å from the synaptic cleft were also reduced, compared with unstimulated control preparations. 6. These results are regarded as support for the hypothesis that the synaptic vesicles in nerve terminals at the mammalian neuromuscular junction represent stores of the transmitter substance, acetylcholine. ImagesABABPlate 2AB PMID:5503879

  15. 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

  16. Differential roles for snapin and synaptotagmin in the synaptic vesicle cycle.

    PubMed

    Yu, Szi-Chieh; Klosterman, Susan M; Martin, Ashley A; Gracheva, Elena O; Richmond, Janet E

    2013-01-01

    Evoked synaptic transmission is dependent on interactions between the calcium sensor Synaptotagmin I and the SNARE complex, comprised of Syntaxin, SNAP-25, and Synaptobrevin. Recent evidence suggests that Snapin may be an important intermediate in this process, through simultaneous interactions of Snapin dimers with SNAP-25 and Synaptotagmin. In support of this model, cultured neurons derived from embryonically lethal Snapin null mutant mice exhibit desynchronized release and a reduced readily releasable vesicle pool. Based on evidence that a dimerization-defective Snapin mutation specifically disrupts priming, Snapin is hypothesized to stabilize primed vesicles by structurally coupling Synaptotagmin and SNAP-25. To explore this model in vivo we examined synaptic transmission in viable, adult C. elegans Snapin (snpn-1) mutants. The kinetics of synaptic transmission were unaffected at snpn-1 mutant neuromuscular junctions (NMJs), but the number of docked, fusion competent vesicles was significantly reduced. However, analyses of snt-1 and snt-1;snpn-1 double mutants suggest that the docking role of SNPN-1 is independent of Synaptotagmin. Based on these results we propose that the primary role of Snapin in C. elegans is to promote vesicle priming, consistent with the stabilization of SNARE complex formation through established interactions with SNAP-25 upstream of the actions of Synaptotagmin in calcium-sensing and endocytosis.

  17. Diffusional spread and confinement of newly exocytosed synaptic vesicle proteins

    PubMed Central

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

    2015-01-01

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

  18. Vesicular Release of Glutamate Utilizes the Proton Gradient Between the Vesicle and Synaptic Cleft

    PubMed Central

    Brown, Jon T.; Weatherall, Kate L.; Corria, Laura R.; Chater, Thomas E.; Isaac, John T.; Marrion, Neil V.

    2009-01-01

    Glutamate is released from synaptic vesicles following formation of a fusion pore, connecting the vesicle interior with the synaptic cleft. Release is proposed to result from either full fusion of the vesicle with the terminal membrane or by ‘kiss-and-run,’ where release occurs through the fusion pore. ‘Kiss-and-run’ seems implausible as passive diffusion of glutamate through the pore is too slow to account for the rapidity of release. Vesicular accumulation of glutamate is driven by a proton gradient, resulting in the co-release of protons during exocytosis. We tested whether the proton gradient between the vesicle and cleft contributes to glutamate exocytosis. Collapse of the gradient reduced hippocampal glutamatergic transmission, an effect that was not associated with presynaptic changes in excitability, transmitter release probability, or postsynaptic sensitivity. These data indicate that approximately half of glutamate release utilizes the proton gradient between vesicle and cleft, suggesting a significant proportion of release by ‘kiss-and-run.’ PMID:21423501

  19. Characterizing Synaptic Vesicle Proteins Using Synaptosomal Fractions and Cultured Hippocampal Neurons

    PubMed Central

    DiGiovanni, Jerome; Sun, Tao; Sheng, Zu-Hang

    2012-01-01

    Cloning and characterization of synaptic vesicle proteins and their binding counterparts on the presynaptic plasma membrane have greatly advanced our understanding of the molecular mechanisms involved in the synaptic vesicle cycle and neurotransmitter release. This unit discusses multidisciplinary approaches to characterize proteins from synaptosome-enriched subcellular fractions and localize them within cultured neurons. The first approach regroups methods used to isolate synaptic vesicles from rat brain synaptosomal preparations, allowing for specific biochemical investigation of synaptic vesicle proteins. The second is a detailed procedure for pre-embedding immunogold staining and electron microscopic observation, which permits the morphological identification of proteins in individual vesicles at intact synapses. Additionally, this chapter proposes methods for light microscopic examination of hippocampal neurons. It includes procedures for embryonic and postnatal hippocampal neuron culture and describes an immunocytochemical staining protocol used to investigate synaptic vesicle protein localization with respect to other proteins or subcellular structures. PMID:22470148

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

    PubMed

    Afuwape, Olusoji A T; Kavalali, Ege T

    2016-01-01

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

  1. An essential role of Rab5 in uniformity of synaptic vesicle size.

    PubMed

    Shimizu, Hideyuki; Kawamura, Satoru; Ozaki, Koichi

    2003-09-01

    Rab5 small GTPase is a famous regulator of endocytic vesicular transport from plasma membrane to early endosomes. In neurons, Rab5 is found not only on endocytic vesicles in cell bodies but also on synaptic vesicles in nerve terminals. However, the function of Rab5 on synaptic vesicles remains unclear. Here, we elucidate the function of Rab5 on synaptic vesicles with in vivo and in vitro experiments using Drosophila photoreceptor cells. Functional inhibition of Rab5 with Rab5N142I, a dominant negative version of Drosophila Rab5, induced enlargement of synaptic vesicles. This enlargement was, however, suppressed by enhancing synaptic vesicle recycling under light illumination. In addition, synaptic vesicles prepared from Rab5N142I-expressing flies exhibited homotypic fusion in vitro. These results indicate that Rab5 functions to keep the size of synaptic vesicles uniform by preventing their homotypic fusion. By contrast, Rab5 was not involved in the endocytic reformation of synaptic vesicles, contrary to expectation from its conventional function. Furthermore, we electrophysiologically and behaviourally showed that the function of Rab5 is essential for efficient signal transmission across synapses.

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

    PubMed

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

    2016-01-25

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

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

    PubMed Central

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

    2016-01-01

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

  4. AP2 hemicomplexes contribute independently to synaptic vesicle endocytosis

    PubMed Central

    Gu, Mingyu; Liu, Qiang; Watanabe, Shigeki; Sun, Lin; Hollopeter, Gunther; Grant, Barth D; Jorgensen, Erik M

    2013-01-01

    The clathrin adaptor complex AP2 is thought to be an obligate heterotetramer. We identify null mutations in the α subunit of AP2 in the nematode Caenorhabditis elegans. α-adaptin mutants are viable and the remaining μ2/β hemicomplex retains some function. Conversely, in μ2 mutants, the alpha/sigma2 hemicomplex is localized and is partially functional. α-μ2 double mutants disrupt both halves of the complex and are lethal. The lethality can be rescued by expression of AP2 components in the skin, which allowed us to evaluate the requirement for AP2 subunits at synapses. Mutations in either α or μ2 subunits alone reduce the number of synaptic vesicles by about 30%; however, simultaneous loss of both α and μ2 subunits leads to a 70% reduction in synaptic vesicles and the presence of large vacuoles. These data suggest that AP2 may function as two partially independent hemicomplexes. DOI: http://dx.doi.org/10.7554/eLife.00190.001 PMID:23482940

  5. 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.

  6. Adaptor protein complexes 1 and 3 are essential for generation of synaptic vesicles from activity-dependent bulk endosomes

    PubMed Central

    Cheung, Giselle; Cousin, Michael Alan

    2012-01-01

    Activity-dependent bulk endocytosis is the dominant synaptic vesicle retrieval mode during high intensity stimulation in central nerve terminals. A key event in this endocytosis mode is the generation of new vesicles from bulk endosomes, which replenish the reserve vesicle pool. We have identified an essential requirement for both adaptor protein complexes 1 and 3 in this process by employing morphological and optical tracking of bulk endosome-derived synaptic vesicles in rat primary neuronal cultures. We show that brefeldin A inhibits synaptic vesicle generation from bulk endosomes, and that both brefeldin A and shRNA knockdown of either adaptor protein 1 or 3 subunits inhibit reserve pool replenishment from bulk endosomes. Conversely, no plasma membrane function was found for adaptor proteins 1 or 3 in either bulk endosome formation or clathrin-mediated endocytosis. Simultaneous knockdown of both adaptor protein 1 and 3 indicated that they generated the same population of SVs. Thus adaptor protein complex 1 and 3 play an essential dual role in generation of synaptic vesicles during activity-dependent bulk endocytosis. PMID:22539861

  7. 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.

  8. 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

  9. 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

  10. Synucleins regulate the kinetics of synaptic vesicle endocytosis.

    PubMed

    Vargas, Karina J; Makani, Sachin; Davis, Taylor; Westphal, Christopher H; Castillo, Pablo E; Chandra, Sreeganga S

    2014-07-09

    Genetic and pathological studies link α-synuclein to the etiology of Parkinson's disease (PD), but the normal function of this presynaptic protein remains unknown. α-Synuclein, an acidic lipid binding protein, shares high sequence identity with β- and γ-synuclein. Previous studies have implicated synucleins in synaptic vesicle (SV) trafficking, although the precise site of synuclein action continues to be unclear. Here we show, using optical imaging, electron microscopy, and slice electrophysiology, that synucleins are required for the fast kinetics of SV endocytosis. Slowed endocytosis observed in synuclein null cultures can be rescued by individually expressing mouse α-, β-, or γ-synuclein, indicating they are functionally redundant. Through comparisons to dynamin knock-out synapses and biochemical experiments, we suggest that synucleins act at early steps of SV endocytosis. Our results categorize α-synuclein with other familial PD genes known to regulate SV endocytosis, implicating this pathway in PD.

  11. 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.

  12. 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

  13. Genetic analysis of synaptotagmin-7 function in synaptic vesicle exocytosis.

    PubMed

    Maximov, Anton; Lao, Ye; Li, Hongmei; Chen, Xiaocheng; Rizo, Josep; Sørensen, Jakob B; Südhof, Thomas C

    2008-03-11

    Synaptotagmin-7 is a candidate Ca(2+) sensor for exocytosis that is at least partly localized to synapses. Similar to synaptotagmin-1, which functions as a Ca(2+) sensor for fast synaptic vesicle (SV) exocytosis, synaptotagmin-7 contains C(2)A and C(2)B domains that exhibit Ca(2+)-dependent phospholipid binding. However, synaptotagmin-7 cannot replace synaptotagmin-1 as a Ca(2+) sensor for fast SV exocytosis, raising questions about the physiological significance of its Ca(2+)-binding properties. Here, we examine how synaptotagmin-7 binds Ca(2+) and test whether this Ca(2+) binding regulates Ca(2+)-triggered SV exocytosis. We show that the synaptotagmin-7 C(2)A domain exhibits a Ca(2+)-binding mode similar to that of the synaptotagmin-1 C(2)A domain, suggesting that the synaptotagmin-1 and -7 C(2) domains generally employ comparable Ca(2+)-binding mechanisms. We then generated mutant mice that lack synaptotagmin-7 or contain point mutations inactivating Ca(2+) binding either to both C(2) domains of synaptotagmin-7 or only to its C(2)B domain. Synaptotagmin-7-mutant mice were viable and fertile. Inactivation of Ca(2+) binding to both C(2) domains caused an approximately 70% reduction in synaptotagmin-7 levels, whereas inactivation of Ca(2+) binding to only the C(2)B domain did not alter synaptotagmin-7 levels. The synaptotagmin-7 deletion did not change fast synchronous release, slow asynchronous release, or short-term synaptic plasticity of release of neurotransmitters. Thus, our results show that Ca(2+) binding to the synaptotagmin-7 C(2) domains is physiologically important for stabilizing synaptotagmin-7, but that Ca(2+) binding by synaptotagmin-7 likely does not regulate SV exocytosis, consistent with a role for synaptotagmin-7 in other forms of Ca(2+)-dependent synaptic exocytosis.

  14. Genetic analysis of synaptotagmin-7 function in synaptic vesicle exocytosis

    PubMed Central

    Maximov, Anton; Lao, Ye; Li, Hongmei; Chen, Xiaocheng; Rizo, Josep; Sørensen, Jakob B.; Südhof, Thomas C.

    2008-01-01

    Synaptotagmin-7 is a candidate Ca2+ sensor for exocytosis that is at least partly localized to synapses. Similar to synaptotagmin-1, which functions as a Ca2+ sensor for fast synaptic vesicle (SV) exocytosis, synaptotagmin-7 contains C2A and C2B domains that exhibit Ca2+-dependent phospholipid binding. However, synaptotagmin-7 cannot replace synaptotagmin-1 as a Ca2+ sensor for fast SV exocytosis, raising questions about the physiological significance of its Ca2+-binding properties. Here, we examine how synaptotagmin-7 binds Ca2+ and test whether this Ca2+ binding regulates Ca2+-triggered SV exocytosis. We show that the synaptotagmin-7 C2A domain exhibits a Ca2+-binding mode similar to that of the synaptotagmin-1 C2A domain, suggesting that the synaptotagmin-1 and -7 C2 domains generally employ comparable Ca2+-binding mechanisms. We then generated mutant mice that lack synaptotagmin-7 or contain point mutations inactivating Ca2+ binding either to both C2 domains of synaptotagmin-7 or only to its C2B domain. Synaptotagmin-7-mutant mice were viable and fertile. Inactivation of Ca2+ binding to both C2 domains caused an ≈70% reduction in synaptotagmin-7 levels, whereas inactivation of Ca2+ binding to only the C2B domain did not alter synaptotagmin-7 levels. The synaptotagmin-7 deletion did not change fast synchronous release, slow asynchronous release, or short-term synaptic plasticity of release of neurotransmitters. Thus, our results show that Ca2+ binding to the synaptotagmin-7 C2 domains is physiologically important for stabilizing synaptotagmin-7, but that Ca2+ binding by synaptotagmin-7 likely does not regulate SV exocytosis, consistent with a role for synaptotagmin-7 in other forms of Ca2+-dependent synaptic exocytosis. PMID:18308933

  15. 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.

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

    PubMed Central

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

    2016-01-01

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

  17. 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.

  18. Isoosmotic isolation of rat brain synaptic vesicles, some of which contain tyrosine hydroxylase.

    PubMed

    Tsudzuki, Toshihiro; Tsujita, Maki

    2004-08-01

    Rat brain synaptic vesicles were isoosmotically isolated and examined for Mg(2+)-ATPase [EC 3.6.1.3.] and tyrosine hydroxylase [EC 1.14.16.2.] associated with the synaptic vesicles. Synaptosomes in 0.32 M sucrose were disrupted by freezing and thawing treatment, and the cytosol fraction was fractionated on a Sephacryl S-500 column with a mean exclusion size of 200 nm. Peak I at the void volume was a mixture of large vesicular membranes, small amounts of synaptic vesicles and coated vesicles, etc. Peak II consisted of non- and granulated synaptic vesicles of 35-40 nm diameter, and peak III of soluble proteins. The synaptic vesicles in peak II reacted with antibodies against the H(+)-ATPase A-subunit, vesicular acetylcholine transporter, and vesicular monoamine transporter. However, they showed little Mg(2+)-ATPase activity. Tyrosine hydroxylase was observed in either peak II or III on blotting with an anti-tyrosine hydroxylase antibody. These results imply that tyrosine hydroxylase exists in soluble and bound forms to synaptic vesicles in nerve terminals.

  19. [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.

  20. A biochemical and functional protein complex involving dopamine synthesis and transport into synaptic vesicles.

    PubMed

    Cartier, Etienne A; Parra, Leonardo A; Baust, Tracy B; Quiroz, Marisol; Salazar, Gloria; Faundez, Victor; Egaña, Loreto; Torres, Gonzalo E

    2010-01-15

    Synaptic transmission depends on neurotransmitter pools stored within vesicles that undergo regulated exocytosis. In the brain, the vesicular monoamine transporter-2 (VMAT(2)) is responsible for the loading of dopamine (DA) and other monoamines into synaptic vesicles. Prior to storage within vesicles, DA synthesis occurs at the synaptic terminal in a two-step enzymatic process. First, the rate-limiting enzyme tyrosine hydroxylase (TH) converts tyrosine to di-OH-phenylalanine. Aromatic amino acid decarboxylase (AADC) then converts di-OH-phenylalanine into DA. Here, we provide evidence that VMAT(2) physically and functionally interacts with the enzymes responsible for DA synthesis. In rat striata, TH and AADC co-immunoprecipitate with VMAT(2), whereas in PC 12 cells, TH co-immunoprecipitates with the closely related VMAT(1) and with overexpressed VMAT(2). GST pull-down assays further identified three cytosolic domains of VMAT(2) involved in the interaction with TH and AADC. Furthermore, in vitro binding assays demonstrated that TH directly interacts with VMAT(2). Additionally, using fractionation and immunoisolation approaches, we demonstrate that TH and AADC associate with VMAT(2)-containing synaptic vesicles from rat brain. These vesicles exhibited specific TH activity. Finally, the coupling between synthesis and transport of DA into vesicles was impaired in the presence of fragments involved in the VMAT(2)/TH/AADC interaction. Taken together, our results indicate that DA synthesis can occur at the synaptic vesicle membrane, where it is physically and functionally coupled to VMAT(2)-mediated transport into vesicles.

  1. 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.

  2. The Vesicle Protein SAM-4 Regulates the Processivity of Synaptic Vesicle Transport

    PubMed Central

    Zheng, Qun; Ahlawat, Shikha; Schaefer, Anneliese; Mahoney, Tim; Koushika, Sandhya P.; Nonet, Michael L.

    2014-01-01

    Axonal transport of synaptic vesicles (SVs) is a KIF1A/UNC-104 mediated process critical for synapse development and maintenance yet little is known of how SV transport is regulated. Using C. elegans as an in vivo model, we identified SAM-4 as a novel conserved vesicular component regulating SV transport. Processivity, but not velocity, of SV transport was reduced in sam-4 mutants. sam-4 displayed strong genetic interactions with mutations in the cargo binding but not the motor domain of unc-104. Gain-of-function mutations in the unc-104 motor domain, identified in this study, suppress the sam-4 defects by increasing processivity of the SV transport. Genetic analyses suggest that SAM-4, SYD-2/liprin-α and the KIF1A/UNC-104 motor function in the same pathway to regulate SV transport. Our data support a model in which the SV protein SAM-4 regulates the processivity of SV transport. PMID:25329901

  3. The vesicle protein SAM-4 regulates the processivity of synaptic vesicle transport.

    PubMed

    Zheng, Qun; Ahlawat, Shikha; Schaefer, Anneliese; Mahoney, Tim; Koushika, Sandhya P; Nonet, Michael L

    2014-10-01

    Axonal transport of synaptic vesicles (SVs) is a KIF1A/UNC-104 mediated process critical for synapse development and maintenance yet little is known of how SV transport is regulated. Using C. elegans as an in vivo model, we identified SAM-4 as a novel conserved vesicular component regulating SV transport. Processivity, but not velocity, of SV transport was reduced in sam-4 mutants. sam-4 displayed strong genetic interactions with mutations in the cargo binding but not the motor domain of unc-104. Gain-of-function mutations in the unc-104 motor domain, identified in this study, suppress the sam-4 defects by increasing processivity of the SV transport. Genetic analyses suggest that SAM-4, SYD-2/liprin-α and the KIF1A/UNC-104 motor function in the same pathway to regulate SV transport. Our data support a model in which the SV protein SAM-4 regulates the processivity of SV transport.

  4. Interaction of Approved Drugs with Synaptic Vesicle Protein 2A.

    PubMed

    Danish, Azeem; Namasivayam, Vigneshwaran; Schiedel, Anke C; Müller, Christa E

    2017-04-01

    Levetiracetam (LEV) and its recently approved derivative brivaracetam are anti-epileptic drugs with a unique mechanism of action. The synaptic vesicle protein 2A (SV2A) was previously identified as their main target. In the current study, we tested a collection of 500 approved drugs for interaction with the human SV2A protein expressed in Chinese hamster ovary cells. Competition binding studies were performed using cell lysates with high SV2A expression and [(3) H]brivaracetam as a radioligand. A hit rate of 3% was obtained, defined as compounds that inhibited radioligand binding by more than 90% at a screening concentration of 20 μM. Subsequent concentration-inhibition curves revealed the antihistaminic prodrug loratadine (Ki  = 1.16 μM) and the antimalarial drug quinine (Ki  = 2.03 μM) to be the most potent SV2A protein ligands of the investigated drug library. Both compounds were similarly potent as LEV (Ki  = 1.74 μM), providing structurally novel scaffolds for SV2A ligands. A pharmacophore model was established, which indicated steric and electronic conformities of brivaracetam with the new SV2A ligands, and preliminary structure-activity relationships were determined. The anti-convulsive effects of the natural product quinine may - at least in part - be explained by interaction with SV2A. Loratadine and quinine represent new lead structures for anti-epileptic drug development.

  5. Distinct yet overlapping roles of Rab GTPases on synaptic vesicles

    PubMed Central

    Pavlos, Nathan J

    2011-01-01

    Exo-endocytotic cycling of synaptic vesicles (SVs) is one of the most intensely studied membrane trafficking pathways. It is governed by sets of conserved proteins including Rab GTPases. Long considered to define the identity and composition of a subcellular organelle, it has become increasingly evident that multiple Rabs co-exist on intracellular compartments, each contributing to its membrane organization and specialised function. Indeed, we have recently demonstrated that at least 11 distinct Rab proteins co-exist on highly purified SVs. These include Rabs involved in exocytosis (Rab3a/b/c and Rab27b) and intermediates of SV recycling such as early endosomes (Rab4, Rab5, Rab10, Rab11b and Rab14). Interestingly, we found that while two of these proteins, namely Rab3a and Rab27b, exhibited differential cycling dynamics on SV membranes; they played complementary roles during Ca2+-triggered neurotransmitter release. The implications of these findings in the SV trafficking cycle are discussed. PMID:21776405

  6. 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.

  7. Synapse-Assembly Proteins Maintain Synaptic Vesicle Cluster Stability and Regulate Synaptic Vesicle Transport in Caenorhabditis elegans

    PubMed Central

    Edwards, Stacey L.; Yorks, Rosalina M.; Morrison, Logan M.; Hoover, Christopher M.; Miller, Kenneth G.

    2015-01-01

    The functional integrity of neurons requires the bidirectional active transport of synaptic vesicles (SVs) in axons. The kinesin motor KIF1A transports SVs from somas to stable SV clusters at synapses, while dynein moves them in the opposite direction. However, it is unclear how SV transport is regulated and how SVs at clusters interact with motor proteins. We addressed these questions by isolating a rare temperature-sensitive allele of Caenorhabditis elegans unc-104 (KIF1A) that allowed us to manipulate SV levels in axons and dendrites. Growth at 20° and 14° resulted in locomotion rates that were ∼3 and 50% of wild type, respectively, with similar effects on axonal SV levels. Corresponding with the loss of SVs from axons, mutants grown at 14° and 20° showed a 10- and 24-fold dynein-dependent accumulation of SVs in their dendrites. Mutants grown at 14° and switched to 25° showed an abrupt irreversible 50% decrease in locomotion and a 50% loss of SVs from the synaptic region 12-hr post-shift, with no further decreases at later time points, suggesting that the remaining clustered SVs are stable and resistant to retrograde removal by dynein. The data further showed that the synapse-assembly proteins SYD-1, SYD-2, and SAD-1 protected SV clusters from degradation by motor proteins. In syd-1, syd-2, and sad-1 mutants, SVs accumulate in an UNC-104-dependent manner in the distal axon region that normally lacks SVs. In addition to their roles in SV cluster stability, all three proteins also regulate SV transport. PMID:26354975

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

    PubMed Central

    Faundez, Victor V.; Kelly, Regis B.

    2000-01-01

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

  9. Regulation of synaptic vesicle docking by different classes of macromolecules in active zone material.

    PubMed

    Szule, Joseph A; Harlow, Mark L; Jung, Jae Hoon; De-Miguel, Francisco F; Marshall, Robert M; McMahan, Uel J

    2012-01-01

    The docking of synaptic vesicles at active zones on the presynaptic plasma membrane of axon terminals is essential for their fusion with the membrane and exocytosis of their neurotransmitter to mediate synaptic impulse transmission. Dense networks of macromolecules, called active zone material, (AZM) are attached to the presynaptic membrane next to docked vesicles. Electron tomography has shown that some AZM macromolecules are connected to docked vesicles, leading to the suggestion that AZM is somehow involved in the docking process. We used electron tomography on the simply arranged active zones at frog neuromuscular junctions to characterize the connections of AZM to docked synaptic vesicles and to search for the establishment of such connections during vesicle docking. We show that each docked vesicle is connected to 10-15 AZM macromolecules, which fall into four classes based on several criteria including their position relative to the presynaptic membrane. In activated axon terminals fixed during replacement of docked vesicles by previously undocked vesicles, undocked vesicles near vacated docking sites on the presynaptic membrane have connections to the same classes of AZM macromolecules that are connected to docked vesicles in resting terminals. The number of classes and the total number of macromolecules to which the undocked vesicles are connected are inversely proportional to the vesicles' distance from the presynaptic membrane. We conclude that vesicle movement toward and maintenance at docking sites on the presynaptic membrane are directed by an orderly succession of stable interactions between the vesicles and distinct classes of AZM macromolecules positioned at different distances from the membrane. Establishing the number, arrangement and sequence of association of AZM macromolecules involved in vesicle docking provides an anatomical basis for testing and extending concepts of docking mechanisms provided by biochemistry.

  10. Tissue-type plasminogen activator induces synaptic vesicle endocytosis in cerebral cortical neurons.

    PubMed

    Yepes, M; Wu, F; Torre, E; Cuellar-Giraldo, D; Jia, D; Cheng, L

    2016-04-05

    The release of the serine proteinase tissue-type plasminogen activator (tPA) from the presynaptic terminal of cerebral cortical neurons plays a central role in the development of synaptic plasticity, adaptation to metabolic stress and neuronal survival. Our earlier studies indicate that by inducing the recruitment of the cytoskeletal protein βII-spectrin and voltage-gated calcium channels to the active zone, tPA promotes Ca(2+)-dependent translocation of synaptic vesicles (SVs) to the synaptic release site where they release their load of neurotransmitters into the synaptic cleft. Here we used a combination of in vivo and in vitro experiments to investigate whether this effect leads to depletion of SVs in the presynaptic terminal. Our data indicate that tPA promotes SV endocytosis via a mechanism that does not require the conversion of plasminogen into plasmin. Instead, we show that tPA induces calcineurin-mediated dynamin I dephosphorylation, which is followed by dynamin I-induced recruitment of the actin-binding protein profilin II to the presynaptic membrane, and profilin II-induced F-actin formation. We report that this tPA-induced sequence of events leads to the association of newly formed SVs with F-actin clusters in the endocytic zone. In summary, the data presented here indicate that following the exocytotic release of neurotransmitters tPA activates the mechanism whereby SVs are retrieved from the presynaptic membrane and endocytosed to replenish the pool of vesicles available for a new cycle of exocytosis. Together, these results indicate that in murine cerebral cortical neurons tPA plays a central role coupling SVs exocytosis and endocytosis.

  11. Synaptic vesicle pool-specific modification of neurotransmitter release by intravesicular free radical generation.

    PubMed

    Afuwape, Olusoji A T; Wasser, Catherine R; Schikorski, Thomas; Kavalali, Ege T

    2017-02-15

    Synaptic transmission is mediated by the release of neurotransmitters from synaptic vesicles in response to stimulation or through the spontaneous fusion of a synaptic vesicle with the presynaptic plasma membrane. There is growing evidence that synaptic vesicles undergoing spontaneous fusion versus those fusing in response to stimuli are functionally distinct. In this study, we acutely probe the effects of intravesicular free radical generation on synaptic vesicles that fuse spontaneously or in response to stimuli. By targeting vesicles that preferentially release spontaneously, we can dissociate the effects of intravesicular free radical generation on spontaneous neurotransmission from evoked neurotransmission and vice versa. Taken together, these results further advance our knowledge of the synapse and the nature of the different synaptic vesicle pools mediating neurotransmission. Earlier studies suggest that spontaneous and evoked neurotransmitter release processes are maintained by synaptic vesicles which are segregated into functionally distinct pools. However, direct interrogation of the link between this putative synaptic vesicle pool heterogeneity and neurotransmission has been difficult. To examine this link, we tagged vesicles with horseradish peroxidase (HRP) - a haem-containing plant enzyme - or antibodies against synaptotagmin-1 (syt1). Filling recycling vesicles in hippocampal neurons with HRP and subsequent treatment with hydrogen peroxide (H2 O2 ) modified the properties of neurotransmitter release depending on the route of HRP uptake. While strong depolarization-induced uptake of HRP suppressed evoked release and augmented spontaneous release, HRP uptake during mild activity selectively impaired evoked release, whereas HRP uptake at rest solely potentiated spontaneous release. Expression of a luminal HRP-tagged syt1 construct and subsequent H2 O2 application resulted in a similar increase in spontaneous release and suppression as well as

  12. ADP ribosylation factor 1 is required for synaptic vesicle budding in PC12 cells.

    PubMed

    Faúndez, V; Horng, J T; Kelly, R B

    1997-08-11

    Carrier vesicle generation from donor membranes typically progresses through a GTP-dependent recruitment of coats to membranes. Here we explore the role of ADP ribosylation factor (ARF) 1, one of the GTP-binding proteins that recruit coats, in the production of neuroendocrine synaptic vesicles (SVs) from PC12 cell membranes. Brefeldin A (BFA) strongly and reversibly inhibited SV formation in vivo in three different PC12 cell lines expressing vesicle-associated membrane protein-T Antigen derivatives. Other membrane traffic events remained unaffected by the drug, and the BFA effects were not mimicked by drugs known to interfere with formation of other classes of vesicles. The involvement of ARF proteins in the budding of SVs was addressed in a cell-free reconstitution system (Desnos, C., L. Clift-O'Grady, and R.B. Kelly. 1995. J. Cell Biol. 130:1041-1049). A peptide spanning the effector domain of human ARF1 (2-17) and recombinant ARF1 mutated in its GTPase activity, both inhibited the formation of SVs of the correct size. During in vitro incubation in the presence of the mutant ARFs, the labeled precursor membranes acquired different densities, suggesting that the two ARF mutations block at different biosynthetic steps. Cell-free SV formation in the presence of a high molecular weight, ARF-depleted fraction from brain cytosol was significantly enhanced by the addition of recombinant myristoylated native ARF1. Thus, the generation of SVs from PC12 cell membranes requires ARF and uses its GTPase activity, probably to regulate coating phenomena.

  13. ADP Ribosylation Factor 1 Is Required for Synaptic Vesicle Budding in PC12 Cells

    PubMed Central

    Faúndez, Victor; Horng, Jim-Tong; Kelly, Regis B.

    1997-01-01

    Carrier vesicle generation from donor membranes typically progresses through a GTP-dependent recruitment of coats to membranes. Here we explore the role of ADP ribosylation factor (ARF) 1, one of the GTP-binding proteins that recruit coats, in the production of neuroendocrine synaptic vesicles (SVs) from PC12 cell membranes. Brefeldin A (BFA) strongly and reversibly inhibited SV formation in vivo in three different PC12 cell lines expressing vesicle-associated membrane protein–T Antigen derivatives. Other membrane traffic events remained unaffected by the drug, and the BFA effects were not mimicked by drugs known to interfere with formation of other classes of vesicles. The involvement of ARF proteins in the budding of SVs was addressed in a cell-free reconstitution system (Desnos, C., L. Clift-O'Grady, and R.B. Kelly. 1995. J. Cell Biol. 130:1041–1049). A peptide spanning the effector domain of human ARF1 (2–17) and recombinant ARF1 mutated in its GTPase activity, both inhibited the formation of SVs of the correct size. During in vitro incubation in the presence of the mutant ARFs, the labeled precursor membranes acquired different densities, suggesting that the two ARF mutations block at different biosynthetic steps. Cell-free SV formation in the presence of a high molecular weight, ARF-depleted fraction from brain cytosol was significantly enhanced by the addition of recombinant myristoylated native ARF1. Thus, the generation of SVs from PC12 cell membranes requires ARF and uses its GTPase activity, probably to regulate coating phenomena. PMID:9245782

  14. Heterogeneity in synaptic vesicle release at neuromuscular synapses of mice expressing synaptopHluorin.

    PubMed

    Wyatt, Ryan M; Balice-Gordon, Rita J

    2008-01-02

    Mammalian neuromuscular junctions are useful model synapses to study the relationship between synaptic structure and function, although these have rarely been studied together at the same synapses. To do this, we generated transgenic lines of mice in which the thy1.2 promoter drives expression of synaptopHluorin (spH) as a means of optically measuring synaptic vesicle distribution and release. SpH is colocalized with other synaptic vesicle proteins in presynaptic terminals and does not alter normal synaptic function. Nerve stimulation leads to readily detectable and reproducible fluorescence changes in motor axon terminals that vary with stimulus frequency and, when compared with electrophysiological recordings, are reliable indicators of neurotransmitter release. Measurements of fluorescence intensity changes reveal a surprising amount of heterogeneity in synaptic vesicle release throughout individual presynaptic motor axon terminals. Some discrete terminal regions consistently displayed a greater rate and extent of release than others, regardless of stimulation frequency. The amount of release at a particular site is highly correlated to the relative abundance of synaptic vesicles there, indicating that a relatively constant fraction of the total vesicular pool, approximately 30%, is released in response to activity. These studies reveal previously unknown relationships between synaptic structure and function at mammalian neuromuscular junctions and demonstrate the usefulness of spH expressing mice as a tool for studying neuromuscular synapses in adults, as well as during development and diseases that affect neuromuscular synaptic function.

  15. Activity-dependent facilitation of Synaptojanin and synaptic vesicle recycling by the Minibrain kinase

    PubMed Central

    Chen, Chun-Kan; Bregere, Catherine; Paluch, Jeremy; Lu, Jason; Dickman, Dion K.; Chang, Karen T.

    2014-01-01

    Phosphorylation has emerged as a crucial regulatory mechanism in the nervous system to integrate the dynamic signaling required for proper synaptic development, function, and plasticity, particularly during changes in neuronal activity. Here we present evidence that Minibrain (Mnb; also known as Dyrk1A), a serine/threonine kinase implicated in autism spectrum disorder and Down syndrome, is required presynaptically for normal synaptic growth and rapid synaptic vesicle endocytosis at the Drosophila neuromuscular junction (NMJ). We find that Mnb-dependent phosphorylation of synaptojanin (Synj) is required, in vivo, for complex endocytic protein interactions and to enhance Synj activity. Neuronal stimulation drives Mnb mobilization to endocytic zones and triggers Mnb-dependent phosphorylation of Synj. Our data identify Mnb as a synaptic kinase that promotes efficient synaptic vesicle recycling by dynamically calibrating Synj function at the Drosophila NMJ, and in turn endocytic capacity, to adapt to conditions of high synaptic activity. PMID:24977345

  16. Activity-dependent facilitation of Synaptojanin and synaptic vesicle recycling by the Minibrain kinase.

    PubMed

    Chen, Chun-Kan; Bregere, Catherine; Paluch, Jeremy; Lu, Jason F; Dickman, Dion K; Chang, Karen T

    2014-06-30

    Phosphorylation has emerged as a crucial regulatory mechanism in the nervous system to integrate the dynamic signalling required for proper synaptic development, function and plasticity, particularly during changes in neuronal activity. Here we present evidence that Minibrain (Mnb; also known as Dyrk1A), a serine/threonine kinase implicated in autism spectrum disorder and Down syndrome, is required presynaptically for normal synaptic growth and rapid synaptic vesicle endocytosis at the Drosophila neuromuscular junction (NMJ). We find that Mnb-dependent phosphorylation of Synaptojanin (Synj) is required, in vivo, for complex endocytic protein interactions and to enhance Synj activity. Neuronal stimulation drives Mnb mobilization to endocytic zones and triggers Mnb-dependent phosphorylation of Synj. Our data identify Mnb as a synaptic kinase that promotes efficient synaptic vesicle recycling by dynamically calibrating Synj function at the Drosophila NMJ, and in turn endocytic capacity, to adapt to conditions of high synaptic activity.

  17. Regulation of Synaptic Vesicle Docking by Different Classes of Macromolecules in Active Zone Material

    PubMed Central

    Szule, Joseph A.; Harlow, Mark L.; Jung, Jae Hoon; De-Miguel, Francisco F.; Marshall, Robert M.; McMahan, Uel J.

    2012-01-01

    The docking of synaptic vesicles at active zones on the presynaptic plasma membrane of axon terminals is essential for their fusion with the membrane and exocytosis of their neurotransmitter to mediate synaptic impulse transmission. Dense networks of macromolecules, called active zone material, (AZM) are attached to the presynaptic membrane next to docked vesicles. Electron tomography has shown that some AZM macromolecules are connected to docked vesicles, leading to the suggestion that AZM is somehow involved in the docking process. We used electron tomography on the simply arranged active zones at frog neuromuscular junctions to characterize the connections of AZM to docked synaptic vesicles and to search for the establishment of such connections during vesicle docking. We show that each docked vesicle is connected to 10–15 AZM macromolecules, which fall into four classes based on several criteria including their position relative to the presynaptic membrane. In activated axon terminals fixed during replacement of docked vesicles by previously undocked vesicles, undocked vesicles near vacated docking sites on the presynaptic membrane have connections to the same classes of AZM macromolecules that are connected to docked vesicles in resting terminals. The number of classes and the total number of macromolecules to which the undocked vesicles are connected are inversely proportional to the vesicles’ distance from the presynaptic membrane. We conclude that vesicle movement toward and maintenance at docking sites on the presynaptic membrane are directed by an orderly succession of stable interactions between the vesicles and distinct classes of AZM macromolecules positioned at different distances from the membrane. Establishing the number, arrangement and sequence of association of AZM macromolecules involved in vesicle docking provides an anatomical basis for testing and extending concepts of docking mechanisms provided by biochemistry. PMID:22438915

  18. Inhibition of protein kinase C affects on mode of synaptic vesicle exocytosis due to cholesterol depletion

    SciTech Connect

    Petrov, Alexey M. Zakyrjanova, Guzalija F. Yakovleva, Anastasia A. Zefirov, Andrei L.

    2015-01-02

    Highlights: • We examine the involvement of PKC in MCD induced synaptic vesicle exocytosis. • PKC inhibitor does not decrease the effect MCD on MEPP frequency. • PKC inhibitor prevents MCD induced FM1-43 unloading. • PKC activation may switch MCD induced exocytosis from kiss-and-run to a full mode. • Inhibition of phospholipase C does not lead to similar change in exocytosis. - Abstract: Previous studies demonstrated that depletion of membrane cholesterol by 10 mM methyl-beta-cyclodextrin (MCD) results in increased spontaneous exocytosis at both peripheral and central synapses. Here, we investigated the role of protein kinase C in the enhancement of spontaneous exocytosis at frog motor nerve terminals after cholesterol depletion using electrophysiological and optical methods. Inhibition of the protein kinase C by myristoylated peptide and chelerythrine chloride prevented MCD-induced increases in FM1-43 unloading, whereas the frequency of spontaneous postsynaptic events remained enhanced. The increase in FM1-43 unloading still could be observed if sulforhodamine 101 (the water soluble FM1-43 quencher that can pass through the fusion pore) was added to the extracellular solution. This suggests a possibility that exocytosis of synaptic vesicles under these conditions could occur through the kiss-and-run mechanism with the formation of a transient fusion pore. Inhibition of phospholipase C did not lead to similar change in MCD-induced exocytosis.

  19. Interactions between synaptic vesicle fusion proteins explored by atomic force microscopy.

    PubMed

    Yersin, A; Hirling, H; Steiner, P; Magnin, S; Regazzi, R; Hüni, B; Huguenot, P; De los Rios, P; Dietler, G; Catsicas, S; Kasas, S

    2003-07-22

    Measuring the biophysical properties of macromolecular complexes at work is a major challenge of modern biology. The protein complex composed of vesicle-associated membrane protein 2, synaptosomal-associated protein of 25 kDa, and syntaxin 1 [soluble N-ethyl-maleimide-sensitive factor attachment protein receptor (SNARE) complex] is essential for docking and fusion of neurotransmitter-filled synaptic vesicles with the presynaptic membrane. To better understand the fusion mechanisms, we reconstituted the synaptic SNARE complex in the imaging chamber of an atomic force microscope and measured the interaction forces between its components. Each protein was tested against the two others, taken either individually or as binary complexes. This approach allowed us to determine specific interaction forces and dissociation kinetics of the SNAREs and led us to propose a sequence of interactions. A theoretical model based on our measurements suggests that a minimum of four complexes is probably necessary for fusion to occur. We also showed that the regulatory protein neuronal Sec1 injected into the atomic force microscope chamber prevented the complex formation. Finally, we measured the effect of tetanus toxin protease on the SNARE complex and its activity by on-line registration during tetanus toxin injection. These experiments provide a basis for the functional study of protein microdomains and also suggest opportunities for sensitive screening of drugs that can modulate protein-protein interactions.

  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. Synaptotagmin-1- and Synaptotagmin-7-Dependent Fusion Mechanisms Target Synaptic Vesicles to Kinetically Distinct Endocytic Pathways.

    PubMed

    Li, Ying C; Chanaday, Natali L; Xu, Wei; Kavalali, Ege T

    2017-02-08

    Synaptic vesicle recycling is essential for maintaining normal synaptic function. The coupling of exocytosis and endocytosis is assumed to be Ca(2+) dependent, but the exact role of Ca(2+) and its key effector synaptotagmin-1 (syt1) in regulation of endocytosis is poorly understood. Here, we probed the role of syt1 in single- as well as multi-vesicle endocytic events using high-resolution optical recordings. Our experiments showed that the slowed endocytosis phenotype previously reported after syt1 loss of function can also be triggered by other manipulations that promote asynchronous release such as Sr(2+) substitution and complexin loss of function. The link between asynchronous release and slowed endocytosis was due to selective targeting of fused synaptic vesicles toward slow retrieval by the asynchronous release Ca(2+) sensor synaptotagmin-7. In contrast, after single synaptic vesicle fusion, syt1 acted as an essential determinant of synaptic vesicle endocytosis time course by delaying the kinetics of vesicle retrieval in response to increasing Ca(2+) levels. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Control of neurotransmitter release by an internal gel matrix in synaptic vesicles.

    PubMed

    Reigada, David; Díez-Pérez, Ismael; Gorostiza, Pau; Verdaguer, Albert; Gómez de Aranda, Inmaculada; Pineda, Oriol; Vilarrasa, Jaume; Marsal, Jordi; Blasi, Joan; Aleu, Jordi; Solsona, Carles

    2003-03-18

    Neurotransmitters are stored in synaptic vesicles, where they have been assumed to be in free solution. Here we report that in Torpedo synaptic vesicles, only 5% of the total acetylcholine (ACh) or ATP content is free, and that the rest is adsorbed to an intravesicular proteoglycan matrix. This matrix, which controls ACh and ATP release by an ion-exchange mechanism, behaves like a smart gel. That is, it releases neurotransmitter and changes its volume when challenged with small ionic concentration change. Immunodetection analysis revealed that the synaptic vesicle proteoglycan SV2 is the core of the intravesicular matrix and is responsible for immobilization and release of ACh and ATP. We suggest that in the early steps of vesicle fusion, this internal matrix regulates the availability of free diffusible ACh and ATP, and thus serves to modulate the quantity of transmitter released.

  3. Matching kinetics of synaptic vesicle recycling and enhanced neurotransmitter influx by Ca2+ in brain plasma membrane vesicles.

    PubMed

    Kovács, I; Szárics, E; Nyitrai, G; Blandl, T; Kardos, J

    1998-11-01

    Using native plasma membrane vesicle suspensions from the rat cerebral cortex under conditions designed to alter intravesicular [Ca2+], we found that Ca2+ induced 47 +/- 5% more influx of [3H]GABA, [3H]D-aspartate and [3H]glycine at 37 degrees C with half-times 1.7 +/- 0.5, 1.3 +/- 0.4 and 1.3 +/- 0.4 min, respectively. We labelled GABA transporter sites with the uptake inhibitor, [3H]-(R,S)-N-[4,4-bis(3-methyl-2-thienyl)but-3-en-1-yl]nipecotic acid and found that Ca2+ induced a partial dissociation of the bound inhibitor from GABA transporter sites with a similar half-time. By means of rapid kinetic techniques applied to native plasma membrane vesicle suspensions, containing synaptic vesicles stained with the amphipathic fluorescent styryl membrane probe N-(3-triethylammoniumpropyl)-4-[4-(dibutylamino)styryl]pyrid inium dibromide, we have measured the progress of the release and reuptake of synaptic vesicles in response to Ca2+ and high-[K+] depolarization in the 0.0004-100 s range of time. Synaptic vesicle exocytosis, strongly influenced by external [Ca2+], appeared with the kinetics accelerated by depolarization. These results are consistent with the potential involvement of Ca2+ in taking low-affinity transporters to the plasma membrane surface via exocytosis.

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

    PubMed

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

    2010-01-01

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

  5. Acute increase of α-synuclein inhibits synaptic vesicle recycling evoked during intense stimulation

    PubMed Central

    Busch, David J.; Oliphint, Paul A.; Walsh, Rylie B.; Banks, Susan M. L.; Woods, Wendy S.; George, Julia M.; Morgan, Jennifer R.

    2014-01-01

    Parkinson's disease is associated with multiplication of the α-synuclein gene and abnormal accumulation of the protein. In animal models, α-synuclein overexpression broadly impairs synaptic vesicle trafficking. However, the exact steps of the vesicle trafficking pathway affected by excess α-synuclein and the underlying molecular mechanisms remain unknown. Therefore we acutely increased synuclein levels at a vertebrate synapse and performed a detailed ultrastructural analysis of the effects on presynaptic membranes. At stimulated synapses (20 Hz), excess synuclein caused a loss of synaptic vesicles and an expansion of the plasma membrane, indicating an impairment of vesicle recycling. The N-terminal domain (NTD) of synuclein, which folds into an α-helix, was sufficient to reproduce these effects. In contrast, α-synuclein mutants with a disrupted N-terminal α-helix (T6K and A30P) had little effect under identical conditions. Further supporting this model, another α-synuclein mutant (A53T) with a properly folded NTD phenocopied the synaptic vesicle recycling defects observed with wild type. Interestingly, the vesicle recycling defects were not observed when the stimulation frequency was reduced (5 Hz). Thus excess α-synuclein impairs synaptic vesicle recycling evoked during intense stimulation via a mechanism that requires a properly folded N-terminal α-helix. PMID:25273557

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

    PubMed

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

    2014-11-24

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

  7. A quantitative analytic pipeline for evaluating neuronal activities by high throughput synaptic vesicle imaging

    PubMed Central

    Fan, Jing; Xia, Xiaofeng; Li, Ying; Dy, Jennifer G.

    2012-01-01

    Synaptic vesicle dynamics play an important role in the study of neuronal and synaptic activities of neurodegradation diseases ranging from the epidemic Alzheimer’s disease to the rare Rett syndrome. A high-throughput assay with a large population of neurons would be useful and efficient to characterize neuronal activity based on the dynamics of synaptic vesicles for the study of mechanisms or to discover drug candidates for neurodegenerative and neurodevelopmental disorders. However, the massive amounts of image data generated via high throughput screening require enormous manual processing time and effort, restricting the practical use of such an assay. This paper presents an automated analytic system to process and interpret the huge data set generated by such assays. Our system enables the automated detection, segmentation, quantification, and measurement of neuron activities based on the synaptic vesicle assay. To overcome challenges such as noisy background, inhomogeneity, and tiny object size, we first employ MSVST (Multi-Scale Variance Stabilizing Transform) to obtain a denoised and enhanced map of the original image data. Then, we propose an adaptive thresholding strategy to solve the inhomogeneity issue, based on the local information, and to accurately segment synaptic vesicles. We design algorithms to address the issue of tiny objects-of-interest overlapping. Several post-processing criteria are defined to filter false positives. A total of 152 features are extracted for each detected vesicle. A score is defined for each synaptic vesicle image to quantify the neuron activity. We also compare the unsupervised strategy with the supervised method. Our experiments on hippocampal neuron assays showed that the proposed system can automatically detect vesicles and quantify their dynamics for evaluating neuron activities. The availability of such an automated system will open opportunities for investigation of synaptic neuropathology and identification of

  8. Distinct Functions of Syntaxin-1 in Neuronal Maintenance, Synaptic Vesicle Docking, and Fusion in Mouse Neurons.

    PubMed

    Vardar, Gülçin; Chang, Shuwen; Arancillo, Marife; Wu, Yuan-Ju; Trimbuch, Thorsten; Rosenmund, Christian

    2016-07-27

    Neurotransmitter release requires the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes by SNARE proteins syntaxin-1 (Stx1), synaptosomal-associated protein 25 (SNAP-25), and synaptobrevin-2 (Syb2). In mammalian systems, loss of SNAP-25 or Syb2 severely impairs neurotransmitter release; however, complete loss of function studies for Stx1 have been elusive due to the functional redundancy between Stx1 isoforms Stx1A and Stx1B and the embryonic lethality of Stx1A/1B double knock-out (DKO) mice. Here, we studied the roles of Stx1 in neuronal maintenance and neurotransmitter release in mice with constitutive or conditional deletion of Stx1B on an Stx1A-null background. Both constitutive and postnatal loss of Stx1 severely compromised neuronal viability in vivo and in vitro, indicating an obligatory role of Stx1 for maintenance of developing and mature neurons. Loss of Munc18-1, a high-affinity binding partner of Stx1, also showed severely impaired neuronal viability, but with a slower time course compared with Stx1A/1B DKO neurons, and exogenous Stx1A or Stx1B expression significantly delayed Munc18-1-dependent lethality. In addition, loss of Stx1 completely abolished fusion-competent vesicles and severely impaired vesicle docking, demonstrating its essential roles in neurotransmission. Putative partial SNARE complex assembly with the SNARE motif mutant Stx1A(AV) (A240V, V244A) was not sufficient to rescue neurotransmission despite full recovery of vesicle docking and neuronal survival. Together, these data suggest that Stx1 has independent functions in neuronal maintenance and neurotransmitter release and complete SNARE complex formation is required for vesicle fusion and priming, whereas partial SNARE complex formation is sufficient for vesicle docking and neuronal maintenance. Syntaxin-1 (Stx1) is a component of the synaptic vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE

  9. Transport vesicle formation in plant cells.

    PubMed

    Hwang, Inhwan; Robinson, David G

    2009-12-01

    In protein trafficking, transport vesicles bud from donor compartments and carry cargo proteins to target compartments with which they fuse. Thus, vesicle formation is an essential step in protein trafficking. As for mammals, plant cells contain the three major types of vesicles: COPI, COPII, and CCV and the major molecular players in vesicle-mediated protein transport are also present. However, plant cells generally contain more isoforms of the coat proteins, ARF GTPases and their regulatory proteins, as well as SNAREs. In addition, plants have established some unique subfamilies, which may reflect plant cell-specific conditions such as the absence of an ER-Golgi intermediate compartment and the combined activities of the TGN and early endosome. Thus, even though we are still at an early stage in understanding the physiological function of these proteins, it is already clear that vesicle-mediated protein transport in plant cells displays both similarities as well as differences in animal cells.

  10. ELECTRON MICROSCOPE OBSERVATIONS ON SYNAPTIC VESICLES IN SYNAPSES OF THE RETINAL RODS AND CONES

    PubMed Central

    De Robertis, Eduardo; Franchi, Carlos M.

    1956-01-01

    The submicroscopic organization of the rod and cone synapses of the albino rabbit has been investigated with the use of the electron microscope. The most common rod synapse consists of an enlarged expansion of the rod fiber (the so called spherule) into which the dendritic postsynaptic fiber of the bipolar cell penetrates and digitates. The membrane surrounding the terminal consists of a double layer, the external of which is interpreted as belonging to the intervening glial cells. The synaptic membrane has a pre- and a postsynaptic layer with a total thickness of 180 to 300 A. The presynaptic layer is frequently denser and is intimately associated with the adjacent synaptic vesicles. The synaptic membrane shows processes constituted by foldings of the presynaptic layer. The entire spherule is filled with synaptic vesicles varying in diameter between 200 and 650 A with a mean of 386 A. In addition, the spherule contains a few large vacuoles near the rod fiber, interpreted as endoplasmic reticulum, and a matrix in which with high resolution a fine filamentous material can be observed. The postsynaptic fiber is homogeneous and usually does not show synaptic vesicles. In animals maintained in complete darkness for 24 hours vesicles appear to accumulate near the synaptic membrane and its processes. After 9 days there is a sharp decrease in size of the synaptic vesicles. A special rod synapse in which the dendritic postsynaptic expansion penetrates directly into the rod cell body has been identified. In line with Cajal's classification this type of synapse could be considered as a somatodendritic one. The cone synapse has a much larger terminal with a more complex relationship with the postsynaptic fiber. However, the same components recognized in the rod synapse can be observed. In animals maintained for 9 days in complete darkness there is also a considerable diminution in size of the synaptic vesicles. PMID:13331963

  11. 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

  12. 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

  13. Acute destruction of the synaptic ribbon reveals a role for the ribbon in vesicle priming

    PubMed Central

    Snellman, Josefin; Mehta, Bhupesh; Babai, Norbert; Bartoletti, Theodore M.; Akmentin, Wendy; Francis, Adam; Matthews, Gary; Thoreson, Wallace; Zenisek, David

    2011-01-01

    In vision, balance, and hearing, sensory receptor cells translate sensory stimuli into electrical signals whose amplitude is graded with stimulus intensity. The output synapses of these sensory neurons must provide fast signaling to follow rapidly changing stimuli, while also transmitting graded information covering a wide range of stimulus intensity and sustained for long time periods. To meet these demands, specialized machinery for transmitter release—the synaptic ribbon—has evolved at the synaptic outputs of these neurons. Here we show that acute disruption of synaptic ribbons by photodamage to the ribbon dramatically reduces both sustained and transient components of neurotransmitter release in mouse bipolar cells and salamander cones, without affecting the ultrastructure of the ribbon or its ability to localize synaptic vesicles to the active zone. Our results indicate that ribbons mediate slow as well as fast signaling at sensory synapses, and support an additional role for the synaptic ribbon in priming vesicles for exocytosis at active zones. PMID:21785435

  14. UNC-41/Stonin Functions with AP2 to Recycle Synaptic Vesicles in Caenorhabditis elegans

    PubMed Central

    Watanabe, Shigeki; Hobson, Robert J.; McManus, John R.; Mathews, Eleanor A.; Jorgensen, Erik M.; Rand, James B.

    2012-01-01

    The recycling of synaptic vesicles requires the recovery of vesicle proteins and membrane. Members of the stonin protein family (Drosophila Stoned B, mammalian stonin 2) have been shown to link the synaptic vesicle protein synaptotagmin to the endocytic machinery. Here we characterize the unc-41 gene, which encodes the stonin ortholog in the nematode Caenorhabditis elegans. Transgenic expression of Drosophila stonedB rescues unc-41 mutant phenotypes, demonstrating that UNC-41 is a bona fide member of the stonin family. In unc-41 mutants, synaptotagmin is present in axons, but is mislocalized and diffuse. In contrast, UNC-41 is localized normally in synaptotagmin mutants, demonstrating a unidirectional relationship for localization. The phenotype of snt-1 unc-41 double mutants is stronger than snt-1 mutants, suggesting that UNC-41 may have additional, synaptotagmin-independent functions. We also show that unc-41 mutants have defects in synaptic vesicle membrane endocytosis, including a ∼50% reduction of vesicles in both acetylcholine and GABA motor neurons. These endocytic defects are similar to those observed in apm-2 mutants, which lack the µ2 subunit of the AP2 adaptor complex. However, no further reduction in synaptic vesicles was observed in unc-41 apm-2 double mutants, suggesting that UNC-41 acts in the same endocytic pathway as µ2 adaptin. PMID:22808098

  15. Artificially imposed electrical potentials drive L-glutamate uptake into synaptic vesicles of bovine cerebral cortex.

    PubMed Central

    Shioi, J; Ueda, T

    1990-01-01

    L-Glutamate is a major excitatory neurotransmitter in the central nervous system. MgATP-dependent glutamate uptake and H(+)-pumping ATPase activity were reported in highly purified synaptic vesicles [Naito & Ueda (1983) J. Biol. Chem. 258, 696-699; Shioi, Naito & Ueda (1989) Biochem. J. 258, 499-504], and it is hypothesized that an electrochemical H+ gradient across the vesicle membrane, the so-called protonmotive force, elicits the neurotransmitter uptake. An inside-positive diffusion potential across the vesicle membrane was established with valinomycin plus Rb+. This artificial electrical potential promoted the uptake of glutamate, but not aspartate, in the synaptic vesicles prepared from bovine cerebral cortex. The uptake was inhibited by the protonmotive-force dissipators carbonyl cyanide p-trifluoro-methoxyphenylhydrazone or nigericin, and was enhanced by concomitant imposition of a pH jump (alkalinization) in the external medium. Subcellular and subvesicular distributions showed the uptake system to be predominantly associated with small synaptic vesicles. The results support the hypothesis that glutamate uptake into synaptic vesicles is coupled with a H+ efflux down the electrochemical potential gradient, which is generated by H(+)-pumping ATPase. Images Fig. 3. PMID:1970243

  16. 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

  17. 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.

  18. Two distinct populations of synaptic-like vesicles from rat brain

    PubMed Central

    Thoidis, Galini; Chen, Peng; Pushkin, Alexander V.; Vallega, Gino; Leeman, Susan E.; Fine, Richard E.; Kandror, Konstantin V.

    1998-01-01

    In nonneuronal cells, several plasma membrane proteins such as exofacial enzymes, receptors, and ion channels recycle between their intracellular compartment(s) and the cell surface via an endosomal pathway. In neurons, however, this pathway has not been extensively characterized. In particular, it remains unclear whether or not it is related to the recycling of small synaptic vesicles, the major pathway of membrane traffic in nerve terminals. To approach this problem, we purified and studied a vesicular fraction from rat brain synaptosomes. Two distinct populations of vesicles with different buoyant densities and sedimentation coefficients were detected in this fraction by sucrose gradient centrifugation and Western blot analysis of the individual proteins. Both populations contain proteins that are markers of synaptic vesicles, namely, SV2, synaptotagmin, synaptophysin, secretory carrier membrane proteins (SCAMPs), synaptobrevin, and rab3a. A striking difference between the two populations is the presence of arginine aminopeptidase activity (a previously suggested marker for the regulated endosomal recycling pathway) exclusively in the lighter less-dense vesicles. The same two vesicular populations were also detected in the preparation of clathrin-coated vesicles isolated from whole rat brain or purified synaptosomes after removal of their clathrin coats by incubation at pH 8.5. We conclude, therefore, that both types of vesicles recycle in synaptosomes via a clathrin-mediated pathway. These data present experimental evidence for biochemical heterogeneity of synaptic-like vesicles in rat brain. PMID:9419350

  19. AP-1/σ1B-adaptin mediates endosomal synaptic vesicle recycling, learning and memory

    PubMed Central

    Glyvuk, Nataliya; Tsytsyura, Yaroslav; Geumann, Constanze; D'Hooge, Rudi; Hüve, Jana; Kratzke, Manuel; Baltes, Jennifer; Böning, Daniel; Klingauf, Jürgen; Schu, Peter

    2010-01-01

    Synaptic vesicle recycling involves AP-2/clathrin-mediated endocytosis, but it is not known whether the endosomal pathway is also required. Mice deficient in the tissue-specific AP-1–σ1B complex have impaired synaptic vesicle recycling in hippocampal synapses. The ubiquitously expressed AP-1–σ1A complex mediates protein sorting between the trans-Golgi network and early endosomes. Vertebrates express three σ1 subunit isoforms: A, B and C. The expressions of σ1A and σ1B are highest in the brain. Synaptic vesicle reformation in cultured neurons from σ1B-deficient mice is reduced upon stimulation, and large endosomal intermediates accumulate. The σ1B-deficient mice have reduced motor coordination and severely impaired long-term spatial memory. These data reveal a molecular mechanism for a severe human X-chromosome-linked mental retardation. PMID:20203623

  20. Synaptotagmin-12, a synaptic vesicle phosphoprotein that modulates spontaneous neurotransmitter release

    PubMed Central

    Maximov, Anton; Shin, Ok-Ho; Liu, Xinran; Südhof, Thomas C.

    2007-01-01

    Central synapses exhibit spontaneous neurotransmitter release that is selectively regulated by cAMP-dependent protein kinase A (PKA). We now show that synaptic vesicles contain synaptotagmin-12, a synaptotagmin isoform that differs from classical synaptotagmins in that it does not bind Ca2+. In synaptic vesicles, synaptotagmin-12 forms a complex with synaptotagmin-1 that prevents synaptotagmin-1 from interacting with SNARE complexes. We demonstrate that synaptotagmin-12 is phosphorylated by cAMP-dependent PKA on serine97, and show that expression of synaptotagmin-12 in neurons increases spontaneous neurotransmitter release by approximately threefold, but has no effect on evoked release. Replacing serine97 by alanine abolishes synaptotagmin-12 phosphorylation and blocks its effect on spontaneous release. Our data suggest that spontaneous synaptic-vesicle exocytosis is selectively modulated by a Ca2+-independent synaptotagmin isoform, synaptotagmin-12, which is controlled by cAMP-dependent phosphorylation. PMID:17190793

  1. Synaptotagmin-12, a synaptic vesicle phosphoprotein that modulates spontaneous neurotransmitter release.

    PubMed

    Maximov, Anton; Shin, Ok-Ho; Liu, Xinran; Südhof, Thomas C

    2007-01-01

    Central synapses exhibit spontaneous neurotransmitter release that is selectively regulated by cAMP-dependent protein kinase A (PKA). We now show that synaptic vesicles contain synaptotagmin-12, a synaptotagmin isoform that differs from classical synaptotagmins in that it does not bind Ca(2+). In synaptic vesicles, synaptotagmin-12 forms a complex with synaptotagmin-1 that prevents synaptotagmin-1 from interacting with SNARE complexes. We demonstrate that synaptotagmin-12 is phosphorylated by cAMP-dependent PKA on serine(97), and show that expression of synaptotagmin-12 in neurons increases spontaneous neurotransmitter release by approximately threefold, but has no effect on evoked release. Replacing serine(97) by alanine abolishes synaptotagmin-12 phosphorylation and blocks its effect on spontaneous release. Our data suggest that spontaneous synaptic-vesicle exocytosis is selectively modulated by a Ca(2+)-independent synaptotagmin isoform, synaptotagmin-12, which is controlled by cAMP-dependent phosphorylation.

  2. Synaptic Plasticity and Memory Formation

    DTIC Science & Technology

    1994-05-31

    The name " Ampakines " has been used to describe this family; when more is known about structure-activity relationships, it should be possible to...regarding the physiological effects of the drugs. Excised patch studies have shown that Ampakines prolong the duration of AMPA receptor-mediated...also revealed that Ampakines produce the expected facilitation and prolongation of synaptic responses in situ; these drugs are thus the first compounds

  3. 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

  4. Neurotransmitter Release: The Last Millisecond in the Life of a Synaptic Vesicle

    PubMed Central

    Südhof, Thomas C.

    2013-01-01

    During an action potential, Ca2+ entering a presynaptic terminal triggers synaptic vesicle exocytosis and neurotransmitter release in less than a millisecond. How does Ca2+ stimulate release so rapidly and precisely? Work over the last decades revealed that Ca2+-binding to synaptotagmin triggers release by stimulating synaptotagmin-binding to a core machinery composed of SNARE and SM proteins that mediates membrane fusion during exocytosis. Complexin adaptor proteins assist synaptotagmin by activating and clamping this core fusion machinery. Synaptic vesicles containing synaptotagmin are positioned at the active zone, the site of vesicle fusion, by a protein complex containing RIM proteins. RIM proteins simultaneously activate docking and priming of synaptic vesicles and recruit Ca2+-channels to active zones, thereby connecting in a single complex primed synaptic vesicles to Ca2+-channels. This architecture allows direct flow of Ca2+-ions from Ca2+-channels to synaptotagmin, which then triggers fusion, thus mediating tight millisecond coupling of an action potential to neurotransmitter release. PMID:24183019

  5. 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.

  6. Activity Induces Fmr1-Sensitive Synaptic Capture of Anterograde Circulating Neuropeptide Vesicles

    PubMed Central

    Cavolo, Samantha L.; Bulgari, Dinara; Deitcher, David L.

    2016-01-01

    Synaptic neuropeptide and neurotrophin stores are maintained by constitutive bidirectional capture of dense-core vesicles (DCVs) as they circulate in and out of the nerve terminal. Activity increases DCV capture to rapidly replenish synaptic neuropeptide stores following release. However, it is not known whether this is due to enhanced bidirectional capture. Here experiments at the Drosophila neuromuscular junction, where DCVs contain neuropeptides and a bone morphogenic protein, show that activity-dependent replenishment of synaptic neuropeptides following release is evident after inhibiting the retrograde transport with the dynactin disruptor mycalolide B or photobleaching DCVs entering a synaptic bouton by retrograde transport. In contrast, photobleaching anterograde transport vesicles entering a bouton inhibits neuropeptide replenishment after activity. Furthermore, tracking of individual DCVs moving through boutons shows that activity selectively increases capture of DCVs undergoing anterograde transport. Finally, upregulating fragile X mental retardation 1 protein (Fmr1, also called FMRP) acts independently of futsch/MAP-1B to abolish activity-dependent, but not constitutive, capture. Fmr1 also reduces presynaptic neuropeptide stores without affecting activity-independent delivery and evoked release. Therefore, presynaptic motoneuron neuropeptide storage is increased by a vesicle capture mechanism that is distinguished from constitutive bidirectional capture by activity dependence, anterograde selectivity, and Fmr1 sensitivity. These results show that activity recruits a separate mechanism than used at rest to stimulate additional synaptic capture of DCVs for future release of neuropeptides and neurotrophins. SIGNIFICANCE STATEMENT Synaptic release of neuropeptides and neurotrophins depends on presynaptic accumulation of dense-core vesicles (DCVs). At rest, DCVs are captured bidirectionally as they circulate through Drosophila motoneuron terminals by

  7. Reduced Synaptic Vesicle Recycling during Hypoxia in Cultured Cortical Neurons

    PubMed Central

    Fedorovich, Sergei; Hofmeijer, Jeannette; van Putten, Michel J. A. M.; le Feber, Joost

    2017-01-01

    Improvement of neuronal recovery in the ischemic penumbra, an area around the core of a brain infarct with some remaining perfusion, has a large potential for the development of therapy against acute ischemic stroke. However, mechanisms that lead to either recovery or secondary damage in the penumbra largely remain unclear. Recent studies in cultured networks of cortical neurons showed that failure of synaptic transmission (referred to as synaptic failure) is a critical factor in the penumbral area, but the mechanisms that lead to synaptic failure are still under investigation. Here we used a Styryl dye, FM1-43, to quantify endocytosis and exocytosis in cultures of rat cortical neurons under normoxic and hypoxic conditions. Hypoxia in cultured cortical networks rapidly depressed endocytosis and, to a lesser extent, exocytosis. These findings support electrophysiological findings that synaptic failure occurs quickly after the induction of hypoxia, and confirms that the failing processes are at least in part presynaptic. PMID:28261063

  8. Synaptic vesicle populations in saccular hair cells reconstructed by electron tomography.

    PubMed

    Lenzi, D; Runyeon, J W; Crum, J; Ellisman, M H; Roberts, W M

    1999-01-01

    We used electron tomography to map the three-dimensional architecture of the ribbon-class afferent synapses in frog saccular hair cells. The synaptic body (SB) at each synapse was nearly spherical (468 +/- 65 nm diameter; mean +/- SD) and was covered by a monolayer of synaptic vesicles (34.3 nm diameter; 8.8% coefficient of variation), many of them tethered to it by approximately 20-nm-long filaments, at an average density of 55% of close-packed (376 +/- 133 vesicles per SB). These vesicles could support approximately 900 msec of exocytosis at the reported maximal rate, which the cells can sustain for at least 2 sec, suggesting that replenishment of vesicles on the SB is not rate limiting. Consistent with this interpretation, prolonged K+ depolarization did not deplete vesicles on the SB. The monolayer of SB-associated vesicles remained after cell lysis in the presence of 4 mM Ca2+, indicating that the association is tight and Ca2+-resistant. The space between the SB and the plasma membrane contained numerous vesicles, many of which ( approximately 32 per synapse) were in contact with the plasma membrane. This number of docked vesicles could support maximal exocytosis for at most approximately 70 msec. Additional docked vesicles were seen within a few hundred nanometers of the synapse and occasionally at greater distances. The presence of omega profiles on the plasma membrane around active zones, in the same locations as coated pits and coated vesicles labeled with an extracellular marker, suggests that local membrane recycling may contribute to the three- to 14-fold greater abundance of vesicles in the cytoplasm (not associated with the SB) near synapses than in nonsynaptic regions.

  9. 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.

  10. Exo-endocytotic recycling of synaptic vesicles in developing processes of cultured hippocampal neurons

    PubMed Central

    1992-01-01

    In mature neurons synaptic vesicles (SVs) undergo cycles of exo- endocytosis at synapses. It is currently unknown whether SV exocytosis and recycling occurs also in developing axons prior to synapse formation. To address this question, we have developed an immunocytochemical assay to reveal SV exo-endocytosis in hippocampal neurons developing in culture. In this assay antibodies directed against the lumenal domain of synaptotagmin I (Syt I), an intrinsic membrane protein of SVs, are used to reveal exposure of SV membranes at the cell surface. Addition of antibodies to the culture medium of living neurons for 1 hr at 37 degrees C resulted in their rapid and specific internalization by all neuronal processes and, particularly, by axons. Double immunofluorescence and electron microscopy immunocytochemistry indicated that the antibodies were retained within SVs in cell processes and underwent cycles of exo-endocytosis in parallel with SV membranes. In contrast, another endocytotic marker, wheat germ agglutinin, was rapidly cleared from the processes and transported to the cell body. Antibody-labeled SVs were still present in axons several days after antibody loading and became clustered at presynaptic sites in parallel with synaptogenesis. These results demonstrate that SVs undergo multiple cycles of exo-endocytosis in developing neuronal processes irrespective of the presence of synaptic contacts. PMID:1577861

  11. Synaptic vesicle endocytosis mediates the entry of tetanus neurotoxin into hippocampal neurons

    PubMed Central

    Matteoli, Michela; Verderio, Claudia; Rossetto, Ornella; Iezzi, Numa; Coco, Silvia; Schiavo, Giampietro; Montecucco, Cesare

    1996-01-01

    Tetanus neurotoxin causes the spastic paralysis of tetanus by blocking neurotransmitter release at inhibitory synapses of the spinal cord. This is due to the penetration of the toxin inside the neuronal cytosol where it cleaves specifically VAMP/synaptobrevin, an essential component of the neuroexocytosis apparatus. Here we show that tetanus neurotoxin is internalized inside the lumen of small synaptic vesicles following the process of vesicle reuptake. Vesicle acidification is essential for the toxin translocation in the cytosol, which results in the proteolytic cleavage of VAMP/synaptobrevin and block of exocytosis. PMID:8917587

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. Haloarchaea and the Formation of Gas Vesicles

    PubMed Central

    Pfeifer, Felicitas

    2015-01-01

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

  18. A novel non-canonical Notch signaling regulates expression of synaptic vesicle proteins in excitatory neurons

    PubMed Central

    Hayashi, Yukari; Nishimune, Hiroshi; Hozumi, Katsuto; Saga, Yumiko; Harada, Akihiro; Yuzaki, Michisuke; Iwatsubo, Takeshi; Kopan, Raphael; Tomita, Taisuke

    2016-01-01

    Notch signaling plays crucial roles for cellular differentiation during development through γ-secretase-dependent intramembrane proteolysis followed by transcription of target genes. Although recent studies implicate that Notch regulates synaptic plasticity or cognitive performance, the molecular mechanism how Notch works in mature neurons remains uncertain. Here we demonstrate that a novel Notch signaling is involved in expression of synaptic proteins in postmitotic neurons. Levels of several synaptic vesicle proteins including synaptophysin 1 and VGLUT1 were increased when neurons were cocultured with Notch ligands-expressing NIH3T3 cells. Neuron-specific deletion of Notch genes decreased these proteins, suggesting that Notch signaling maintains the expression of synaptic vesicle proteins in a cell-autonomous manner. Unexpectedly, cGMP-dependent protein kinase (PKG) inhibitor, but not γ-secretase inhibitor, abolished the elevation of synaptic vesicle proteins, suggesting that generation of Notch intracellular domain is dispensable for this function. These data uncover a ligand-dependent, but γ-secretase-independent, non-canonical Notch signaling involved in presynaptic protein expression in postmitotic neurons. PMID:27040987

  19. β-Hydroxybutyrate supports synaptic vesicle cycling but reduces endocytosis and exocytosis in rat brain synaptosomes.

    PubMed

    Hrynevich, Sviatlana V; Waseem, Tatyana V; Hébert, Audrey; Pellerin, Luc; Fedorovich, Sergei V

    2016-02-01

    The ketogenic diet is used as a prophylactic treatment for different types of brain diseases, such as epilepsy or Alzheimer's disease. In such a diet, carbohydrates are replaced by fats in everyday food, resulting in an elevation of blood-borne ketone bodies levels. Despite clinical applications of this treatment, the molecular mechanisms by which the ketogenic diet exerts its beneficial effects are still uncertain. In this study, we investigated the effect of replacing glucose by the ketone body β-hydroxybutyrate as the main energy substrate on synaptic vesicle recycling in rat brain synaptosomes. First, we observed that exposing presynaptic terminals to nonglycolytic energy substrates instead of glucose did not alter the plasma membrane potential. Next, we found that synaptosomes were able to maintain the synaptic vesicle cycle monitored with the fluorescent dye acridine orange when glucose was replaced by β-hydroxybutyrate. However, in presence of β-hydroxybutyrate, synaptic vesicle recycling was modified with reduced endocytosis. Replacing glucose by pyruvate also led to a reduced endocytosis. Addition of β-hydroxybutyrate to glucose-containing incubation medium was without effect. Reduced endocytosis in presence of β-hydroxybutyrate as sole energy substrate was confirmed using the fluorescent dye FM2-10. Also we found that replacement of glucose by ketone bodies leads to inhibition of exocytosis, monitored by FM2-10. However this reduction was smaller than the effect on endocytosis under the same conditions. Using both acridine orange in synaptosomes and the genetically encoded sensor synaptopHluorin in cortical neurons, we observed that replacing glucose by β-hydroxybutyrate did not modify the pH gradient of synaptic vesicles. In conclusion, the nonglycolytic energy substrates β-hydroxybutyrate and pyruvate are able to support synaptic vesicle recycling. However, they both reduce endocytosis. Reduction of both endocytosis and exocytosis together with

  20. Rab3 reversibly recruits rabphilin to synaptic vesicles by a mechanism analogous to raf recruitment by ras.

    PubMed Central

    Stahl, B; Chou, J H; Li, C; Südhof, T C; Jahn, R

    1996-01-01

    GTP activates the interaction between the synaptic vesicle proteins rabphilin and rab3. This raises the question of whether rabphilin is a resident vesicle protein that recruits rab3 in a stage-dependent fashion, or if it is instead an effector protein recruited by rab3. We now show that rabphilin, like rab3, dissociates from synaptic vesicles after exocytosis in a manner requiring both Ca2+ and membrane fusion. Rabphilin interacts with GTP-rab3 via a N-terminal domain comprising a novel Zn2+(-)finger motif, and this interaction is essential for rabphilin binding to synaptic vesicles. Thus, in the same way that ras recruits raf to the plasma membrane, rab3 reversibly recruits rabphilin to synaptic vesicles in a stage-dependent manner. These results reveal an unexpected similarity between the molecular mechanisms by which small G protein function in recruiting effector proteins to membranes during membrane traffic and signal transduction. Images PMID:8617225

  1. 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

  2. Synaptic vesicles in motor synapses change size and distribution during the day.

    PubMed

    Ruiz, Santiago; Ferreiro, Maria Jose; Casanova, Gabriela; Olivera, Alvaro; Cantera, Rafael

    2010-01-01

    The morphology of Drosophila motor terminals changes along the day with a circadian rhythm controlled by the biological clock. Here, we used electron microscopy to investigate the size, number, and distribution of synaptic vesicles, at intervals of 6 h during 2 consecutive days, under light-dark (LD) or the first 2 days in constant darkness (DD). We found changes in the size and distribution of vesicles located either at the active zone or in the reserve pool, indicating a circadian rhythm of synapse reorganization. Vesicles at the active zone were generally smaller than those in the reserve pool in LD and DD conditions. The size of active zones vesicles decreased twice in LD, corresponding with times of more intense locomotion activity, but only once in DD conditions.

  3. LRRK2 controls synaptic vesicle storage and mobilization within the recycling pool.

    PubMed

    Piccoli, Giovanni; Condliffe, Steven B; Bauer, Matthias; Giesert, Florian; Boldt, Karsten; De Astis, Silvia; Meixner, Andrea; Sarioglu, Hakan; Vogt-Weisenhorn, Daniela M; Wurst, Wolfgang; Gloeckner, Christian Johannes; Matteoli, Michela; Sala, Carlo; Ueffing, Marius

    2011-02-09

    Mutations in leucine-rich repeat kinase 2 (LRRK2) are the single most common cause of inherited Parkinson's disease. Little is known about its involvement in the pathogenesis of Parkinson's disease mainly because of the lack of knowledge about the physiological role of LRRK2. To determine the function of LRRK2, we studied the impact of short hairpin RNA-mediated silencing of LRRK2 expression in cortical neurons. Paired recording indicated that LRRK2 silencing affects evoked postsynaptic currents. Furthermore, LRRK2 silencing induces at the presynaptic site a redistribution of vesicles within the bouton, altered recycling dynamics, and increased vesicle kinetics. Accordingly, LRRK2 protein is present in the synaptosomal compartment of cortical neurons in which it interacts with several proteins involved in vesicular recycling. Our results suggest that LRRK2 modulates synaptic vesicle trafficking and distribution in neurons and in consequence participates in regulating the dynamics between vesicle pools inside the presynaptic bouton.

  4. Profilin2 contributes to synaptic vesicle exocytosis, neuronal excitability, and novelty-seeking behavior

    PubMed Central

    Pilo Boyl, Pietro; Di Nardo, Alessia; Mulle, Christophe; Sassoè-Pognetto, Marco; Panzanelli, Patrizia; Mele, Andrea; Kneussel, Matthias; Costantini, Vivian; Perlas, Emerald; Massimi, Marzia; Vara, Hugo; Giustetto, Maurizio; Witke, Walter

    2007-01-01

    Profilins are actin binding proteins essential for regulating cytoskeletal dynamics, however, their function in the mammalian nervous system is unknown. Here, we provide evidence that in mouse brain profilin1 and profilin2 have distinct roles in regulating synaptic actin polymerization with profilin2 preferring a WAVE-complex-mediated pathway. Mice lacking profilin2 show a block in synaptic actin polymerization in response to depolarization, which is accompanied by increased synaptic excitability of glutamatergic neurons due to higher vesicle exocytosis. These alterations in neurotransmitter release correlate with a hyperactivation of the striatum and enhanced novelty-seeking behavior in profilin2 mutant mice. Our results highlight a novel, profilin2-dependent pathway, regulating synaptic physiology, neuronal excitability, and complex behavior. PMID:17541406

  5. Netrin instructs synaptic vesicle clustering through Rac GTPase, MIG-10, and the actin cytoskeleton

    PubMed Central

    Stavoe, Andrea K.H.; Colón-Ramos, Daniel A.

    2012-01-01

    Netrin is a chemotrophic factor known to regulate a number of neurodevelopmental processes, including cell migration, axon guidance, and synaptogenesis. Although the role of Netrin in synaptogenesis is conserved throughout evolution, the mechanisms by which it instructs synapse assembly are not understood. Here we identify a mechanism by which the Netrin receptor UNC-40/DCC instructs synaptic vesicle clustering in vivo. UNC-40 localized to presynaptic regions in response to Netrin. We show that UNC-40 interacted with CED-5/DOCK180 and instructed CED-5 presynaptic localization. CED-5 in turn signaled through CED-10/Rac1 and MIG-10/Lamellipodin to organize the actin cytoskeleton in presynaptic regions. Localization of this signaling pathway to presynaptic regions was necessary for synaptic vesicle clustering during synapse assembly but not for the subcellular localization of active zone proteins. Thus, vesicle clustering and localization of active zone proteins are instructed by separate pathways downstream of Netrin. Our data indicate that signaling modules known to organize the actin cytoskeleton during guidance can be co-opted to instruct synaptic vesicle clustering. PMID:22451697

  6. Synaptic vesicle docking: sphingosine regulates syntaxin1 interaction with Munc18.

    PubMed

    Camoletto, Paola G; Vara, Hugo; Morando, Laura; Connell, Emma; Marletto, Fabio P; Giustetto, Maurizio; Sassoè-Pognetto, Marco; Van Veldhoven, Paul P; Ledesma, Maria Dolores

    2009-01-01

    Consensus exists that lipids must play key functions in synaptic activity but precise mechanistic information is limited. Acid sphingomyelinase knockout mice (ASMko) are a suitable model to address the role of sphingolipids in synaptic regulation as they recapitulate a mental retardation syndrome, Niemann Pick disease type A (NPA), and their neurons have altered levels of sphingomyelin (SM) and its derivatives. Electrophysiological recordings showed that ASMko hippocampi have increased paired-pulse facilitation and post-tetanic potentiation. Consistently, electron microscopy revealed reduced number of docked vesicles. Biochemical analysis of ASMko synaptic membranes unveiled higher amounts of SM and sphingosine (Se) and enhanced interaction of the docking molecules Munc18 and syntaxin1. In vitro reconstitution assays demonstrated that Se changes syntaxin1 conformation enhancing its interaction with Munc18. Moreover, Se reduces vesicle docking in primary neurons and increases paired-pulse facilitation when added to wt hippocampal slices. These data provide with a novel mechanism for synaptic vesicle control by sphingolipids and could explain cognitive deficits of NPA patients.

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

    NASA Astrophysics Data System (ADS)

    Parsons, Stanley M.; Koenigsberger, Robert

    1980-10-01

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

  8. 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

  9. 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

  10. Botulinum neurotoxin type A inhibits synaptic vesicle 2 expression in breast cancer cell lines

    PubMed Central

    Bandala, C; Cortés-Algara, AL; Mejía-Barradas, CM; Ilizaliturri-Flores, I; Dominguez-Rubio, R; Bazán-Méndez, CI; Floriano-Sánchez, E; Luna-Arias, JP; Anaya-Ruiz, M; Lara-Padilla, E

    2015-01-01

    Aim: It is known that botulinum neurotoxin type A (BoNTA) improves some kinds of cancer (e.g. prostate) and that synaptic vesicle glycoprotein 2 (SV2) is the molecular target of this neurotoxin. Besides having potential therapeutic value, this glycoprotein has recently been proposed as a molecular marker for several types of cancer. Although the mechanisms of cancer development and the improvement found with botulinum treatment are not well understood, the formation of the botulinum-SV2 complex may influence the presence and distribution of SV2 and the function of vesicles. To date, there are no reports on the possible effect of botulinum on breast cancer of unknown causes, which have a great impact on women’s health. Thus we determined the presence of SV2 in three breast cancer cell lines and the alterations found with botulinum application. Materials and methods: With and without adding 10 units of botulinum, SV2 protein expression was determined by optical densitometry in T47D, MDA-MB-231 and MDA-MB-453 cell lines and the distribution of SV2 was observed with immunochemistry (hematoxylin staining). Results: The SV2 protein was abundant in the cancer cells herein tested, and maximally so in T47D. In all three cancer cell lines botulinum diminished SV2 expression, which was found mostly in the cell periphery. Conclusion: SV2 could be a molecular marker in breast cancer. Its expression and distribution is regulated by botulinum, suggesting an interesting control mechanism for SV2 expression and a possible alternative therapy. Further studies are needed in this sense. PMID:26339411

  11. An Endocytic Scaffolding Protein together with Synapsin Regulates Synaptic Vesicle Clustering in the Drosophila Neuromuscular Junction.

    PubMed

    Winther, Åsa M E; Vorontsova, Olga; Rees, Kathryn A; Näreoja, Tuomas; Sopova, Elena; Jiao, Wei; Shupliakov, Oleg

    2015-11-04

    Many endocytic proteins accumulate in the reserve pool of synaptic vesicles (SVs) in synapses and relocalize to the endocytic periactive zone during neurotransmitter release. Currently little is known about their functions outside the periactive zone. Here we show that in the Drosophila neuromuscular junction (NMJ), the endocytic scaffolding protein Dap160 colocalizes during the SV cycle and forms a functional complex with the SV-associated phosphoprotein synapsin, previously implicated in SV clustering. This direct interaction is strongly enhanced under phosphorylation-promoting conditions and is essential for proper localization of synapsin at NMJs. In a dap160 rescue mutant lacking the interaction between Dap160 and synapsin, perturbed reclustering of SVs during synaptic activity is observed. Our data indicate that in addition to the function in endocytosis, Dap160 is a component of a network of protein-protein interactions that serves for clustering of SVs in conjunction with synapsin. During the SV cycle, Dap160 interacts with synapsin dispersed from SVs and helps direct synapsin back to vesicles. The proteins function in synergy to achieve efficient clustering of SVs in the reserve pool. We provide the first evidence for the function of the SH3 domain interaction in synaptic vesicle (SV) organization at the synaptic active zone. Using Drosophila neuromuscular junction as a model synapse, we describe the molecular mechanism that enables the protein implicated in SV clustering, synapsin, to return to the pool of vesicles during neurotransmitter release. We also identify the endocytic scaffolding complex that includes Dap160 as a regulator of the events linking exocytosis and endocytosis in synapses. Copyright © 2015 the authors 0270-6474/15/3514756-15$15.00/0.

  12. 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

  13. Developmental changes in the localization of the synaptic vesicle protein rab3A in rat brain.

    PubMed

    Stettler, O; Moya, K L; Zahraoui, A; Tavitian, B

    1994-09-01

    Rab3A is a protein associated with the membrane of synaptic vesicles and is involved in the control of the targeting or docking of these vesicles at the presynaptic membrane for the release of neurotransmitters. Here, we have examined the expression and localization of this protein during the development of the rat brain. Relative to total protein, the concentration of rab3A greatly increased during brain development. Both the intracellular localization of the protein and its cerebral distribution showed an age-dependent shift. In contrast to other synaptic vesicle proteins, rab3A was heavily concentrated in cell bodies when immature neurons were migrating and during early differentiation. Later, the protein disappeared from perikarya and had a diffuse distribution in the neuropil, indicating a redistribution to nerve terminals, its exclusive localization in the adult. In the developing somatosensory cortex, rab3A delimited the modular organization of the barrels well after the afferents have arrived but just around the time that mature synaptic activity has been observed. In the hippocampus, rab3A defined a novel "blob-like" organization of the mossy fibre terminals and its appearance in terminal fields closely preceded the known onset of long-term potentiation. The appearance of rab3A in specific terminal fields during the period of increased physiological activity suggests that this small GTP-binding protein may be an important late element in the establishment of the mature characteristics of the presynaptic terminal.

  14. Differences in the osmotic fragility of recycling and reserve synaptic vesicles from the cholinergic electromotor nerve terminals of Torpedo and their possible significance for vesicle recycling.

    PubMed

    Giompres, P E; Whittaker, V P

    1984-03-14

    In this study we demonstrate differences in the osmotic fragility of two metabolically and physically heterogeneous synaptic vesicle populations from stimulated electromotor nerve terminals. When synaptic vesicles isolated on sucrose density gradients are submitted to solutions of decreasing osmolarity 50% of VP2-type vesicles lysed at (mean + S.E. (number of experiments] 332 +/- 14 (4) mosM and 50% of VP1-type vesicles lysed at 573 +/- 8 (3) mosM. These results indicate that recycling vesicles are more resistant to hypo-osmotic lysis and they are consistent with our earlier conclusion that changes in water content on recycling are secondary to changes in the content of the osmotically active small-molecular-mass constituents acetylcholine and ATP.

  15. Molecular Machines Determining the Fate of Endocytosed Synaptic Vesicles in Nerve Terminals

    PubMed Central

    Fassio, Anna; Fadda, Manuela; Benfenati, Fabio

    2016-01-01

    The cycle of a synaptic vesicle (SV) within the nerve terminal is a step-by-step journey with the final goal of ensuring the proper synaptic strength under changing environmental conditions. The SV cycle is a precisely regulated membrane traffic event in cells and, because of this, a plethora of membrane-bound and cytosolic proteins are devoted to assist SVs in each step of the journey. The cycling fate of endocytosed SVs determines both the availability for subsequent rounds of release and the lifetime of SVs in the terminal and is therefore crucial for synaptic function and plasticity. Molecular players that determine the destiny of SVs in nerve terminals after a round of exo-endocytosis are largely unknown. Here we review the functional role in SV fate of phosphorylation/dephosphorylation of SV proteins and of small GTPases acting on membrane trafficking at the synapse, as they are emerging as key molecules in determining the recycling route of SVs within the nerve terminal. In particular, we focus on: (i) the cyclin-dependent kinase-5 (cdk5) and calcineurin (CN) control of the recycling pool of SVs; (ii) the role of small GTPases of the Rab and ADP-ribosylation factor (Arf) families in defining the route followed by SV in their nerve terminal cycle. These regulatory proteins together with their synaptic regulators and effectors, are molecular nanomachines mediating homeostatic responses in synaptic plasticity and potential targets of drugs modulating the efficiency of synaptic transmission. PMID:27242505

  16. β-Pix modulates actin-mediated recruitment of synaptic vesicles to synapses.

    PubMed

    Sun, Yu; Bamji, Shernaz X

    2011-11-23

    Presynaptic compartments are formed through the recruitment of preassembled clusters of proteins to points of cell-cell contact, however, the molecular mechanism(s) underlying this process remains unclear. We demonstrate that clusters of polymerized actin can recruit and maintain synaptic vesicles to discrete sites along the axon, and that cadherin/β-catenin/scribble/β-pix complexes play an important role in this event. Previous work has demonstrated that β-catenin and scribble are important for the clustering of vesicles at synapses. We demonstrate that β-pix, a Rac/Cdc42 guanine nucleotide exchange factor (GEF), forms a complex with cadherin, β-catenin, and scribble at synapses and enhances localized actin polymerization in rat hippocampal neurons. In cells expressing β-pix siRNA or dominant-negative β-pix that lacks its GEF activity, actin polymerization at synapses is dramatically reduced, and synaptic vesicle localization is disrupted. This β-pix phenotype can be rescued by cortactin overexpression, suggesting that β-pix-mediated actin polymerization at synapses regulates vesicle localization.

  17. Circulation and turnover of synaptic vesicle membrane in cultured fetal mammalian spinal cord neurons

    PubMed Central

    1975-01-01

    Intact neurons in cultures of fetal rodent spinal cord explants show stimulation-dependent uptake of horseradish peroxidase (HRP) into many small vesicles and occasional tubules and multivesicular bodies (MVB) at presynaptic terminals. Presynaptic terminals were allowed to take up HRP during 1 h of strychnine-enhanced stimulation of synaptic transmitter release and then "chased" in tracer-free medium either with strychnine or with 10 mM Mg++ which depresses transmitter release. Tracer-containing vesicles are lost from terminals under both chase conditions; the loss is more rapid (4-8 h) with strychnine than with 10 mM Mg++ (8-16 h). There is a parallel decrease in the numbers of labeled MVB's at terminals. Loss of tracer with 10 mM Mg++ does not appear to be due to the membrane rearrangements (exocytosis coupled to endocytosis) that presumably lead to initial tracer uptake; terminals exposed to HRP and Mg++ for up to 16 h show little tracer uptake into vesicles. Nor is the decrease likely to the due to loss of HRP enzyme activity; HRP is very stable in solution. During the chases there is a striking accumulation of HRP in perikarya that is far more extensive in cultures initially exposed to tracer with strychnine than 10 mM Mg++ regardless of chase conditions. Much of the tracer ends up in large dense bodies. These findings suggest that synaptic vesicle membrane turnover involves retrograde axonal transport of membrane to neuronal perikarya for further processing, including lysosomal degradation. The more rapid (4-8 h) loss of tracer-containing vesicles with strychnine may reflect vesicle membrane reutilization for exocytosis. PMID:1176531

  18. The regulation of synaptic vesicle recycling by cGMP-dependent protein kinase type II in cerebellar granule cells under strong and sustained stimulation.

    PubMed

    Collado-Alsina, Andrea; Ramírez-Franco, Jorge; Sánchez-Prieto, José; Torres, Magdalena

    2014-06-25

    From the early periods of neurogenesis and migration, up until synaptogenesis, both nitric oxide (NO) and its downstream messenger, cGMP, are thought to influence the development of neurons. The NO/cGMP/cGMP-dependent protein kinase (cGK) pathway regulates the clustering and recruitment of synaptic proteins and vesicles to the synapse, adjusting the exoendocytic cycle to the intensity of activity and accelerating endocytosis following large-scale exocytosis. Here, we show that blockage of the N-methyl-D-aspartate receptor impairs the cycling of synaptic vesicles in a subset of boutons on cerebellar granule cells, an effect that was reversed by increasing cGMP. Furthermore, we demonstrate that presynaptic cGK type II (cGKII) plays a major role in this process. Using the FM1-43 dye to track vesicle recycling, we found that knockdown of cGKII and/or the application of a cGK inhibitor reduced the efficiency of synaptic vesicle recycling to a similar extent. Likewise, in cerebellar granule cells transfected with vGlut1-pHluorin to follow the exoendocytotic cycle, application of a cGK inhibitor slowed vesicle endocytosis when exocytosis was accelerated through strong and sustained stimulation. Additionally, ultrastructural analysis showed that cGKII knockdown or inhibition favored the formation of endosomal-like structures after strong and sustained stimulation. We conclude that cGKII controls the homeostatic balance of vesicle exocytosis and endocytosis in synaptic boutons of rat cerebellar granule cells.

  19. 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

  20. High-Throughput All-Optical Analysis of Synaptic Transmission and Synaptic Vesicle Recycling in Caenorhabditis elegans

    PubMed Central

    Wabnig, Sebastian; Liewald, Jana Fiona; Yu, Szi-chieh; Gottschalk, Alexander

    2015-01-01

    Synaptic vesicles (SVs) undergo a cycle of biogenesis and membrane fusion to release transmitter, followed by recycling. How exocytosis and endocytosis are coupled is intensively investigated. We describe an all-optical method for identification of neurotransmission genes that can directly distinguish SV recycling factors in C. elegans, by motoneuron photostimulation and muscular RCaMP Ca2+ imaging. We verified our approach on mutants affecting synaptic transmission. Mutation of genes affecting SV recycling (unc-26 synaptojanin, unc-41 stonin, unc-57 endophilin, itsn-1 intersectin, snt-1 synaptotagmin) showed a distinct ‘signature’ of muscle Ca2+ dynamics, induced by cholinergic motoneuron photostimulation, i.e. faster rise, and earlier decrease of the signal, reflecting increased synaptic fatigue during ongoing photostimulation. To facilitate high throughput, we measured (3–5 times) ~1000 nematodes for each gene. We explored if this method enables RNAi screening for SV recycling genes. Previous screens for synaptic function genes, based on behavioral or pharmacological assays, allowed no distinction of the stage of the SV cycle in which a protein might act. We generated a strain enabling RNAi specifically only in cholinergic neurons, thus resulting in healthier animals and avoiding lethal phenotypes resulting from knockdown elsewhere. RNAi of control genes resulted in Ca2+ measurements that were consistent with results obtained in the respective genomic mutants, albeit to a weaker extent in most cases, and could further be confirmed by opto-electrophysiological measurements for mutants of some of the genes, including synaptojanin. We screened 95 genes that were previously implicated in cholinergic transmission, and several controls. We identified genes that clustered together with known SV recycling genes, exhibiting a similar signature of their Ca2+ dynamics. Five of these genes (C27B7.7, erp-1, inx-8, inx-10, spp-10) were further assessed in respective

  1. Synaptic vesicles control the time course of neurotransmitter secretion via a Ca2+/H+ antiport

    PubMed Central

    Cordeiro, J Miguel; Gonçalves, Paula P; Dunant, Yves

    2011-01-01

    We investigated the physiological role of the vesicular Ca2+/H+ antiport in rapid synaptic transmission using the Torpedo electric organ (a modified neuromuscular system). By inhibiting V-type H+-transporting ATPase (V-ATPase), bafilomycin A1 dissipates the H+ gradient of synaptic vesicles, thereby abolishing the Ca2+/H+ antiport driving force. In electrophysiology experiments, bafilomycin A1 significantly prolonged the duration of the evoked electroplaque potential. A biochemical assay for acetylcholine (ACh) release showed that the effect of bafilomycin A1 was presynaptic. Indeed, bafilomycin A1 increased the amount of radio-labelled ACh released in response to paired-pulse stimulation. Bafilomycin A1 also enhanced Ca2+-dependent ACh release from isolated nerve terminals (synaptosomes). The bafilomycin-induced electroplaque potential lengthening did not arise from cholinesterase inhibition, since eserine (which also prolonged the electroplaque potential) strongly decreased evoked ACh release. Bafilomycin A1 augmented the amount of calcium accumulating in nerve terminals following a short tetanic stimulation and delayed subsequent calcium extrusion. By reducing stimulation-dependent calcium accumulation in synaptic vesicles, bafilomycin A1 diminished the corresponding depletion of vesicular ACh, as tested using both intact tissue and isolated synaptic vesicles. Strontium ions inhibit the vesicular Ca2+/H+ antiport, while activating transmitter release at concentrations one order of magnitude higher than Ca2+ does. In the presence of Sr2+ the time course of the electroplaque potential was also prolonged but, unlike bafilomycin A1, Sr2+ enhanced facilitation in paired-pulse experiments. It is therefore proposed that the vesicular Ca2+/H+ antiport function is to shorten ‘phasic’ transmitter release, allowing the synapse to transmit briefer impulses and so to work at higher frequencies. PMID:21059764

  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. Tension-induced pore formation and leakage in adhering vesicles

    NASA Astrophysics Data System (ADS)

    Lenz, P.; Johnson, J. M.; Chan, Y.-H. M.; Boxer, S. G.

    2006-08-01

    The influence of inclusion-induced tension on pore formation is studied theoretically and experimentally. It is shown that fluorescently labeled lipids can enhance pore formation and induce leakage of adhering vesicles. These effects are more pronounced for smaller vesicles. The theoretical predictions are confirmed by experimental two-color fluorescent data. Finally, the influence of the pore formation dynamics on rupture processes of vesicles is analyzed yielding a new picture of the transition to bilayer disks.

  4. 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

  5. Fife organizes synaptic vesicles and calcium channels for high-probability neurotransmitter release

    PubMed Central

    Rao, Monica; Ukken, Fiona

    2017-01-01

    The strength of synaptic connections varies significantly and is a key determinant of communication within neural circuits. Mechanistic insight into presynaptic factors that establish and modulate neurotransmitter release properties is crucial to understanding synapse strength, circuit function, and neural plasticity. We previously identified Drosophila Piccolo-RIM-related Fife, which regulates neurotransmission and motor behavior through an unknown mechanism. Here, we demonstrate that Fife localizes and interacts with RIM at the active zone cytomatrix to promote neurotransmitter release. Loss of Fife results in the severe disruption of active zone cytomatrix architecture and molecular organization. Through electron tomographic and electrophysiological studies, we find a decrease in the accumulation of release-ready synaptic vesicles and their release probability caused by impaired coupling to Ca2+ channels. Finally, we find that Fife is essential for the homeostatic modulation of neurotransmission. We propose that Fife organizes active zones to create synaptic vesicle release sites within nanometer distance of Ca2+ channel clusters for reliable and modifiable neurotransmitter release. PMID:27998991

  6. Tissue-type plasminogen activator triggers the synaptic vesicle cycle in cerebral cortical neurons

    PubMed Central

    Wu, Fang; Torre, Enrique; Cuellar-Giraldo, David; Cheng, Lihong; Yi, Hong; Bichler, Edyta K; García, Paul S; Yepes, Manuel

    2015-01-01

    The active zone (AZ) is a thickening of the presynaptic membrane where exocytosis takes place. Chemical synapses contain neurotransmitter-loaded synaptic vesicles (SVs) that at rest are tethered away from the synaptic release site, but after the presynaptic inflow of Ca+2 elicited by an action potential translocate to the AZ to release their neurotransmitter load. We report that tissue-type plasminogen activator (tPA) is stored outside the AZ of cerebral cortical neurons, either intermixed with small clear-core vesicles or in direct contact with the presynaptic membrane. We found that cerebral ischemia-induced release of neuronal tPA, or treatment with recombinant tPA, recruits the cytoskeletal protein βII-spectrin to the AZ and promotes the binding of SVs to βII-spectrin, enlarging the population of SVs in proximity to the synaptic release site. This effect does not require the generation of plasmin and is followed by the recruitment of voltage gated calcium channels (VGCC) to the presynaptic terminal that leads to Ca+2-dependent synapsin I phosphorylation, freeing SVs to translocate to the AZ to deliver their neurotransmitter load. Our studies indicate that tPA activates the SV cycle and induces the structural and functional changes in the synapse that are required for successful neurotransmission. PMID:26126868

  7. Synaptic vesicle protein synaptoporin is differently expressed by subpopulations of mouse hippocampal neurons.

    PubMed

    Singec, Ilyas; Knoth, Rolf; Ditter, Margarethe; Hagemeyer, Christoph E; Rosenbrock, Holger; Frotscher, Michael; Volk, Benedikt

    2002-10-14

    In the hippocampus, the synaptic vesicle protein synaptoporin (SPO) has been reported to be exclusively enriched in the granule cell axons, the mossy fibers. In this study, we show that in adult rats and mice SPO immunoreactivity (IR) is also detectable in strata oriens, radiatum, and lacunosum-moleculare of CA1-CA3, as well as perisomatically in the hippocampus proper and fascia dentata. In situ hybridization confirmed that SPO mRNA was present in granule cells and CA3 pyramidal cells but not in CA1 pyramidal cells. Importantly, cells scattered throughout the hippocampal layers resembling the distribution of interneurons were found to synthesize high amounts of SPO mRNA, too. Thus, these findings indicate that SPO expression in the hippocampus was underestimated until now. Moreover, double-labeling immunohistochemistry and confocal microscopy revealed selective colocalization of SPO and glutamate decarboxylase (GAD 65), a marker for gamma-aminobutyric acid (GABA)ergic terminals. To identify SPO expressing interneurons, in situ hybridization was combined with immunocytochemistry against parvalbumin (PV), calbindin (CB), calretinin (CR), cholecystokinin (CCK), and vasoactive intestinal polypeptide (VIP). We found that SPO transcripts were differentially expressed by various interneuron subpopulations in the hippocampus of C57Bl/6 mice (PV 44.2%, CB 46.3%, CR 19.3%, CCK 38.6%, VIP 59.9%). Immunoelectron microscopy for SPO labeled synaptic vesicle profiles in distinct symmetric and asymmetric synapses. In conclusion, our data demonstrate that hippocampal principal cells and interneurons display a variety of synaptic vesicles that are likely to contribute to the functional characteristics of their output synapses.

  8. Clathrin and synaptic vesicle endocytosis: studies at the squid giant synapse

    PubMed Central

    Augustine, G.J.; Morgan, J.R.; Villalba-Galea, C.A.; Jin, S.; Prasad, K.; Lafer, E.M.

    2015-01-01

    The role of clathrin-mediated endocytosis in SV (synaptic vesicle) recycling has been studied by combining molecular biology, physiology and electron microscopy at the squid giant synapse. Procedures that prevent clathrin from assembling into membrane coats, such as impairment of binding of the AP180 and AP-2 adaptor proteins, completely prevent membrane budding during endocytosis. These procedures also reduce exocytosis, presumably an indirect effect of a reduction in the number of SVs following block of endocytosis. Disrupting the binding of auxilin to Hsc70 (heat-shock cognate 70) prevents clathrin-coated vesicles from uncoating and also disrupts SV recycling. Taken together, these results indicate that a clathrin-dependent pathway is the primary means of SV recycling at this synapse under physiological conditions. PMID:16417485

  9. Clathrin and synaptic vesicle endocytosis: studies at the squid giant synapse.

    PubMed

    Augustine, G J; Morgan, J R; Villalba-Galea, C A; Jin, S; Prasad, K; Lafer, E M

    2006-02-01

    The role of clathrin-mediated endocytosis in SV (synaptic vesicle) recycling has been studied by combining molecular biology, physiology and electron microscopy at the squid giant synapse. Procedures that prevent clathrin from assembling into membrane coats, such as impairment of binding of the AP180 and AP-2 adaptor proteins, completely prevent membrane budding during endocytosis. These procedures also reduce exocytosis, presumably an indirect effect of a reduction in the number of SVs following block of endocytosis. Disrupting the binding of auxilin to Hsc70 (heat-shock cognate 70) prevents clathrin-coated vesicles from uncoating and also disrupts SV recycling. Taken together, these results indicate that a clathrin-dependent pathway is the primary means of SV recycling at this synapse under physiological conditions.

  10. Evidence that the ZNT3 protein controls the total amount of elemental zinc in synaptic vesicles

    USGS Publications Warehouse

    Linkous, D.H.; Flinn, J.M.; Koh, J.Y.; Lanzirotti, A.; Bertsch, P.M.; Jones, B.F.; Giblin, L.J.; Frederickson, C.J.

    2008-01-01

    The ZNT3 protein decorates the presynaptic vesicles of central neurons harboring vesicular zinc, and deletion of this protein removes staining for zinc. However, it has been unclear whether only histochemically reactive zinc is lacking or if, indeed, total elemental zinc is missing from neurons lacking the Slc30a3 gene, which encodes the ZNT3 protein. The limitations of conventional histochemical procedures have contributed to this enigma. However, a novel technique, microprobe synchrotron X-ray fluorescence, reveals that the normal 2- to 3-fold elevation of zinc concentration normally present in the hippocampal mossy fibers is absent in Slc30a3 knockout (ZNT3) mice. Thus, the ZNT3 protein evidently controls not only the "stainability" but also the actual mass of zinc in mossy-fiber synaptic vesicles. This work thus confirms the metal-transporting role of the ZNT3 protein in the brain. ?? The Histochemical Society, Inc.

  11. Evidence That the ZNT3 Protein Controls the Total Amount of Elemental Zinc in Synaptic Vesicles

    SciTech Connect

    Linkous,D.; Flinn, J.; Koh, J.; Lanzirotti, A.; Bertsch, P.; Jones, B.; Giblin, L.; Fredrickson, C.

    2008-01-01

    The ZNT3 protein decorates the presynaptic vesicles of central neurons harboring vesicular zinc, and deletion of this protein removes staining for zinc. However, it has been unclear whether only histochemically reactive zinc is lacking or if, indeed, total elemental zinc is missing from neurons lacking the Slc30a3 gene, which encodes the ZNT3 protein. The limitations of conventional histochemical procedures have contributed to this enigma. However, a novel technique, microprobe synchrotron X-ray fluorescence, reveals that the normal 2- to 3-fold elevation of zinc concentration normally present in the hippocampal mossy fibers is absent in Slc30a3 knockout (ZNT3) mice. Thus, the ZNT3 protein evidently controls not only the 'stainability' but also the actual mass of zinc in mossy-fiber synaptic vesicles. This work thus confirms the metal-transporting role of the ZNT3 protein in the brain.

  12. Activity-Dependent Degradation of Synaptic Vesicle Proteins Requires Rab35 and the ESCRT Pathway

    PubMed Central

    Sheehan, Patricia; Zhu, Mei; Beskow, Anne; Vollmer, Cyndel

    2016-01-01

    Synaptic vesicle (SV) pools must maintain a functional repertoire of proteins to efficiently release neurotransmitter. The accumulation of old or damaged proteins on SV membranes is linked to synaptic dysfunction and neurodegeneration. However, despite the importance of SV protein turnover for neuronal health, the molecular mechanisms underlying this process are largely unknown. Here, we have used dissociated rat hippocampal neurons to investigate the pathway for SV protein degradation. We find that neuronal activity drives the degradation of a subset of SV proteins and that the endosomal sorting complex required for transport (ESCRT) machinery and SV-associated GTPase Rab35 are key elements of this use-dependent degradative pathway. Specifically, neuronal activity induces Rab35 activation and binding to the ESCRT-0 protein Hrs, which we have identified as a novel Rab35 effector. These actions recruit the downstream ESCRT machinery to SV pools, thereby initiating SV protein degradation via the ESCRT pathway. Our findings show that the Rab35/ESCRT pathway facilitates the activity-dependent removal of specific proteins from SV pools, thereby maintaining presynaptic protein homeostasis. SIGNIFICANCE STATEMENT Synaptic transmission is mediated by the release of chemical neurotransmitters from synaptic vesicles (SVs). This tightly regulated process requires a functional pool of SVs, necessitating cellular mechanisms for removing old or damaged proteins that could impair SV cycling. Here, we show that a subset of SV proteins is degraded in an activity-dependent manner and that key steps in this degradative pathway are the activation of the small GTPase Rab35 and the subsequent recruitment of the endosomal sorting complex required for transport (ESCRT) machinery to SV pools. Further, we demonstrate that ESCRT-0 component Hrs is an effector of Rab35, thus providing novel mechanistic insight into the coupling of neuronal activity with SV protein degradation and the

  13. VAT-1 from Torpedo electric organ forms a high-molecular-mass protein complex within the synaptic vesicle membrane.

    PubMed

    Linial, M

    1993-08-15

    VAT-1 is an abundant 41-kDa protein from Torpedo cholinergic synaptic vesicles. Most of VAT-1 immunoreactivity (70%) is localized to the synaptic vesicle membrane while the rest (30%) copurifies with larger membranous fragments. VAT-1 forms a high-molecular-mass complex within the synaptic vesicle membrane. The Stokes radius of the VAT-1 complex is 4.85 nm and the sedimentation coefficient is 8.0 x 10(-13) S. Using these values, the calculated apparent mass of the VAT-1 complex is 176 kDa and the friction coefficient is consistent with that for a globular protein. Electrophoresis of solubilized synaptic vesicle proteins following cross-linking resulted in a 40-kDa ladder which was detected by VAT-1 antibodies. This is in accord with VAT-1 protein complex being composed primarily of VAT-1 subunits. The hydrodynamic characteristics of the VAT-1 protein complex suggest that it is composed of three or four VAT-1 subunits. Synaptophysin, an abundant component of Torpedo synaptic vesicle membranes, which has a similar apparent size as VAT-1, is not part of the VAT-1 protein complex. Interactions between the subunits within the protein complex do not depend on disulfide bonds or on lowering the ionic strength. However, partial dissociation of VAT-1 subunits from the complex occurs by chelating calcium ions.

  14. 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.

  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. THE EFFECT OF NITRIC OXIDE ON SYNAPTIC VESICLE PROTON GRADIENT AND MITOCHONDRIAL POTENTIAL OF BRAIN NERVE TERMINALS.

    PubMed

    Tarasenko, A S

    2015-01-01

    The effect of nitric oxide on synaptic vesicle proton gradient and membrane potential of rat brain nerve terminals was studied. It has been shown that nitric oxide in the form of S-nitrosothiols at nanomolar concentrations had no effect on the studied parameters, but caused a rapid dissipation of synaptic vesicle proton gradient and depolarization of mitochondrial membrane in the presence of a SH-reducing compound such as dithiothreitol. Both processes were reversible and the rate of H(+)-gradient restoration depended on the redox potential of nerve terminals, namely the molar ratio of reductant/oxidant. This facts, as well as insensitivity of the studied processes to the inhibitor of NO-sensitive guanylate cyclase such as ODQ, allow suggesting that post-translational modification of thiol residues of the mitochondrial and synaptic vesicle proteins underlies the effect of nitric oxide on the key functional parameters ofpresynaptic nerve terminals.

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

    PubMed Central

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

    2010-01-01

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

  18. Simultaneous lipid and content mixing assays for in vitro reconstitution studies of synaptic vesicle fusion.

    PubMed

    Liu, Xiaoxia; Seven, Alpay Burak; Xu, Junjie; Esser, Victoria; Su, Lijing; Ma, Cong; Rizo, Josep

    2017-09-01

    This protocol describes reconstitution assays to study how the neurotransmitter release machinery triggers Ca(2+)-dependent synaptic vesicle fusion. The assays monitor fusion between proteoliposomes containing the synaptic vesicle SNARE synaptobrevin (with or without the Ca(2+) sensor synaptotagmin-1) and proteoliposomes initially containing the plasma membrane SNAREs syntaxin-1 and soluble NSF attachment protein (SNAP)-25. Lipid mixing (from fluorescence de-quenching of Marina-Blue-labeled lipids) and content mixing (from development of fluorescence resonance energy transfer (FRET) between phycoerythrin-biotin (PhycoE-Biotin) and Cy5-streptavidin trapped in the two proteoliposome populations) are measured simultaneously to ensure that true, nonleaky membrane fusion is monitored. This protocol is based on a method developed to study yeast vacuolar fusion. In contrast to other protocols used to study the release machinery, this assay incorporates N-ethylmaleimide sensitive factor (NSF) and α-SNAP, which disassemble syntaxin-1 and SNAP-25 heterodimers. As a result, fusion requires Munc18-1, which binds to the released syntaxin-1, and Munc13-1, which, together with Munc18-1, orchestrates SNARE complex assembly. The protocol can be readily adapted to investigation of other types of intracellular membrane fusion by using appropriate alternative proteins. Total time required for one round of the assay is 4 d.

  19. KIF1Bbeta2, capable of interacting with CHP, is localized to synaptic vesicles.

    PubMed

    Nakamura, Norihiro; Miyake, Yoshihide; Matsushita, Masafumi; Tanaka, Shingo; Inoue, Hiroki; Kanazawa, Hiroshi

    2002-09-01

    Kinesin family proteins are microtubule-dependent molecular motors involved in the intracellular motile process. Using a Ca2+ -binding protein, CHP (calcineurin B homologous protein), as a bait for yeast two hybrid screening, we identified a novel kinesin-related protein, KIF1Bbeta2. KIF1Bbeta2 is a member of the KIF1 subfamily of kinesin-related proteins, and consists of an amino terminal KIF1B-type motor domain followed by a tail region highly similar to that of KIF1A. CHP binds to regions adjacent to the motor domains of KIF1Bbeta2 and KIF1B, but not to those of the other KIF1 family members, KIF1A and KIF1C. Immunostaining of neuronal cells showed that a significant portion of KIF1Bbeta2 is co-localized with synaptophysin, a marker protein for synaptic vesicles, but not with a mitochondria-staining dye. Subcellular fractionation analysis indicated the co-localization of KIF1Bbeta2 with synaptophysin. These results suggest that KIF1Bbeta2, a novel CHP-interacting molecular motor, mediates the transport of synaptic vesicles in neuronal cells.

  20. ATM protein is located on presynaptic vesicles and its deficit leads to failures in synaptic plasticity.

    PubMed

    Vail, Graham; Cheng, Aifang; Han, Yu Ray; Zhao, Teng; Du, Shengwang; Loy, Michael M T; Herrup, Karl; Plummer, Mark R

    2016-07-01

    Ataxia telangiectasia is a multisystemic disorder that includes a devastating neurodegeneration phenotype. The ATM (ataxia-telangiectasia mutated) protein is well-known for its role in the DNA damage response, yet ATM is also found in association with cytoplasmic vesicular structures: endosomes and lysosomes, as well as neuronal synaptic vesicles. In keeping with this latter association, electrical stimulation of the Schaffer collateral pathway in hippocampal slices from ATM-deficient mice does not elicit normal long-term potentiation (LTP). The current study was undertaken to assess the nature of this deficit. Theta burst-induced LTP was reduced in Atm(-/-) animals, with the reduction most pronounced at burst stimuli that included 6 or greater trains. To assess whether the deficit was associated with a pre- or postsynaptic failure, we analyzed paired-pulse facilitation and found that it too was significantly reduced in Atm(-/-) mice. This indicates a deficit in presynaptic function. As further evidence that these synaptic effects of ATM deficiency were presynaptic, we used stochastic optical reconstruction microscopy. Three-dimensional reconstruction revealed that ATM is significantly more closely associated with Piccolo (a presynaptic marker) than with Homer1 (a postsynaptic marker). These results underline how, in addition to its nuclear functions, ATM plays an important functional role in the neuronal synapse where it participates in the regulation of presynaptic vesicle physiology. Copyright © 2016 the American Physiological Society.

  1. The iTRAPs: Guardians of Synaptic Vesicle Cargo Retrieval During Endocytosis

    PubMed Central

    Gordon, Sarah L.; Cousin, Michael A.

    2016-01-01

    The reformation of synaptic vesicles (SVs) during endocytosis is essential for the maintenance of neurotransmission in central nerve terminals. Newly formed SVs must be generated with the correct protein cargo in the correct stoichiometry to be functional for exocytosis. Classical clathrin adaptor protein complexes play a key role in sorting and clustering synaptic vesicle cargo in this regard. However it is becoming increasingly apparent that additional “fail-safe” mechanisms exist to ensure the accurate retrieval of essential cargo molecules. For example, the monomeric adaptor proteins AP180/CALM and stonin-2 are required for the efficient retrieval of synaptobrevin II (sybII) and synaptotagmin-1 respectively. Furthermore, recent studies have revealed that sybII and synaptotagmin-1 interact with other SV cargoes to ensure a high fidelity of retrieval. These cargoes are synaptophysin (for sybII) and SV2A (for synaptotagmin-1). In this review, we summarize current knowledge regarding the retrieval mechanisms for both sybII and synaptotagmin-1 during endocytosis. We also define and set criteria for a new functional group of SV molecules that facilitate the retrieval of their interaction partners. We have termed these molecules intrinsic trafficking partners (iTRAPs) and we discuss how the function of this group impacts on presynaptic performance in both health and disease. PMID:26903854

  2. Guanine derivatives modulate L-glutamate uptake into rat brain synaptic vesicles.

    PubMed

    Tasca, Carla I; Santos, Tiago G; Tavares, Rejane G; Battastini, Ana M O; Rocha, João B T; Souza, Diogo O

    2004-05-01

    Glutamate uptake into synaptic vesicles is driven by a proton electrochemical gradient generated by a vacuolar H(+)-ATPase and stimulated by physiological concentrations of chloride. This uptake plays an important role in glutamatergic transmission. We show here that vesicular glutamate uptake is selectively inhibited by guanine derivatives, in a time- and concentration-dependent manner. Guanosine, GMP, GDP, guanosine-5'-O-2-thiodiphosphate, GTP, or 5'-guanylylimidodiphosphate (GppNHp) inhibited glutamate uptake in 1.5 and 3 min incubations, however, when incubating for 10 min, only GTP or GppNHp displayed such inhibition. By increasing ATP concentrations, the inhibitory effect of GTP was no longer observed, but GppNHp still inhibited glutamate uptake. In the absence of ATP, vesicular ATPase can hydrolyze GTP in order to drive glutamate uptake. However, 5mM GppNHp inhibited ATP hydrolysis by synaptic vesicle preparations. GTP or GppNHp decreased the proton electrochemical gradient, whereas the other guanine derivatives did not. Glutamate saturation curves were assayed in order to evaluate the specificity of inhibition of the vesicular glutamate carrier by the guanine derivatives. The maximum velocity of the initial rate of glutamate uptake was decreased by all guanine derivatives. These results indicate that, although GppNHp can inhibit ATPase activity, guanine derivatives are more likely to be acting through interaction with vesicular glutamate carrier.

  3. Synaptic-like vesicles and candidate transduction channels in mechanosensory terminals.

    PubMed

    Bewick, Guy S

    2015-08-01

    This article summarises progress to date over an exciting and very enjoyable first 15 years of collaboration with Bob Banks. Our collaboration began when I contacted him with (to me) an unexpected observation that a dye used to mark recycling synaptic vesicle membrane at efferent terminals also labelled muscle spindle afferent terminals. This observation led to the re-discovery of a system of small clear vesicles present in all vertebrate primary mechanosensory nerve terminals. These synaptic-like vesicles (SLVs) have been, and continue to be, the major focus of our work. This article describes our characterisation of the properties and functional significance of these SLVs, combining our complementary skills: Bob's technical expertise and encyclopaedic knowledge of mechanosensation with my experience of synaptic vesicles and the development of the styryl pyridinium dyes, of which the most widely used is FM1-43. On the way we have found that SLVs seem to be part of a constitutive glutamate secretory system necessary to maintain the stretch-sensitivity of spindle endings. The glutamate activates a highly unusual glutamate receptor linked to phospholipase D activation, which we have termed the PLD-mGluR. It has a totally distinct pharmacology first described in the hippocampus nearly 20 years ago but, like the SLVs that were first described over 50 years ago, has since been little researched. Yet, our evidence and literature searches suggest this glutamate/SLV/PLD-mGluR system is a ubiquitous feature of mechanosensory endings and, at least for spindles, is essential for maintaining mechanosensory function. This article summarises how this system integrates with the classical model of mechanosensitive channels in spindles and other mechanosensory nerve terminals, including hair follicle afferents and baroreceptors controlling blood pressure. Finally, in this time when there is an imperative to show translational relevance, I describe how this fascinating system might

  4. Synaptic-like vesicles and candidate transduction channels in mechanosensory terminals

    PubMed Central

    Bewick, Guy S

    2015-01-01

    This article summarises progress to date over an exciting and very enjoyable first 15 years of collaboration with Bob Banks. Our collaboration began when I contacted him with (to me) an unexpected observation that a dye used to mark recycling synaptic vesicle membrane at efferent terminals also labelled muscle spindle afferent terminals. This observation led to the re-discovery of a system of small clear vesicles present in all vertebrate primary mechanosensory nerve terminals. These synaptic-like vesicles (SLVs) have been, and continue to be, the major focus of our work. This article describes our characterisation of the properties and functional significance of these SLVs, combining our complementary skills: Bob’s technical expertise and encyclopaedic knowledge of mechanosensation with my experience of synaptic vesicles and the development of the styryl pyridinium dyes, of which the most widely used is FM1-43. On the way we have found that SLVs seem to be part of a constitutive glutamate secretory system necessary to maintain the stretch-sensitivity of spindle endings. The glutamate activates a highly unusual glutamate receptor linked to phospholipase D activation, which we have termed the PLD-mGluR. It has a totally distinct pharmacology first described in the hippocampus nearly 20 years ago but, like the SLVs that were first described over 50 years ago, has since been little researched. Yet, our evidence and literature searches suggest this glutamate/SLV/PLD-mGluR system is a ubiquitous feature of mechanosensory endings and, at least for spindles, is essential for maintaining mechanosensory function. This article summarises how this system integrates with the classical model of mechanosensitive channels in spindles and other mechanosensory nerve terminals, including hair follicle afferents and baroreceptors controlling blood pressure. Finally, in this time when there is an imperative to show translational relevance, I describe how this fascinating system

  5. Lead-dependent deposits in diverse synaptic vesicles: suggestive evidence for the presence of anionic binding sites

    SciTech Connect

    Sulzer, D.; Piscopo, I.; Ungar, F.; Holtzman, E.

    1987-09-01

    We have observed electron dense deposits dependent on incubation of aldehyde-fixed tissues with lead ions within synaptic vesicles of several types of neurons that differ in the neurotransmitters utilized and in the secretory granules of the adrenal medulla. Evidently, vesicle components that can interact with lead ions are widespread. A plausible explanation for the occurrence of the deposits is the presence of anionic binding sites within the vesicles. This would agree well with other biochemical, cytochemical, and immunocytochemical evidence, such as that indicating the presence of sulfated macromolecules in certain synaptic vesicles. Anionic binding sites could play significant roles by participating in processes such as Ca/sup 2 +/ storage, stabilization of pH gradients, or the control of osmotic phenomena.

  6. Regulation of synaptic vesicles pools within motor nerve terminals during short-term facilitation and neuromodulation.

    PubMed

    Logsdon, S; Johnstone, A F M; Viele, K; Cooper, R L

    2006-02-01

    The reserve pool (RP) and readily releasable pool (RRP) of synaptic vesicles within presynaptic nerve terminals were physiologically differentiated into distinctly separate functional groups. This was accomplished in glutamatergic nerve terminals by blocking the glutamate transporter with dl-threo-beta-benzyloxyaspartate (TBOA; 10 microM) during electrical stimulation with either 40 Hz of 10 pulses within a train or 20- or 50-Hz continuous stimulation. The 50-Hz continuous stimulation decreased the excitatory postsynaptic potential amplitude 60 min faster than for the 20-Hz continuous stimulation in the presence of TBOA (P < 0.05). There was no significant difference between the train stimulation and 20-Hz continuous stimulation in the run-down time in the presence of TBOA. After TBOA-induced synaptic depression, the excitatory postsynaptic potentials were rapidly (<1 min) revitalized by exposure to serotonin (5-HT, 1 microM) in every preparation tested (P < 0.05). At this glutamatergic nerve terminal, 5-HT promotes an increase probability of vesicular docking and fusion. Quantal recordings made directly at nerve terminals revealed smaller quantal sizes with TBOA exposure with a marked increase in quantal size as well as a continual appearance of smaller quanta upon 5-HT treatment after TBOA-induced depression. Thus 5-HT was able to recruit vesicles from the RP that were not rapidly depleted by acute TBOA treatment and electrical stimulation. The results support the notion that the RRP is selectively activated during rapid electrical stimulation sparing the RP; however, the RP can be recruited by the neuromodulator 5-HT. This suggests at least two separate kinetic and distinct regulatory paths for vesicle recycling within the presynaptic nerve terminal.

  7. 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.

  8. Synaptic Vesicle Glycoprotein 2A Ligands in the Treatment of Epilepsy and Beyond.

    PubMed

    Löscher, Wolfgang; Gillard, Michel; Sands, Zara A; Kaminski, Rafal M; Klitgaard, Henrik

    2016-11-01

    The synaptic vesicle glycoprotein SV2A belongs to the major facilitator superfamily (MFS) of transporters and is an integral constituent of synaptic vesicle membranes. SV2A has been demonstrated to be involved in vesicle trafficking and exocytosis, processes crucial for neurotransmission. The anti-seizure drug levetiracetam was the first ligand to target SV2A and displays a broad spectrum of anti-seizure activity in various preclinical models. Several lines of preclinical and clinical evidence, including genetics and protein expression changes, support an important role of SV2A in epilepsy pathophysiology. While the functional consequences of SV2A ligand binding are not fully elucidated, studies suggest that subsequent SV2A conformational changes may contribute to seizure protection. Conversely, the recently discovered negative SV2A modulators, such as UCB0255, counteract the anti-seizure effect of levetiracetam and display procognitive properties in preclinical models. More broadly, dysfunction of SV2A may also be involved in Alzheimer's disease and other types of cognitive impairment, suggesting potential novel therapies for levetiracetam and its congeners. Furthermore, emerging data indicate that there may be important roles for two other SV2 isoforms (SV2B and SV2C) in the pathogenesis of epilepsy, as well as other neurodegenerative diseases. Utilization of recently developed SV2A positron emission tomography ligands will strengthen and reinforce the pharmacological evidence that SV2A is a druggable target, and will provide a better understanding of its role in epilepsy and other neurological diseases, aiding in further defining the full therapeutic potential of SV2A modulation.

  9. 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.

  10. Synaptic vesicle glycoprotein 2A (SV2A) regulates kindling epileptogenesis via GABAergic neurotransmission

    PubMed Central

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

    2016-01-01

    Synaptic vesicle glycoprotein 2A (SV2A) is a prototype synaptic vesicle protein regulating action potential-dependent neurotransmitters release. SV2A also serves as a specific binding site for certain antiepileptics and is implicated in the treatment of epilepsy. Here, to elucidate the role of SV2A in modulating epileptogenesis, we generated a novel rat model (Sv2aL174Q rat) carrying a Sv2a-targeted missense mutation (L174Q) and analyzed its susceptibilities to kindling development. Although animals homozygous for the Sv2aL174Q mutation exhibited normal appearance and development, they are susceptible to pentylenetetrazole (PTZ) seizures. In addition, development of kindling associated with repeated PTZ treatments or focal stimulation of the amygdala was markedly facilitated by the Sv2aL174Q mutation. Neurochemical studies revealed that the Sv2aL174Q mutation specifically reduced depolarization-induced GABA, but not glutamate, release in the hippocampus without affecting basal release or the SV2A expression level in GABAergic neurons. In addition, the Sv2aL174Q mutation selectively reduced the synaptotagmin1 (Syt1) level among the exocytosis-related proteins examined. The present results demonstrate that dysfunction of SV2A due to the Sv2aL174Q mutation impairs the synaptic GABA release by reducing the Syt1 level and facilitates the kindling development, illustrating the crucial role of SV2A-GABA system in modulating kindling epileptogenesis. PMID:27265781

  11. 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.

  12. Synaptic vesicle clustering requires a distinct MIG-10/Lamellipodin isoform and ABI-1 downstream from Netrin.

    PubMed

    Stavoe, Andrea K H; Nelson, Jessica C; Martínez-Velázquez, Luis A; Klein, Mason; Samuel, Aravinthan D T; Colón-Ramos, Daniel A

    2012-10-01

    The chemotrophic factor Netrin can simultaneously instruct different neurodevelopmental programs in individual neurons in vivo. How neurons correctly interpret the Netrin signal and undergo the appropriate neurodevelopmental response is not understood. Here we identify MIG-10 isoforms as critical determinants of individual cellular responses to Netrin. We determined that distinct MIG-10 isoforms, varying only in their N-terminal motifs, can localize to specific subcellular domains and are differentially required for discrete neurodevelopmental processes in vivo. We identified MIG-10B as an isoform uniquely capable of localizing to presynaptic regions and instructing synaptic vesicle clustering in response to Netrin. MIG-10B interacts with Abl-interacting protein-1 (ABI-1)/Abi1, a component of the WAVE complex, to organize the actin cytoskeleton at presynaptic sites and instruct vesicle clustering through SNN-1/Synapsin. We identified a motif in the MIG-10B N-terminal domain that is required for its function and localization to presynaptic sites. With this motif, we engineered a dominant-negative MIG-10B construct that disrupts vesicle clustering and animal thermotaxis behavior when expressed in a single neuron in vivo. Our findings indicate that the unique N-terminal domains confer distinct MIG-10 isoforms with unique capabilities to localize to distinct subcellular compartments, organize the actin cytoskeleton at these sites, and instruct distinct Netrin-dependent neurodevelopmental programs.

  13. Synaptic vesicle clustering requires a distinct MIG-10/Lamellipodin isoform and ABI-1 downstream from Netrin

    PubMed Central

    Stavoe, Andrea K.H.; Nelson, Jessica C.; Martínez-Velázquez, Luis A.; Klein, Mason; Samuel, Aravinthan D.T.; Colón-Ramos, Daniel A.

    2012-01-01

    The chemotrophic factor Netrin can simultaneously instruct different neurodevelopmental programs in individual neurons in vivo. How neurons correctly interpret the Netrin signal and undergo the appropriate neurodevelopmental response is not understood. Here we identify MIG-10 isoforms as critical determinants of individual cellular responses to Netrin. We determined that distinct MIG-10 isoforms, varying only in their N-terminal motifs, can localize to specific subcellular domains and are differentially required for discrete neurodevelopmental processes in vivo. We identified MIG-10B as an isoform uniquely capable of localizing to presynaptic regions and instructing synaptic vesicle clustering in response to Netrin. MIG-10B interacts with Abl-interacting protein-1 (ABI-1)/Abi1, a component of the WAVE complex, to organize the actin cytoskeleton at presynaptic sites and instruct vesicle clustering through SNN-1/Synapsin. We identified a motif in the MIG-10B N-terminal domain that is required for its function and localization to presynaptic sites. With this motif, we engineered a dominant-negative MIG-10B construct that disrupts vesicle clustering and animal thermotaxis behavior when expressed in a single neuron in vivo. Our findings indicate that the unique N-terminal domains confer distinct MIG-10 isoforms with unique capabilities to localize to distinct subcellular compartments, organize the actin cytoskeleton at these sites, and instruct distinct Netrin-dependent neurodevelopmental programs. PMID:23028145

  14. Mitochondrial support of persistent presynaptic vesicle mobilization with age-dependent synaptic growth after LTP

    PubMed Central

    Smith, Heather L; Bourne, Jennifer N; Cao, Guan; Chirillo, Michael A; Ostroff, Linnaea E; Watson, Deborah J; Harris, Kristen M

    2016-01-01

    Mitochondria support synaptic transmission through production of ATP, sequestration of calcium, synthesis of glutamate, and other vital functions. Surprisingly, less than 50% of hippocampal CA1 presynaptic boutons contain mitochondria, raising the question of whether synapses without mitochondria can sustain changes in efficacy. To address this question, we analyzed synapses from postnatal day 15 (P15) and adult rat hippocampus that had undergone theta-burst stimulation to produce long-term potentiation (TBS-LTP) and compared them to control or no stimulation. At 30 and 120 min after TBS-LTP, vesicles were decreased only in presynaptic boutons that contained mitochondria at P15, and vesicle decrement was greatest in adult boutons containing mitochondria. Presynaptic mitochondrial cristae were widened, suggesting a sustained energy demand. Thus, mitochondrial proximity reflected enhanced vesicle mobilization well after potentiation reached asymptote, in parallel with the apparently silent addition of new dendritic spines at P15 or the silent enlargement of synapses in adults. DOI: http://dx.doi.org/10.7554/eLife.15275.001 PMID:27991850

  15. 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

  16. 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.

  17. Fast retrieval and autonomous regulation of single spontaneously recycling synaptic vesicles

    PubMed Central

    Leitz, Jeremy; Kavalali, Ege T

    2014-01-01

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

  18. Synaptic Vesicle Recycling Is Unaffected in the Ts65Dn Mouse Model of Down Syndrome

    PubMed Central

    Marland, Jamie R. K.; Smillie, Karen J.; Cousin, Michael A.

    2016-01-01

    Down syndrome (DS) is the most common genetic cause of intellectual disability, and arises from trisomy of human chromosome 21. Accumulating evidence from studies of both DS patient tissue and mouse models has suggested that synaptic dysfunction is a key factor in the disorder. The presence of several genes within the DS trisomy that are either directly or indirectly linked to synaptic vesicle (SV) endocytosis suggested that presynaptic dysfunction could underlie some of these synaptic defects. Therefore we determined whether SV recycling was altered in neurons from the Ts65Dn mouse, the best characterised model of DS to date. We found that SV exocytosis, the size of the SV recycling pool, clathrin-mediated endocytosis, activity-dependent bulk endocytosis and SV generation from bulk endosomes were all unaffected by the presence of the Ts65Dn trisomy. These results were obtained using battery of complementary assays employing genetically-encoded fluorescent reporters of SV cargo trafficking, and fluorescent and morphological assays of fluid-phase uptake in primary neuronal culture. The absence of presynaptic dysfunction in central nerve terminals of the Ts65Dn mouse suggests that future research should focus on the established alterations in excitatory / inhibitory balance as a potential route for future pharmacotherapy. PMID:26808141

  19. Isoflurane inhibits synaptic vesicle exocytosis through reduced Ca2+ influx, not Ca2+-exocytosis coupling

    PubMed Central

    Baumgart, Joel P.; Zhou, Zhen-Yu; Hara, Masato; Cook, Daniel C.; Hoppa, Michael B.; Ryan, Timothy A.; Hemmings, Hugh C.

    2015-01-01

    Identifying presynaptic mechanisms of general anesthetics is critical to understanding their effects on synaptic transmission. We show that the volatile anesthetic isoflurane inhibits synaptic vesicle (SV) exocytosis at nerve terminals in dissociated rat hippocampal neurons through inhibition of presynaptic Ca2+ influx without significantly altering the Ca2+ sensitivity of SV exocytosis. A clinically relevant concentration of isoflurane (0.7 mM) inhibited changes in [Ca2+]i driven by single action potentials (APs) by 25 ± 3%, which in turn led to 62 ± 3% inhibition of single AP-triggered exocytosis at 4 mM extracellular Ca2+ ([Ca2+]e). Lowering external Ca2+ to match the isoflurane-induced reduction in Ca2+ entry led to an equivalent reduction in exocytosis. These data thus indicate that anesthetic inhibition of neurotransmitter release from small SVs occurs primarily through reduced axon terminal Ca2+ entry without significant direct effects on Ca2+-exocytosis coupling or on the SV fusion machinery. Isoflurane inhibition of exocytosis and Ca2+ influx was greater in glutamatergic compared with GABAergic nerve terminals, consistent with selective inhibition of excitatory synaptic transmission. Such alteration in the balance of excitatory to inhibitory transmission could mediate reduced neuronal interactions and network-selective effects observed in the anesthetized central nervous system. PMID:26351670

  20. Structural basis for recognition of synaptic vesicle protein 2C by botulinum neurotoxin A

    NASA Astrophysics Data System (ADS)

    Benoit, Roger M.; Frey, Daniel; Hilbert, Manuel; Kevenaar, Josta T.; Wieser, Mara M.; Stirnimann, Christian U.; McMillan, David; Ceska, Tom; Lebon, Florence; Jaussi, Rolf; Steinmetz, Michel O.; Schertler, Gebhard F. X.; Hoogenraad, Casper C.; Capitani, Guido; Kammerer, Richard A.

    2014-01-01

    Botulinum neurotoxin A (BoNT/A) belongs to the most dangerous class of bioweapons. Despite this, BoNT/A is used to treat a wide range of common medical conditions such as migraines and a variety of ocular motility and movement disorders. BoNT/A is probably best known for its use as an antiwrinkle agent in cosmetic applications (including Botox and Dysport). BoNT/A application causes long-lasting flaccid paralysis of muscles through inhibiting the release of the neurotransmitter acetylcholine by cleaving synaptosomal-associated protein 25 (SNAP-25) within presynaptic nerve terminals. Two types of BoNT/A receptor have been identified, both of which are required for BoNT/A toxicity and are therefore likely to cooperate with each other: gangliosides and members of the synaptic vesicle glycoprotein 2 (SV2) family, which are putative transporter proteins that are predicted to have 12 transmembrane domains, associate with the receptor-binding domain of the toxin. Recently, fibroblast growth factor receptor 3 (FGFR3) has also been reported to be a potential BoNT/A receptor. In SV2 proteins, the BoNT/A-binding site has been mapped to the luminal domain, but the molecular details of the interaction between BoNT/A and SV2 are unknown. Here we determined the high-resolution crystal structure of the BoNT/A receptor-binding domain (BoNT/A-RBD) in complex with the SV2C luminal domain (SV2C-LD). SV2C-LD consists of a right-handed, quadrilateral β-helix that associates with BoNT/A-RBD mainly through backbone-to-backbone interactions at open β-strand edges, in a manner that resembles the inter-strand interactions in amyloid structures. Competition experiments identified a peptide that inhibits the formation of the complex. Our findings provide a strong platform for the development of novel antitoxin agents and for the rational design of BoNT/A variants with improved therapeutic properties.

  1. Presynaptic malfunction: the neurotoxic effects of cadmium and lead on the proton gradient of synaptic vesicles and glutamate transport.

    PubMed

    Borisova, Tatiana; Krisanova, Natalia; Sivko, Roman; Kasatkina, Ludmila; Borysov, Arseniy; Griffin, Susan; Wireman, Mike

    2011-08-01

    Exposure to Cd(2+) and Pb(2+) has neurotoxic consequences for human health and may cause neurodegeneration. The study focused on the analysis of the presynaptic mechanisms underlying the neurotoxic effects of non-essential heavy metals Cd(2+) and Pb(2+). It was shown that the preincubation of rat brain nerve terminals with Cd(2+) (200 μM) or Pb(2+) (200 μM) resulted in the attenuation of synaptic vesicles acidification, which was assessed by the steady state level of the fluorescence of pH-sensitive dye acridine orange. A decrease in L-[(14)C]glutamate accumulation in digitonin-permeabilized synaptosomes after the addition of the metals, which reflected lowered L-[(14)C]glutamate accumulation by synaptic vesicles inside of synaptosomes, may be considered in the support of the above data. Using isolated rat brain synaptic vesicles, it was found that 50 μM Cd(2+) or Pb(2+) caused dissipation of their proton gradient, whereas the application of essential heavy metal Mn(2+) did not do it within the range of the concentration of 50-500 μM. Thus, synaptic malfunction associated with the influence of Cd(2+) and Pb(2+) may result from partial dissipation of the synaptic vesicle proton gradient that leads to: (1) a decrease in stimulated exocytosis, which is associated not only with the blockage of voltage-gated Ca(2+) channels, but also with incomplete filling of synaptic vesicles; (2) an attenuation of Na(+)-dependent glutamate uptake. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. Genetic Analysis of a Novel Tubulin Mutation That Redirects Synaptic Vesicle Targeting and Causes Neurite Degeneration in C. elegans

    PubMed Central

    Chen, Yen-Chih; McDonald, Kent L.; Gurling, Mark; Lee, Albert; Garriga, Gian; Pan, Chun-Liang

    2014-01-01

    Neuronal cargos are differentially targeted to either axons or dendrites, and this polarized cargo targeting critically depends on the interaction between microtubules and molecular motors. From a forward mutagenesis screen, we identified a gain-of-function mutation in the C. elegans α-tubulin gene mec-12 that triggered synaptic vesicle mistargeting, neurite swelling and neurodegeneration in the touch receptor neurons. This missense mutation replaced an absolutely conserved glycine in the H12 helix with glutamic acid, resulting in increased negative charges at the C-terminus of α-tubulin. Synaptic vesicle mistargeting in the mutant neurons was suppressed by reducing dynein function, suggesting that aberrantly high dynein activity mistargeted synaptic vesicles. We demonstrated that dynein showed preference towards binding mutant microtubules over wild-type in microtubule sedimentation assay. By contrast, neurite swelling and neurodegeneration were independent of dynein and could be ameliorated by genetic paralysis of the animal. This suggests that mutant microtubules render the neurons susceptible to recurrent mechanical stress induced by muscle activity, which is consistent with the observation that microtubule network was disorganized under electron microscopy. Our work provides insights into how microtubule-dynein interaction instructs synaptic vesicle targeting and the importance of microtubule in the maintenance of neuronal structures against constant mechanical stress. PMID:25392990

  3. Genetic analysis of a novel tubulin mutation that redirects synaptic vesicle targeting and causes neurite degeneration in C. elegans.

    PubMed

    Hsu, Jiun-Min; Chen, Chun-Hao; Chen, Yen-Chih; McDonald, Kent L; Gurling, Mark; Lee, Albert; Garriga, Gian; Pan, Chun-Liang

    2014-11-01

    Neuronal cargos are differentially targeted to either axons or dendrites, and this polarized cargo targeting critically depends on the interaction between microtubules and molecular motors. From a forward mutagenesis screen, we identified a gain-of-function mutation in the C. elegans α-tubulin gene mec-12 that triggered synaptic vesicle mistargeting, neurite swelling and neurodegeneration in the touch receptor neurons. This missense mutation replaced an absolutely conserved glycine in the H12 helix with glutamic acid, resulting in increased negative charges at the C-terminus of α-tubulin. Synaptic vesicle mistargeting in the mutant neurons was suppressed by reducing dynein function, suggesting that aberrantly high dynein activity mistargeted synaptic vesicles. We demonstrated that dynein showed preference towards binding mutant microtubules over wild-type in microtubule sedimentation assay. By contrast, neurite swelling and neurodegeneration were independent of dynein and could be ameliorated by genetic paralysis of the animal. This suggests that mutant microtubules render the neurons susceptible to recurrent mechanical stress induced by muscle activity, which is consistent with the observation that microtubule network was disorganized under electron microscopy. Our work provides insights into how microtubule-dynein interaction instructs synaptic vesicle targeting and the importance of microtubule in the maintenance of neuronal structures against constant mechanical stress.

  4. A novel extraction protocol to probe the role of cholesterol in synaptic vesicle recycling.

    PubMed

    Dason, Jeffrey S; Charlton, Milton P

    2014-01-01

    Cholesterol helps to stabilize membrane fluidity and many membrane proteins interact with cholesterol and are functionally clustered in cholesterol rich "rafts." Synaptic vesicle (SV) membranes are enriched in cholesterol in comparison to other organelles. Attempts to study the function of this high cholesterol content have been hampered by the inability to extract cholesterol from SVs in live presynaptic terminals. Here, we describe a method to extract vesicular cholesterol using a temperature-sensitive Drosophila dynamin mutant, shibire-ts1 (shi), to trap SVs on the plasma membrane. Trapped SVs are more accessible to cholesterol extraction by the cholesterol chelator, methyl-β-cyclodextrin (MβCD). This method can likely be extended to extract other lipids from SVs and could also be used to add lipids. We speculate that this method could be used on mammalian preparations in conjunction with dynamin inhibitors.

  5. Analysis of shape and spatial interaction of synaptic vesicles using data from focused ion beam scanning electron microscopy (FIB-SEM)

    PubMed Central

    Khanmohammadi, Mahdieh; Waagepetersen, Rasmus P.; Sporring, Jon

    2015-01-01

    The spatial interactions of synaptic vesicles in synapses were assessed after a detailed characterization of size, shape, and orientation of the synaptic vesicles. We hypothesized that shape and orientation of the synaptic vesicles are influenced by their movement toward the active zone causing deviations from spherical shape and systematic trends in their orientation. We studied three-dimensional representations of synapses obtained by manual annotation of focused ion beam scanning electron microscopy (FIB-SEM) images of male mouse brain. The configurations of synaptic vesicles were regarded as marked point patterns, where the points are the centers of the vesicles, and the mark of a vesicle is given by its size, shape, and orientation characteristics. Statistics for marked point processes were employed to study spatial interactions between vesicles. We found that the synaptic vesicles in excitatory synapses appeared to be of oblate ellipsoid shape and in inhibitory synapses appeared to be of cigar ellipsoid shape, and followed a systematic pattern regarding their orientation toward the active zone. Moreover, there was strong evidence of spatial alignment in the orientations of pairs of synaptic vesicles, and of repulsion between them only in excitatory synapses, beyond that caused by their physical extent. PMID:26388743

  6. Analysis of shape and spatial interaction of synaptic vesicles using data from focused ion beam scanning electron microscopy (FIB-SEM).

    PubMed

    Khanmohammadi, Mahdieh; Waagepetersen, Rasmus P; Sporring, Jon

    2015-01-01

    The spatial interactions of synaptic vesicles in synapses were assessed after a detailed characterization of size, shape, and orientation of the synaptic vesicles. We hypothesized that shape and orientation of the synaptic vesicles are influenced by their movement toward the active zone causing deviations from spherical shape and systematic trends in their orientation. We studied three-dimensional representations of synapses obtained by manual annotation of focused ion beam scanning electron microscopy (FIB-SEM) images of male mouse brain. The configurations of synaptic vesicles were regarded as marked point patterns, where the points are the centers of the vesicles, and the mark of a vesicle is given by its size, shape, and orientation characteristics. Statistics for marked point processes were employed to study spatial interactions between vesicles. We found that the synaptic vesicles in excitatory synapses appeared to be of oblate ellipsoid shape and in inhibitory synapses appeared to be of cigar ellipsoid shape, and followed a systematic pattern regarding their orientation toward the active zone. Moreover, there was strong evidence of spatial alignment in the orientations of pairs of synaptic vesicles, and of repulsion between them only in excitatory synapses, beyond that caused by their physical extent.

  7. 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

  8. 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.

  9. Subdiffractional tracking of internalized molecules reveals heterogeneous motion states of synaptic vesicles.

    PubMed

    Joensuu, Merja; Padmanabhan, Pranesh; Durisic, Nela; Bademosi, Adekunle T D; Cooper-Williams, Elizabeth; Morrow, Isabel C; Harper, Callista B; Jung, WooRam; Parton, Robert G; Goodhill, Geoffrey J; Papadopulos, Andreas; Meunier, Frédéric A

    2016-10-24

    Our understanding of endocytic pathway dynamics is severely restricted by the diffraction limit of light microscopy. To address this, we implemented a novel technique based on the subdiffractional tracking of internalized molecules (sdTIM). This allowed us to image anti-green fluorescent protein Atto647N-tagged nanobodies trapped in synaptic vesicles (SVs) from live hippocampal nerve terminals expressing vesicle-associated membrane protein 2 (VAMP2)-pHluorin with 36-nm localization precision. Our results showed that, once internalized, VAMP2-pHluorin/Atto647N-tagged nanobodies exhibited a markedly lower mobility than on the plasma membrane, an effect that was reversed upon restimulation in presynapses but not in neighboring axons. Using Bayesian model selection applied to hidden Markov modeling, we found that SVs oscillated between diffusive states or a combination of diffusive and transport states with opposite directionality. Importantly, SVs exhibiting diffusive motion were relatively less likely to switch to the transport motion. These results highlight the potential of the sdTIM technique to provide new insights into the dynamics of endocytic pathways in a wide variety of cellular settings.

  10. Structure parameters of synaptic vesicles quantified by small-angle x-ray scattering.

    PubMed

    Castorph, Simon; Riedel, Dietmar; Arleth, Lise; Sztucki, Michael; Jahn, Reinhard; Holt, Matthew; Salditt, Tim

    2010-04-07

    Synaptic vesicles (SVs) are small, membrane-bound organelles that are found in the synaptic terminal of neurons, and which are crucial in neurotransmission. After a rise in internal [Ca(2+)] during neuronal stimulation, SVs fuse with the plasma membrane releasing their neurotransmitter content, which then signals neighboring neurons. SVs are subsequently recycled and refilled with neurotransmitter for further rounds of release. Recently, tremendous progress has been made in elucidating the molecular composition of SVs, as well as putative protein-protein interactions. However, what is lacking is an empirical description of SV structure at the supramolecular level-which is necessary to enable us to fully understand the processes of membrane fusion, retrieval, and recycling. Using small-angle x-ray scattering, we have directly investigated the size and structure of purified SVs. From this information, we deduced detailed size and density parameters for the protein layers responsible for SV function, as well as information about the lipid bilayer. To achieve a convincing model fit, a laterally anisotropic structure for the protein shell is needed, as a rotationally symmetric density profile does not explain the data. Not only does our model confirm many of the preexisting ideas concerning SV structure, but also for the first time, to our knowledge, it indicates structural refinements, such as the presence of protein microdomains.

  11. Preferential increase in the hippocampal synaptic vesicle protein 2A (SV2A) by pentylenetetrazole kindling.

    PubMed

    Ohno, Yukihiro; Ishihara, Shizuka; Terada, Ryo; Kikuta, Miki; Sofue, Nobumasa; Kawai, Yoshiko; Serikawa, Tadao; Sasa, Masashi

    2009-12-18

    The present study evaluated the expressional levels of synaptic vesicle protein 2A (SV2A) and other secretary machinery proteins (i.e., soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, Munc18-1, N-ethylmaleimide-sensitive factor (NSF) and soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)) in a pentylenetetrazole (PTZ) kindling model. Repeated administration of sub-convulsive PTZ (40 mg/kg, i.p.) progressively increased seizure susceptibility in mice and consistently induced clonic seizures in most animals tested at 15 days after the treatment. Western blot analysis revealed that, among the secretary machinery proteins examined, hippocampal SV2A was selectively elevated by PTZ kindling. PTZ kindling-induced SV2A expression appeared region-specific and the SV2A levels in the cerebral cortex or cerebellum were unaltered. In addition, SV2A expression by PTZ kindling was prominent in the hilar region of the dentate gyrus (DG) where GABAergic interneurons are located, but not in other hippocampal regions (e.g., the stratum lucidum of the CA3 and synaptic layers surrounding CA1 or CA3 pyramidal neurons). These findings suggest that PTZ kindling preferentially elevates SV2A expression in the hippocampus probably as a compensatory mechanism to activate the inhibitory neurotransmission.

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

    PubMed

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

    2014-11-28

    SV2A is a synaptic vesicle membrane protein expressed in neurons and endocrine cells and involved in the regulation of neurotransmitter release. Although the exact function of SV2A still remains elusive, it was identified as the specific binding site for levetiracetam, a second generation antiepileptic drug. Our sequence analysis demonstrates that SV2A has significant homology with several yeast transport proteins belonging to the major facilitator superfamily (MFS). Many of these transporters are involved in sugar transport into yeast cells. Here we present evidence showing, for the first time, that SV2A is a galactose transporter. We expressed human SV2A in hexose transport-deficient EBY.VW4000 yeast cells and demonstrated that these cells are able to grow on galactose-containing medium but not on other fermentable carbon sources. Furthermore, the addition of the SV2A-binding antiepileptic drug levetiracetam to the medium inhibited the galactose-dependent growth of hexose transport-deficient EBY.VW4000 yeast cells expressing human SV2A. Most importantly, direct measurement of galactose uptake in the same strain verified that SV2A is able to transport extracellular galactose inside the cells. The newly identified galactose transport capability of SV2A may have an important role in regulating/modulating synaptic function. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. 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.

  14. 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

  15. 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

  16. 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. Copyright © 2010 Elsevier Inc. All rights reserved.

  17. Vesamicol blocks the recovery, by recycling cholinergic electromotor synaptic vesicles, of the biophysical characteristics of the reserve population.

    PubMed

    Rícný, J; Whittaker, V P

    1993-06-05

    The effect of vesamicol on the ability of recycling cholinergic synaptic vesicles to recover, during a period of post-stimulation rest, the biophysical properties of the reserve pool was studied in prestimulated perfused blocks of the electric organ of the electric ray, Torpedo marmorata, a tissue rich in cholinergic synapses. The effect of the drug was analysed by high-resolution centrifugal density-gradient fractionation in a zonal rotor of the extracted vesicles. The two vesicle fractions were identified by their ATP and acetylcholine content and the recycled vesicles by their acquisition of [3H]acetylcholine derived from [3H]acetate in the perfusate. Vesamicol (10 microM) blocked the uptake of tritiated acetylcholine by recycled vesicles and also prevented them from rejoining the reserve pool. This is consistent with a previously formulated model of the recovery process, whereby the increase in the acetylcholine and ATP content of the recycled vesicles which takes place during a post-stimulus period of rest increases their osmotic load and thus their content of free water. Vesamicol, by blocking acetylcholine uptake, also blocks rehydration of the recycled vesicles and thus the accompanying decrease in their density to the value characteristic of fully charged vesicles.

  18. Formation and size distribution of self-assembled vesicles.

    PubMed

    Huang, Changjin; Quinn, David; Sadovsky, Yoel; Suresh, Subra; Hsia, K Jimmy

    2017-03-14

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

  19. Bmp4 from the optic vesicle specifies murine retina formation.

    PubMed

    Huang, Jie; Liu, Ying; Oltean, Alina; Beebe, David C

    2015-06-01

    Previous studies of mouse embryos concluded that after the optic vesicle evaginates from the ventral forebrain and contacts the surface ectoderm, signals from the ectoderm specify the distal region of the optic vesicle to become retina and signals from the optic vesicle induce the lens. Germline deletion of Bmp4 resulted in failure of lens formation. We performed conditional deletion of Bmp4 from the optic vesicle to test the function of Bmp4 in murine eye development. The optic vesicle evaginated normally and contacted the surface ectoderm. Lens induction did not occur. The optic cup failed to form and the expression of retina-specific genes decreased markedly in the distal optic vesicle. Instead, cells in the prospective retina expressed genes characteristic of the retinal pigmented epithelium. We conclude that Bmp4 is required for retina specification in mice. In the absence of Bmp4, formation of the retinal pigmented epithelium is the default differentiation pathway of the optic vesicle. Differences in the signaling pathways required for specification of the retina and retinal pigmented epithelium in chicken and mouse embryos suggest major changes in signaling during the evolution of the vertebrate eye.

  20. Formation and size distribution of self-assembled vesicles

    PubMed Central

    Huang, Changjin; Quinn, David; Suresh, Subra

    2017-01-01

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

  1. Microfluidic mixing and the formation of nanoscale lipid vesicles.

    PubMed

    Jahn, Andreas; Stavis, Samuel M; Hong, Jennifer S; Vreeland, Wyatt N; DeVoe, Don L; Gaitan, Michael

    2010-04-27

    We investigate the formation of unilamellar lipid vesicles (liposomes) with diameters of tens of nanometers by controlled microfluidic mixing and nanoparticle determination (COMMAND). Our study includes liposome synthesis experiments and numerical modeling of our microfluidic implementation of the batch solvent injection method. We consider microfluidic liposome formation from the perspective of fluid interfaces and convective-diffusive mixing, as we find that bulk fluid flow parameters including hydrodynamically focused alcohol stream width, final alcohol concentration, and shear stress do not primarily determine the vesicle formation process. Microfluidic device geometry in conjunction with hydrodynamic flow focusing strongly influences vesicle size distributions, providing a coarse method to control liposome size, while total flow rate allows fine-tuning the vesicle size in certain focusing regimes. Although microfluidic liposome synthesis is relatively simple to implement experimentally, numerical simulations of the mixing process reveal a complex system of fluid flow and mass transfer determining the formation of nonequilibrium vesicles. These results expand our understanding of the microfluidic environment that controls liposome self-assembly and yield several technological advances for the on-chip synthesis of nanoscale lipid vesicles.

  2. 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.

  3. Differential regulation of polarized synaptic vesicle trafficking and synapse stability in neural circuit rewiring in Caenorhabditis elegans.

    PubMed

    Kurup, Naina; Yan, Dong; Kono, Karina; Jin, Yishi

    2017-06-01

    Neural circuits are dynamic, with activity-dependent changes in synapse density and connectivity peaking during different phases of animal development. In C. elegans, young larvae form mature motor circuits through a dramatic switch in GABAergic neuron connectivity, by concomitant elimination of existing synapses and formation of new synapses that are maintained throughout adulthood. We have previously shown that an increase in microtubule dynamics during motor circuit rewiring facilitates new synapse formation. Here, we further investigate cellular control of circuit rewiring through the analysis of mutants obtained in a forward genetic screen. Using live imaging, we characterize novel mutations that alter cargo binding in the dynein motor complex and enhance anterograde synaptic vesicle movement during remodeling, providing in vivo evidence for the tug-of-war between kinesin and dynein in fast axonal transport. We also find that a casein kinase homolog, TTBK-3, inhibits stabilization of nascent synapses in their new locations, a previously unexplored facet of structural plasticity of synapses. Our study delineates temporally distinct signaling pathways that are required for effective neural circuit refinement.

  4. Vesicle Size Regulates Nanotube Formation in the Cell.

    PubMed

    Su, Qian Peter; Du, Wanqing; Ji, Qinghua; Xue, Boxin; Jiang, Dong; Zhu, Yueyao; Lou, Jizhong; Yu, Li; Sun, Yujie

    2016-04-07

    Intracellular membrane nanotube formation and its dynamics play important roles for cargo transportation and organelle biogenesis. Regarding the regulation mechanisms, while much attention has been paid on the lipid composition and its associated protein molecules, effects of the vesicle size has not been studied in the cell. Giant unilamellar vesicles (GUVs) are often used for in vitro membrane deformation studies, but they are much larger than most intracellular vesicles and the in vitro studies also lack physiological relevance. Here, we use lysosomes and autolysosomes, whose sizes range between 100 nm and 1 μm, as model systems to study the size effects on nanotube formation both in vivo and in vitro. Single molecule observations indicate that driven by kinesin motors, small vesicles (100-200 nm) are mainly transported along the tracks while a remarkable portion of large vesicles (500-1000 nm) form nanotubes. This size effect is further confirmed by in vitro reconstitution assays on liposomes and purified lysosomes and autolysosomes. We also apply Atomic Force Microscopy (AFM) to measure the initiation force for nanotube formation. These results suggest that the size-dependence may be one of the mechanisms for cells to regulate cellular processes involving membrane-deformation, such as the timing of tubulation-mediated vesicle recycling.

  5. The isolation of pure cholinergic synaptic vesicles from the electric organs of elasmobranch fish of the family Torpedinidae

    PubMed Central

    Whittaker, V. P.; Essman, W. B.; Dowe, G. H. C.

    1972-01-01

    1. Zonal centrifuging permitted the separation, on the milligram scale and in a form largely free from contamination by soluble cytoplasmic protein or membrane fragments derived from other structures, of synaptic vesicles from the purely cholinergic terminals of the electric organ of Torpedo. Up to 100g of tissue could be processed in a single run. 2. As much as 46% of the bound acetylcholine from the original tissue preparation was recovered as a single peak of density equivalent to 0.38m-sucrose–0.21m-NaCl and with a concentration of up to 680nmol of acetylcholine/mg of protein. 3. The limiting concentration of acetylcholine in isolated vesicles when allowance had been made for non-vesicular protein appeared to be about 600nmol/mg of protein. 4. Vesicle counts by a `bead-tagging' procedure indicated an acetylcholine content of about 360μmol/ml of vesicles; thus the vesicle protein content would be about 60% (w/v). 5. Calculations showed that the core of the vesicle, accounting for about 55% of the vesicle volume, could be largely filled with acetylcholine and protein. PMID:4638794

  6. Recruitment of resting vesicles into recycling pools supports NMDA receptor-dependent synaptic potentiation in cultured hippocampal neurons

    PubMed Central

    Ratnayaka, Arjuna; Marra, Vincenzo; Bush, Daniel; Burden, Jemima J; Branco, Tiago; Staras, Kevin

    2012-01-01

    Most presynaptic terminals in the central nervous system are characterized by two functionally distinct vesicle populations: a recycling pool, which supports action potential-driven neurotransmitter release via vesicle exocytosis, and a resting pool. The relative proportions of these two pools are highly variable between individual synapses, prompting speculation on their specific relationship, and on the possible functions of the resting pool. Using fluorescence imaging of FM-styryl dyes and synaptophysinI-pHluorin (sypHy) as well as correlative electron microscopy approaches, we show here that Hebbian plasticity-dependent changes in synaptic strength in rat hippocampal neurons can increase the recycling pool fraction at the expense of the resting pool in individual synaptic terminals. This recruitment process depends on NMDA-receptor activation, nitric oxide signalling and calcineurin and is accompanied by an increase in the probability of neurotransmitter release at individual terminals. Blockade of actin-mediated intersynaptic vesicle exchange does not prevent recycling pool expansion demonstrating that vesicle recruitment is intrasynaptic. We propose that the conversion of resting pool vesicles to the functionally recycling pool provides a rapid mechanism to implement long-lasting changes in presynaptic efficacy. PMID:22271866

  7. Photoinduced Vesicle Formation via the Copper-Catalyzed Azide-Alkyne Cycloaddition Reaction.

    PubMed

    Konetski, Danielle; Gong, Tao; Bowman, Christopher N

    2016-08-16

    Synthetic vesicles have a wide range of applications from drug and cosmetic delivery to artificial cell and membrane studies, making simple and controlled formation of vesicles a large focus of the field today. Here, we report the use of the photoinitiated copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction using visible light to introduce spatiotemporal control into the formation of vesicles. Upon the establishment of the spatiotemporal control over vesicle formation, it became possible to adjust initiation conditions to modulate vesicle sizes resulting in the formation of controllably small or large vesicles based on light intensity or giant vesicles when the formation was initiated in flow-free conditions. Additionally, this photoinitiated method enables vesicle formation at a density 400-fold higher than initiation using sodium ascorbate as the catalyst. Together, these advances enable the formation of high-density, controlled size vesicles using low-energy wavelengths while producing enhanced control over the formation characteristics of the vesicle.

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

    NASA Astrophysics Data System (ADS)

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

    2010-10-01

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

  9. 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.

  10. 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

  11. 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

  12. Mitochondrial Calcium Uptake Modulates Synaptic Vesicle Endocytosis in Central Nerve Terminals.

    PubMed

    Marland, Jamie Roslin Keynes; Hasel, Philip; Bonnycastle, Katherine; Cousin, Michael Alan

    2016-01-29

    Presynaptic calcium influx triggers synaptic vesicle (SV) exocytosis and modulates subsequent SV endocytosis. A number of calcium clearance mechanisms are present in central nerve terminals that regulate intracellular free calcium levels both during and after stimulation. During action potential stimulation, mitochondria rapidly accumulate presynaptic calcium via the mitochondrial calcium uniporter (MCU). The role of mitochondrial calcium uptake in modulating SV recycling has been debated extensively, but a definitive conclusion has not been achieved. To directly address this question, we manipulated the expression of the MCU channel subunit in primary cultures of neurons expressing a genetically encoded reporter of SV turnover. Knockdown of MCU resulted in ablation of activity-dependent mitochondrial calcium uptake but had no effect on the rate or extent of SV exocytosis. In contrast, the rate of SV endocytosis was increased in the absence of mitochondrial calcium uptake and slowed when MCU was overexpressed. MCU knockdown did not perturb activity-dependent increases in presynaptic free calcium, suggesting that SV endocytosis may be controlled by calcium accumulation and efflux from mitochondria in their immediate vicinity.

  13. Disassembly of the reconstituted synaptic vesicle membrane fusion complex in vitro.

    PubMed Central

    Hayashi, T; Yamasaki, S; Nauenburg, S; Binz, T; Niemann, H

    1995-01-01

    The interaction of the presynaptic membrane proteins SNAP-25 and syntaxin with the synaptic vesicle protein synaptobrevin (VAMP) plays a key role in the regulated exocytosis of neurotransmitters. Clostridial neurotoxins, which proteolyze these polypeptides, are potent inhibitors of neurotransmission. The cytoplasmic domains of the three membrane proteins join into a tight SDS-resistant complex (Hayashi et al., 1994). Here, we show that this reconstituted complex, as well as heterodimers composed of syntaxin and SNAP-25, can be disassembled by the concerted action of the N-ethylmaleimide-sensitive factor, NSF, and the soluble NSF attachment protein, alpha-SNAP. alpha-SNAP binds to predicted alpha-helical coiled-coil regions of syntaxin and SNAP-25, shown previously to be engaged in their direct interaction. Synaptobrevin, although incapable of binding alpha-SNAP individually, induced a third alpha-SNAP binding site when associated with syntaxin and SNAP-25 into heterotrimers. NSF released prebound alpha-SNAP from full-length syntaxin but not from a syntaxin derivative truncated at the N-terminus. Disassembly of complexes containing this syntaxin mutant was impaired, indicating a critical role for the N-terminal domain in the alpha-SNAP/NSF-mediated dissociation process. Complexes containing C-terminally deleted SNAP-25 derivatives, as generated by botulinal toxins type A and E, were dissociated more efficiently. In contrast, the N-terminal fragment generated from synaptobrevin by botulinal toxin type F produced an SDS-sensitive complex that was poorly dissociated. Images PMID:7774590

  14. A presynaptic role for the cytomatrix protein GIT in synaptic vesicle recycling.

    PubMed

    Podufall, Jasmin; Tian, Rui; Knoche, Elena; Puchkov, Dmytro; Walter, Alexander M; Rosa, Stefanie; Quentin, Christine; Vukoja, Anela; Jung, Nadja; Lampe, Andre; Wichmann, Carolin; Böhme, Mathias; Depner, Harald; Zhang, Yong Q; Schmoranzer, Jan; Sigrist, Stephan J; Haucke, Volker

    2014-06-12

    Neurotransmission involves the exo-endocytic cycling of synaptic vesicles (SVs) within nerve terminals. Exocytosis is facilitated by a cytomatrix assembled at the active zone (AZ). The precise spatial and functional relationship between exocytic fusion of SVs at AZ membranes and endocytic SV retrieval is unknown. Here, we identify the scaffold G protein coupled receptor kinase 2 interacting (GIT) protein as a component of the AZ-associated cytomatrix and as a regulator of SV endocytosis. GIT1 and its D. melanogaster ortholog, dGIT, are shown to directly associate with the endocytic adaptor stonin 2/stoned B. In Drosophila dgit mutants, stoned B and synaptotagmin levels are reduced and stoned B is partially mislocalized. Moreover, dgit mutants show morphological and functional defects in SV recycling. These data establish a presynaptic role for GIT in SV recycling and suggest a connection between the AZ cytomatrix and the endocytic machinery. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Position of UNC-13 in the active zone regulates synaptic vesicle release probability and release kinetics

    PubMed Central

    Zhou, Keming; Stawicki, Tamara M; Goncharov, Alexandr; Jin, Yishi

    2013-01-01

    The presynaptic active zone proteins UNC-13/Munc13s are essential for synaptic vesicle (SV) exocytosis by directly interacting with SV fusion apparatus. An open question is how their association with active zones, hence their position to Ca2+ entry sites, regulates SV release. The N-termini of major UNC-13/Munc13 isoforms contain a non-calcium binding C2A domain that mediates protein homo- or hetero-meric interactions. Here, we show that the C2A domain of Caenorhabditis elegans UNC-13 regulates release probability of evoked release and its precise active zone localization. Kinetics analysis of SV release supports that the proximity of UNC-13 to Ca2+ entry sites, mediated by the C2A-domain containing N-terminus, is critical for accelerating neurotransmitter release. Additionally, the C2A domain is specifically required for spontaneous release. These data reveal multiple roles of UNC-13 C2A domain, and suggest that spontaneous release and the fast phase of evoked release may involve a common pool of SVs at the active zone. DOI: http://dx.doi.org/10.7554/eLife.01180.001 PMID:24220508

  16. Synaptotagmin 7 functions as a Ca2+-sensor for synaptic vesicle replenishment

    PubMed Central

    Liu, Huisheng; Bai, Hua; Hui, Enfu; Yang, Lu; Evans, Chantell S; Wang, Zhao; Kwon, Sung E; Chapman, Edwin R

    2014-01-01

    Synaptotagmin (syt) 7 is one of three syt isoforms found in all metazoans; it is ubiquitously expressed, yet its function in neurons remains obscure. Here, we resolved Ca2+-dependent and Ca2+-independent synaptic vesicle (SV) replenishment pathways, and found that syt 7 plays a selective and critical role in the Ca2+-dependent pathway. Mutations that disrupt Ca2+-binding to syt 7 abolish this function, suggesting that syt 7 functions as a Ca2+-sensor for replenishment. The Ca2+-binding protein calmodulin (CaM) has also been implicated in SV replenishment, and we found that loss of syt 7 was phenocopied by a CaM antagonist. Moreover, we discovered that syt 7 binds to CaM in a highly specific and Ca2+-dependent manner; this interaction requires intact Ca2+-binding sites within syt 7. Together, these data indicate that a complex of two conserved Ca2+-binding proteins, syt 7 and CaM, serve as a key regulator of SV replenishment in presynaptic nerve terminals. DOI: http://dx.doi.org/10.7554/eLife.01524.001 PMID:24569478

  17. Synaptotagmin 7 functions as a Ca2+-sensor for synaptic vesicle replenishment.

    PubMed

    Liu, Huisheng; Bai, Hua; Hui, Enfu; Yang, Lu; Evans, Chantell S; Wang, Zhao; Kwon, Sung E; Chapman, Edwin R

    2014-02-25

    Synaptotagmin (syt) 7 is one of three syt isoforms found in all metazoans; it is ubiquitously expressed, yet its function in neurons remains obscure. Here, we resolved Ca(2+)-dependent and Ca(2+)-independent synaptic vesicle (SV) replenishment pathways, and found that syt 7 plays a selective and critical role in the Ca(2+)-dependent pathway. Mutations that disrupt Ca(2+)-binding to syt 7 abolish this function, suggesting that syt 7 functions as a Ca(2+)-sensor for replenishment. The Ca(2+)-binding protein calmodulin (CaM) has also been implicated in SV replenishment, and we found that loss of syt 7 was phenocopied by a CaM antagonist. Moreover, we discovered that syt 7 binds to CaM in a highly specific and Ca(2+)-dependent manner; this interaction requires intact Ca(2+)-binding sites within syt 7. Together, these data indicate that a complex of two conserved Ca(2+)-binding proteins, syt 7 and CaM, serve as a key regulator of SV replenishment in presynaptic nerve terminals. DOI: http://dx.doi.org/10.7554/eLife.01524.001.

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

    SciTech Connect

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

    1989-05-01

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

  19. Carbonic anhydrase inhibitor acetazolamide shifts synaptic vesicle recycling to a fast mode at the mouse neuromuscular junction.

    PubMed

    Bertone, Nicolas Ivan; Groisman, Ayelén Ivana; Mazzone, Graciela Lujan; Cano, Raquel; Tabares, Lucia; Uchitel, Osvaldo Daniel

    2017-09-05

    Acetazolamide (AZ), a molecule frequently used to treat different neurological syndromes, is an inhibitor of the carbonic anhydrase (CA), an enzyme that regulates pH inside and outside cells. We combined fluorescent FM styryl dyes and electrophysiological techniques at ex vivo levator auris longus neuromuscular junctions (NMJs) from mice to investigate the modulation of synaptic transmission and vesicle recycling by AZ. Transmitter release was minimally affected by AZ, as evidenced by evoked and spontaneous end-plate potential measurements. However, optical evaluation with FM-styryl dyes of vesicle exocytosis elicited by 50 Hz stimuli showed a strong reduction in fluorescence loss in AZ treated NMJ, an effect that was abolished by bathing the NMJ in Hepes. The remaining dye was quenched by bromophenol, a small molecule capable of diffusing inside vesicles. Furthermore, in transgenic mice expressing Synaptophysin-pHluorin (SypHy), the fluorescence responses of motor nerve terminals to a 50 Hz train of stimuli was decrease to a 50% of controls in the presence of AZ. Immunohistochemistry experiments to evaluate the state of the Myosin light chain kinase (MLCK), an enzyme involved in vesicle recycling, demonstrated that MLCK phosphorylation was much stronger in the presence than AZ than in its absence in 50 Hz stimulated NMJs. We postulate that AZ, via cytosol acidification and activation of MLCK, shifts synaptic vesicle recycling to a fast (kiss-and-run) mode, which changes synaptic performance. These changes may contribute to the therapeutic action reported in many neurological syndromes like ataxia, epilepsy, and migraine. © 2017 Wiley Periodicals, Inc.

  20. synaptotagmin mutants reveal essential functions for the C2B domain in Ca2+-triggered fusion and recycling of synaptic vesicles in vivo.

    PubMed

    Littleton, J T; Bai, J; Vyas, B; Desai, R; Baltus, A E; Garment, M B; Carlson, S D; Ganetzky, B; Chapman, E R

    2001-03-01

    Synaptotagmin has been proposed to function as a Ca(2+) sensor that regulates synaptic vesicle exocytosis, whereas the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is thought to form the core of a conserved membrane fusion machine. Little is known concerning the functional relationships between synaptotagmin and SNAREs. Here we report that synaptotagmin can facilitate SNARE complex formation in vitro and that synaptotagmin mutations disrupt SNARE complex formation in vivo. Synaptotagmin oligomers efficiently bind SNARE complexes, whereas Ca(2+) acting via synaptotagmin triggers cross-linking of SNARE complexes into dimers. Mutations in Drosophila that delete the C2B domain of synaptotagmin disrupt clathrin AP-2 binding and endocytosis. In contrast, a mutation that blocks Ca(2+)-triggered conformational changes in C2B and diminishes Ca(2+)-triggered synaptotagmin oligomerization results in a postdocking defect in neurotransmitter release and a decrease in SNARE assembly in vivo. These data suggest that Ca(2+)-driven oligomerization via the C2B domain of synaptotagmin may trigger synaptic vesicle fusion via the assembly and clustering of SNARE complexes.

  1. Distinct changes in neuronal and astrocytic amino acid neurotransmitter metabolism in mice with reduced numbers of synaptic vesicles.

    PubMed

    Bogen, Inger Lise; Risa, Øystein; Haug, Kristin H; Sonnewald, Ursula; Fonnum, Frode; Walaas, S Ivar

    2008-06-01

    The relations between glutamate and GABA concentrations and synaptic vesicle density in nerve terminals were examined in an animal model with 40-50% reduction in synaptic vesicle numbers caused by inactivation of the genes encoding synapsin I and II. Concentrations and synthesis of amino acids were measured in extracts from cerebrum and a crude synaptosomal fraction by HPLC and (13)C nuclear magnetic resonance spectroscopy (NMRS), respectively. Analysis of cerebrum extracts, comprising both neurotransmitter and metabolic pools, showed decreased concentration of GABA, increased concentration of glutamine and unchanged concentration of glutamate in synapsin I and II double knockout (DKO) mice. In contrast, both glutamate and GABA concentrations were decreased in crude synaptosomes isolated from synapsin DKO mice, suggesting that the large metabolic pool of glutamate in the cerebral extracts may overshadow minor changes in the transmitter pool. (13)C NMRS studies showed that the changes in amino acid concentrations in the synapsin DKO mice were caused by decreased synthesis of GABA (20-24%) in cerebral neurons and increased synthesis of glutamine (36%) in astrocytes. In a crude synaptosomal fraction, the glutamate synthesis was reduced (24%), but this reduction could not be detected in cerebrum extracts. We suggest that lack of synaptic vesicles causes down-regulation of neuronal GABA and glutamate synthesis, with a concomitant increase in astrocytic synthesis of glutamine, in order to maintain normal neurotransmitter concentrations in the nerve terminal cytosol.

  2. In Situ Vesicle Formation by Native Chemical Ligation

    PubMed Central

    Brea, Roberto J.; Cole, Christian M.

    2015-01-01

    Phospholipid vesicles are of intense fundamental and practical interest, yet methods for their de novo generation from reactive precursors are limited. A non-enzymatic and chemoselective method to spontaneously generate phospholipid membranes from water-soluble starting materials would be a powerful tool for generating vesicles and studying lipid membranes. Here we describe the use of native chemical ligation (NCL) to rapidly prepare phospholipids spontaneously from thioesters. While NCL is one of the most popular tools for synthesizing proteins and nucleic acids, to our knowledge this is the first example of using NCL to generate phospholipids de novo. The lipids are capable of in situ synthesis and self-assembly into vesicles that can grow to several microns in diameter. The selectivity of the NCL reaction enables compatibility of in situ membrane formation with biological materials such as proteins. This work expands the application of NCL to the formation of phospholipid membranes. PMID:25346090

  3. Appearance and Distribution of Neuronal Cell Surface and Synaptic Vesicle Antigens in the Developing Rat Superior Cervical Ganglion1

    PubMed Central

    Greif, Karen F.; Reichardt, Louis F.

    2009-01-01

    Monoclonal antibodies directed against a neuronal cell surface heparan sulfate proteoglycan and against a synaptic vesicle protein were used to study the postnatal development of ganglionic neurons and synapses in the rat superior cervical ganglion. Antigen levels in developing ganglia were quantitated by radioimmune assays. Localization of antigens in adult and developing ganglia was carried out using peroxidase-antiperoxidase immunocytochemistry at the light microscopic level. Ultrastructural staining patterns in adult ganglia also were studied. The time course of antigen increases parallels those in previous reports on the accumulation of neurotransmitter enzymes within the ganglion. Both synaptic and surface antigens increase postnatally, with the most rapid changes occurring during the 2nd week. Antibodies stain adult tissue in patterns consistent with the expected distribution of antigens: antibodies directed against synaptic vesicles stain synaptic terminals and cell cytoplasm and those directed against surface proteoglycan stain the plasma membranes of neuronal cell bodies and processes. Variable staining of the cell cytoplasm also is observed. No apparent changes in antigen distribution are observed with the light microscope during development. Variations in the time course of the development of antigens associated with different portions of the proteoglycan molecule suggest that the intracellular processing of this molecule may vary during development. PMID:6212651

  4. Vesicle Formation and Endocytosis: Function, Machinery, Mechanisms, and Modeling

    PubMed Central

    Parkar, Nihal S.; Akpa, Belinda S.; Nitsche, Ludwig C.; Wedgewood, Lewis E.; Place, Aaron T.; Sverdlov, Maria S.; Chaga, Oleg

    2009-01-01

    Abstract Vesicle formation provides a means of cellular entry for extracellular substances and for recycling of membrane constituents. Mechanisms governing the two primary endocytic pathways (i.e., caveolae- and clathrin-mediated endocytosis, as well as newly emerging vesicular pathways) have become the focus of intense investigation to improve our understanding of nutrient, hormone, and drug delivery, as well as opportunistic invasion of pathogens. In this review of endocytosis, we broadly discuss the structural and signaling proteins that compose the molecular machinery governing endocytic vesicle formation (budding, invagination, and fission from the membrane), with some regard for the specificity observed in certain cell types and species. Important biochemical functions of endocytosis and diseases caused by their disruption also are discussed, along with the structures of key components of endocytic pathways and their known mechanistic contributions. The mechanisms by which principal components of the endocytic machinery are recruited to the plasma membrane, where they interact to induce vesicle formation, are discussed, together with computational approaches used to simulate simplified versions of endocytosis with the hope of clarifying aspects of vesicle formation that may be difficult to determine experimentally. Finally, we pose several unanswered questions intended to stimulate further research interest in the cell biology and modeling of endocytosis. Antioxid. Redox Signal. 11, 1301–1312. PMID:19113823

  5. Dynamic control of synaptic vesicle replenishment and short-term plasticity by Ca(2+)-calmodulin-Munc13-1 signaling.

    PubMed

    Lipstein, Noa; Sakaba, Takeshi; Cooper, Benjamin H; Lin, Kun-Han; Strenzke, Nicola; Ashery, Uri; Rhee, Jeong-Seop; Taschenberger, Holger; Neher, Erwin; Brose, Nils

    2013-07-10

    Short-term synaptic plasticity, the dynamic alteration of synaptic strength during high-frequency activity, is a fundamental characteristic of all synapses. At the calyx of Held, repetitive activity eventually results in short-term synaptic depression, which is in part due to the gradual exhaustion of releasable synaptic vesicles. This is counterbalanced by Ca(2+)-dependent vesicle replenishment, but the molecular mechanisms of this replenishment are largely unknown. We studied calyces of Held in knockin mice that express a Ca(2+)-Calmodulin insensitive Munc13-1(W464R) variant of the synaptic vesicle priming protein Munc13-1. Calyces of these mice exhibit a slower rate of synaptic vesicle replenishment, aberrant short-term depression and reduced recovery from synaptic depression after high-frequency stimulation. Our data establish Munc13-1 as a major presynaptic target of Ca(2+)-Calmodulin signaling and show that the Ca(2+)-Calmodulin-Munc13-1 complex is a pivotal component of the molecular machinery that determines short-term synaptic plasticity characteristics.

  6. Effect of protic ionic liquid nanostructure on phospholipid vesicle formation.

    PubMed

    Bryant, Saffron J; Wood, Kathleen; Atkin, Rob; Warr, Gregory G

    2017-02-15

    The formation of bilayer-based lyotropic liquid crystals and vesicle dispersions by phospholipids in a range of protic ionic liquids has been investigated by polarizing optical microscopy using isothermal penetration scans, differential scanning calorimetry, and small angle X-ray and neutron scattering. The stability and structure of both lamellar phases and vesicle dispersions is found to depend primarily on the underlying amphiphilic nanostructure of the ionic liquid itself. This finding has significant implications for the use of ionic liquids in soft and biological materials and for biopreservation, and demonstrates how vesicle structure and properties can be controlled through selection of cation and anion. For a given ionic liquid, systematic trends in bilayer thickness, chain-melting temperature and enthalpy increase with phospholipid acyl chain length, paralleling behaviour in aqueous systems.

  7. Loss of synaptotagmin IV results in a reduction in synaptic vesicles and a distortion of the Golgi structure in cultured hippocampal neurons.

    PubMed

    Arthur, C P; Dean, C; Pagratis, M; Chapman, E R; Stowell, M H B

    2010-04-28

    Fusion of synaptic vesicles with the plasma membrane is mediated by the SNARE (soluble NSF attachment receptor) proteins and is regulated by synaptotagmin (syt). There are at least 17 syt isoforms that have the potential to act as modulators of membrane fusion events. Synaptotagmin IV (syt IV) is particularly interesting; it is an immediate early gene that is regulated by seizures and certain classes of drugs, and, in humans, syt IV maps to a region of chromosome 18 associated with schizophrenia and bipolar disease. Syt IV has recently been found to localize to dense core vesicles in hippocampal neurons, where it regulates neurotrophin release. Here we have examined the ultrastructure of cultured hippocampal neurons from wild-type and syt IV -/- mice using electron tomography. Perhaps surprisingly, we observed a potential synaptic vesicle transport defect in syt IV -/- neurons, with the accumulation of large numbers of small clear vesicles (putative axonal transport vesicles) near the trans-Golgi network. We also found an interaction between syt IV and KIF1A, a kinesin known to be involved in vesicle trafficking to the synapse. Finally, we found that syt IV -/- synapses exhibited reduced numbers of synaptic vesicles and a twofold reduction in the proportion of docked vesicles compared to wild-type. The proportion of docked vesicles in syt IV -/- boutons was further reduced, 5-fold, following depolarization. Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

  8. 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.

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

    PubMed Central

    Stanley, Elise F

    2015-01-01

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

  10. Synaptic vesicle protein2A decreases in amygdaloid-kindling pharmcoresistant epileptic rats.

    PubMed

    Shi, Jing; Zhou, Feng; Wang, Li-kun; Wu, Guo-feng

    2015-10-01

    Synaptic vesicle protein 2A (SV2A) involvement has been reported in the animal models of epilepsy and in human intractable epilepsy. The difference between pharmacosensitive epilepsy and pharmacoresistant epilepsy remains poorly understood. The present study aimed to observe the hippocampus SV2A protein expression in amygdale-kindling pharmacoresistant epileptic rats. The pharmacosensitive epileptic rats served as control. Amygdaloid-kindling model of epilepsy was established in 100 healthy adult male Sprague-Dawley rats. The kindled rat model of epilepsy was used to select pharmacoresistance by testing their seizure response to phenytoin and phenobarbital. The selected pharmacoresistant rats were assigned to a pharmacoresistant epileptic group (PRE group). Another 12 pharmacosensitive epileptic rats (PSE group) served as control. Immunohistochemistry, real-time PCR and Western blotting were used to determine SV2A expression in the hippocampus tissue samples from both the PRE and the PSE rats. Immunohistochemistry staining showed that SV2A was mainly accumulated in the cytoplasm of the neurons, as well as along their dendrites throughout all subfields of the hippocampus. Immunoreactive staining level of SV2A-positive cells was 0.483 ± 0.304 in the PRE group and 0.866 ± 0.090 in the PSE group (P < 0.05). Real-time PCR analysis demonstrated that 2(-ΔΔCt) value of SV2A mRNA was 0.30 ± 0.43 in the PRE group and 0.76 ± 0.18 in the PSE group (P < 0.05). Western blotting analysis obtained the similar findings (0.27 ± 0.21 versus 1.12 ± 0.21, P < 0.05). PRE rats displayed a significant decrease of SV2A in the brain. SV2A may be associated with the pathogenesis of intractable epilepsy of the amygdaloid-kindling rats.

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

    PubMed

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

    1998-07-01

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

  12. Functional reconstitution of the. gamma. -aminobutyric acid transporter from synaptic vesicles using artificial ion gradients

    SciTech Connect

    Hell, J.W.; Edelmann, L.; Hartinger, J.; Jahn, R. )

    1991-12-24

    The {gamma}-aminobutyric acid transporter of rat brain synaptic vesicles was reconstituted in proteoliposomes, and its activity was studied in response to artificially created membrane potentials or proton gradients. Changes of the membrane potential were monitored using the dyes oxonol VI and 3,3{prime}-diisopropylthiodicarbocyanine iodide, and changes of the H{sup +} gradient were followed using acridine orange. An inside positive membrane potential was generated by the creation of an inwardly directed K{sup +} gradient and the subsequent addition of valinomycin. Under these conditions, valinomycin evoked uptake of ({sup 3}H)GABA which was saturable. Similarly, ({sup 3}H)glutamate uptake was stimulated by valinomycin, indicating that both transporters can be driven by the membrane potential. Proton gradients were generated by the incubation of K{sup +}-loaded proteoliposomes in a buffer free of K{sup +} or Na{sup +} ions and the subsequent addition of nigericin. Proton gradients were also generated via the endogenous H{sup +} ATPase by incubation of K{sup +}-loaded proteoliposomes in equimolar K{sup +} buffer in the presence of valinomycin. These proton gradients evoked nonspecific, nonsaturable uptake of GABA and {beta}-alanine but not of glycine in proteoliposomes as well as protein-free liposomes. Therefore, transporter activity was monitored using glycine as an alternative substrate. Proton gradients generated by both methods elicited saturable glycine uptake in proteoliposomes. Together, these data confirm that the vesicular GABA transporter can be energized by both the membrane potential and the pH gradient and show that transport can be achieved by artificial gradients independently of the endogenous proton ATPase.

  13. Presynaptic G protein-coupled receptors dynamically modify vesicle fusion, synaptic cleft glutamate concentrations and motor behavior

    PubMed Central

    Gerachshenko, Tatyana; Schwartz, Eric; Bleckert, Adam; Photowala, Huzefa; Seymour, Andrew; Alford, Simon

    2009-01-01

    Understanding how neuromodulators regulate behavior requires investigating their effects on functional neural systems, but also their underlying cellular mechanisms. Utilizing extensively characterized lamprey motor circuits, and the unique access to reticulospinal presynaptic terminals in the intact spinal cord that initiate these behaviours, we have investigated effects of presynaptic G protein-coupled receptors on locomotion from the systems level, to the molecular control of vesicle fusion. 5-HT inhibits neurotransmitter release via a Gβγ interaction with the SNARE complex that promotes kiss-and-run vesicle fusion. In the lamprey spinal cord we demonstrate that while presynaptic 5-HT receptors inhibit evoked neurotransmitter release from reticulospinal command neurons, their activation does not abolish locomotion, but rather modulates locomotor rhythms. Liberation of presynaptic Gβγ causes substantial inhibition of AMPA receptor-mediated synaptic responses, but leaves NMDA receptor-mediated components of neurotransmission largely intact. Because Gβγ binding to the SNARE complex is displaced by Ca2+-synaptotagmin binding, 5-HT-mediated inhibition displays Ca2+ sensitivity. We show that as Ca2+ accumulates presynaptically during physiological bouts of activity, 5-HT/Gβγ-mediated presynaptic inhibition is relieved leading to a frequency-dependent increase in synaptic concentrations of glutamate. This frequency dependent phenomenon mirrors a shift in the vesicle fusion mode and a recovery of AMPA receptor-mediated EPSCs from inhibition without a modification of NMDA receptor EPSCs. We conclude that activation of presynaptic 5-HT GPCRs state-dependently alters vesicle fusion properties to shift the weight of NMDA vs AMPA receptor-mediated responses at excitatory synapses. We have therefore identified a novel mechanism in which modification of vesicle fusion modes may profoundly alter locomotor behaviour. PMID:19692597

  14. 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

  15. The dual phosphatase activity of Synaptojanin1 is required for both efficient synaptic vesicle internalization and re-availability at nerve terminals

    PubMed Central

    Mani, Meera; Lee, Sang Yoon; Lucast, Louise; Cremona, Ottavio; Di Paolo, Gilbert; De Camilli, Pietro; Ryan, Timothy A.

    2013-01-01

    Summary Phosphoinositides have been implicated in synaptic vesicle recycling largely based on studies of enzymes that regulate phosphoinositide synthesis and hydrolysis. One such enzyme is Synaptojanin1, a multifunctional protein conserved from yeast to humans, which contains two phospho-inositol phosphatase domains and a proline-rich domain. Genetic ablation of Synaptojanin1 leads to pleiotropic defects in presynaptic function, including accumulation of free clathrin-coated vesicles and delayed vesicle re-availability, implicating this enzyme in post-endocytic uncoating of vesicles. To further elucidate the role of Synaptojanin1 at nerve terminals, we performed quantitative synaptic vesicle recycling assays in synj1−/− neurons. Our studies show that Synaptojanin1 is also required for normal vesicle endocytosis. Defects in both endocytosis and post-endocytic vesicle re-availability can be fully restored upon reintroduction of Synaptojanin1. However, expression of Synaptojanin1 with mutations abolishing catalytic activity of each phosphatase domain reveals that the dual action of both domains is required for normal synaptic vesicle internalization and re-availability. PMID:18093523

  16. Synaptic clustering within dendrites: an emerging theory of memory formation

    PubMed Central

    Kastellakis, George; Cai, Denise J.; Mednick, Sara C.; Silva, Alcino J.; Poirazi, Panayiota

    2015-01-01

    It is generally accepted that complex memories are stored in distributed representations throughout the brain, however the mechanisms underlying these representations are not understood. Here, we review recent findings regarding the subcellular mechanisms implicated in memory formation, which provide evidence for a dendrite-centered theory of memory. Plasticity-related phenomena which affect synaptic properties, such as synaptic tagging and capture, synaptic clustering, branch strength potentiation and spinogenesis provide the foundation for a model of memory storage that relies heavily on processes operating at the dendrite level. The emerging picture suggests that clusters of functionally related synapses may serve as key computational and memory storage units in the brain. We discuss both experimental evidence and theoretical models that support this hypothesis and explore its advantages for neuronal function. PMID:25576663

  17. Formation and maintenance of neuronal assemblies through synaptic plasticity.

    PubMed

    Litwin-Kumar, Ashok; Doiron, Brent

    2014-11-14

    The architecture of cortex is flexible, permitting neuronal networks to store recent sensory experiences as specific synaptic connectivity patterns. However, it is unclear how these patterns are maintained in the face of the high spike time variability associated with cortex. Here we demonstrate, using a large-scale cortical network model, that realistic synaptic plasticity rules coupled with homeostatic mechanisms lead to the formation of neuronal assemblies that reflect previously experienced stimuli. Further, reverberation of past evoked states in spontaneous spiking activity stabilizes, rather than erases, this learned architecture. Spontaneous and evoked spiking activity contains a signature of learned assembly structures, leading to testable predictions about the effect of recent sensory experience on spike train statistics. Our work outlines requirements for synaptic plasticity rules capable of modifying spontaneous dynamics and shows that this modification is beneficial for stability of learned network architectures.

  18. 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.

  19. Influence of Glucose Deprivation on Membrane Potentials of Plasma Membranes, Mitochondria and Synaptic Vesicles in Rat Brain Synaptosomes.

    PubMed

    Hrynevich, Sviatlana V; Pekun, Tatyana G; Waseem, Tatyana V; Fedorovich, Sergei V

    2015-06-01

    Hypoglycemia can cause neuronal cell death similar to that of glutamate-induced cell death. In the present paper, we investigated the effect of glucose removal from incubation medium on changes of mitochondrial and plasma membrane potentials in rat brain synaptosomes using the fluorescent dyes DiSC3(5) and JC-1. We also monitored pH gradients in synaptic vesicles and their recycling by the fluorescent dye acridine orange. Glucose deprivation was found to cause an inhibition of K(+)-induced Ca(2+)-dependent exocytosis and a shift of mitochondrial and plasma membrane potentials to more positive values. The sensitivity of these parameters to the energy deficit caused by the removal of glucose showed the following order: mitochondrial membrane potential > plasma membrane potential > pH gradient in synaptic vesicles. The latter was almost unaffected by deprivation compared with the control. The pH-dependent dye acridine orange was used to investigate synaptic vesicle recycling. However, the compound's fluorescence was shown to be enhanced also by the mixture of mitochondrial toxins rotenone (10 µM) and oligomycin (5 µg/mL). This means that acridine orange can presumably be partially distributed in the intermembrane space of mitochondria. Glucose removal from the incubation medium resulted in a 3.7-fold raise of acridine orange response to rotenone + oligomycin suggesting a dramatic increase in the mitochondrial pH gradient. Our results suggest that the biophysical characteristics of neuronal presynaptic endings do not favor excessive non-controlled neurotransmitter release in case of hypoglycemia. The inhibition of exocytosis and the increase of the mitochondrial pH gradient, while preserving the vesicular pH gradient, are proposed as compensatory mechanisms.

  20. Palmitoyl protein thioesterase (PPT) localizes into synaptosomes and synaptic vesicles in neurons: implications for infantile neuronal ceroid lipofuscinosis (INCL).

    PubMed

    Lehtovirta, M; Kyttälä, A; Eskelinen, E L; Hess, M; Heinonen, O; Jalanko, A

    2001-01-01

    A deficiency of palmitoyl protein thioesterase (PPT) leads to the neurodegenerative disease infantile neuronal ceroid lipofuscinosis (INCL), which is characterized by an almost complete loss of cortical neurons. PPT expressed in COS-1 cells is recognized by the mannose-6-phosphate receptor (M6PR) and is routed to lysosome, but a substantial fraction of PPT is secreted. We have here determined the neuronal localization of PPT by confocal microscopy, cryoimmunoelectron microscopy and cell fractionation. In mouse primary neurons and brain tissue, PPT is localized in synaptosomes and synaptic vesicles but not in lysosomes. Furthermore, in polarized epithelial Caco-2 cells, PPT is localized exclusively to the basolateral site, in contrast to the classical lysosomal enzyme, aspartylglucosaminidase (AGA), which is localized in the apical site. The current data imply that PPT has a role outside the lysosomes in the brain and may be associated with synaptic functioning. This finding opens a new route to study the neuropathological events associated with INCL.

  1. Endosome-mediated endocytic mechanism replenishes the majority of synaptic vesicles at mature CNS synapses in an activity-dependent manner

    PubMed Central

    Park, Joohyun; Cho, Oh Yeon; Kim, Jung Ah; Chang, Sunghoe

    2016-01-01

    Whether synaptic vesicles (SVs) are recovered via endosome-mediated pathways is a matter of debate; however, recent evidence suggests that clathrin-independent bulk endocytosis (CIE) via endosomes is functional and preferentially replenishes SV pools during strong stimulation. Here, using brefeldin-A (BFA) to block CIE, we found that CIE retrieved a minority of SVs at developing CNS synapses during strong stimulation, but its contribution increased up to 61% at mature CNS synapses. Contrary to previous views, BFA not only blocked SV formation from the endosome but also blocked the endosome formation at the plasma membrane. Adaptor protein 1 and 3 (AP-1/3) have key roles in SV reformation from endosomes during CIE, and AP-1 also affects bulk endosome formation from the plasma membrane. Finally, temporary blocking of chronic or acute neuronal activity with tetrodotoxin in mature neurons redirected most SV retrieval to endosome-independent pathways. These results show that during high neuronal activity, CIE becomes the major endocytic pathway at mature CNS synapses. Moreover, mature neurons use clathrin-mediated endocytosis and the CIE pathway to different extents depending on their previous activity; this may result in activity-dependent alterations of the SV composition which ultimately influence transmitter release and contribute to synaptic plasticity. PMID:27534442

  2. Endosome-mediated endocytic mechanism replenishes the majority of synaptic vesicles at mature CNS synapses in an activity-dependent manner.

    PubMed

    Park, Joohyun; Cho, Oh Yeon; Kim, Jung Ah; Chang, Sunghoe

    2016-08-18

    Whether synaptic vesicles (SVs) are recovered via endosome-mediated pathways is a matter of debate; however, recent evidence suggests that clathrin-independent bulk endocytosis (CIE) via endosomes is functional and preferentially replenishes SV pools during strong stimulation. Here, using brefeldin-A (BFA) to block CIE, we found that CIE retrieved a minority of SVs at developing CNS synapses during strong stimulation, but its contribution increased up to 61% at mature CNS synapses. Contrary to previous views, BFA not only blocked SV formation from the endosome but also blocked the endosome formation at the plasma membrane. Adaptor protein 1 and 3 (AP-1/3) have key roles in SV reformation from endosomes during CIE, and AP-1 also affects bulk endosome formation from the plasma membrane. Finally, temporary blocking of chronic or acute neuronal activity with tetrodotoxin in mature neurons redirected most SV retrieval to endosome-independent pathways. These results show that during high neuronal activity, CIE becomes the major endocytic pathway at mature CNS synapses. Moreover, mature neurons use clathrin-mediated endocytosis and the CIE pathway to different extents depending on their previous activity; this may result in activity-dependent alterations of the SV composition which ultimately influence transmitter release and contribute to synaptic plasticity.

  3. Working Memory Impairment in Calcineurin Knock-out Mice Is Associated with Alterations in Synaptic Vesicle Cycling and Disruption of High-Frequency Synaptic and Network Activity in Prefrontal Cortex

    PubMed Central

    Cottrell, Jeffrey R.; Levenson, Jonathan M.; Kim, Sung Hyun; Gibson, Helen E.; Richardson, Kristen A.; Sivula, Michael; Li, Bing; Ashford, Crystle J.; Heindl, Karen A.; Babcock, Ryan J.; Rose, David M.; Hempel, Chris M.; Wiig, Kjesten A.; Laeng, Pascal; Levin, Margaret E.; Ryan, Timothy A.

    2013-01-01

    Working memory is an essential component of higher cognitive function, and its impairment is a core symptom of multiple CNS disorders, including schizophrenia. Neuronal mechanisms supporting working memory under normal conditions have been described and include persistent, high-frequency activity of prefrontal cortical neurons. However, little is known about the molecular and cellular basis of working memory dysfunction in the context of neuropsychiatric disorders. To elucidate synaptic and neuronal mechanisms of working memory dysfunction, we have performed a comprehensive analysis of a mouse model of schizophrenia, the forebrain-specific calcineurin knock-out mouse. Biochemical analyses of cortical tissue from these mice revealed a pronounced hyperphosphorylation of synaptic vesicle cycling proteins known to be necessary for high-frequency synaptic transmission. Examination of the synaptic vesicle cycle in calcineurin-deficient neurons demonstrated an impairment of vesicle release enhancement during periods of intense stimulation. Moreover, brain slice and in vivo electrophysiological analyses showed that loss of calcineurin leads to a gene dose-dependent disruption of high-frequency synaptic transmission and network activity in the PFC, correlating with selective working memory impairment. Finally, we showed that levels of dynamin I, a key presynaptic protein and calcineurin substrate, are significantly reduced in prefrontal cortical samples from schizophrenia patients, extending the disease relevance of our findings. Our data provide support for a model in which impaired synaptic vesicle cycling represents a critical node for disease pathologies underlying the cognitive deficits in schizophrenia. PMID:23825400

  4. 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

  5. Interactome of the amyloid precursor protein APP in brain reveals a protein network involved in synaptic vesicle turnover and a close association with Synaptotagmin-1.

    PubMed

    Kohli, Bernhard M; Pflieger, Delphine; Mueller, Lukas N; Carbonetti, Giovanni; Aebersold, Ruedi; Nitsch, Roger M; Konietzko, Uwe

    2012-08-03

    Knowledge of the protein networks interacting with the amyloid precursor protein (APP) in vivo can shed light on the physiological function of APP. To date, most proteins interacting with the APP intracellular domain (AICD) have been identified by Yeast Two Hybrid screens which only detect direct interaction partners. We used a proteomics-based approach by biochemically isolating tagged APP from the brains of transgenic mice and subjecting the affinity-purified complex to mass spectrometric (MS) analysis. Using two different quantitative MS approaches, we compared the protein composition of affinity-purified samples isolated from wild-type mice versus transgenic mice expressing tagged APP. This enabled us to assess truly enriched proteins in the transgenic sample and yielded an overlapping set of proteins containing the major proteins involved in synaptic vesicle endo- and exocytosis. Confocal microscopy analyses of cotransfected primary neurons showed colocalization of APP with synaptic vesicle proteins in vesicular structures throughout the neurites. We analyzed the interaction of APP with these proteins using pulldown experiments from transgenic mice or cotransfected cells followed by Western blotting. Synaptotagmin-1 (Stg1), a resident synaptic vesicle protein, was found to directly bind to APP. We fused Citrine and Cerulean to APP and the candidate proteins and measured fluorescence resonance energy transfer (FRET) in differentiated SH-SY5Y cells. Differentially tagged APPs showed clear sensitized FRET emission, in line with the described dimerization of APP. Among the candidate APP-interacting proteins, again only Stg1 was in close proximity to APP. Our results strongly argue for a function of APP in synaptic vesicle turnover in vivo. Thus, in addition to the APP cleavage product Aβ, which influences synaptic transmission at the postsynapse, APP interacts with the calcium sensor of synaptic vesicles and might thus play a role in the regulation of synaptic

  6. HDAC2 negatively regulates memory formation and synaptic plasticity

    PubMed Central

    Guan, Ji-Song; Haggarty, Stephen J.; Giacometti, Emanuela; Dannenberg, Jan-Hermen; Joseph, Nadine; Gao, Jun; Nieland, Thomas J.F.; Zhou, Ying; Wang, Xinyu; Mazitschek, Ralph; Bradner, James E.; DePinho, Ronald A.; Jaenisch, Rudolf; Tsai, Li-Huei

    2012-01-01

    Chromatin modifications, especially histone-tail acetylation, have been implicated in memory formation. Increased histone-tail acetylation induced by inhibitors of histone deacetylases (HDACis) facilitates learning and memory in wildtype mice as well as in mouse models of neurodegeneration. Harnessing the therapeutic potential of HDACi requires knowledge of the specific HDAC family member(s) linked to cognitive enhancement. Here we show that neuron-specific overexpression of HDAC2, but not HDAC1, reduced dendritic spine density, synapse number, synaptic plasticity, and memory formation. Conversely, HDAC2 deficiency resulted in increased synapse number and memory facilitation, similar to chronic HDACi treatment in mice. Notably, reduced synapse number and learning impairment of HDAC2-overexpressing mice were ameliorated by chronic HDACi treatment. Correspondingly, HDACi treatment failed to further facilitate memory formation in HDAC2-deficient mice. Furthermore, analysis of promoter occupancy revealed association of HDAC2 with the promoters of genes implicated in synaptic plasticity and memory formation. Together, our results suggest that HDAC2 plays a role in modulating synaptic plasticity and long-lasting changes of neural circuits, which in turn negatively regulates learning and memory. These observations encourage the development and testing of HDAC2-selective inhibitors for human diseases associated with memory impairment. PMID:19424149

  7. Uptake of the neurotoxin, 4-methylphenylpyridinium, into chromaffin granules and synaptic vesicles: a proton gradient drives its uptake through monoamine transporter.

    PubMed

    Moriyama, Y; Amakatsu, K; Futai, M

    1993-09-01

    Energy dependence for uptake of 4-methyphenylpyridinium (MPP+), a neurotoxin causing Parkinsonism-like symptoms, by adrenal chromaffin granule membrane vesicles and brain synaptic vesicles was studied. The compound was actively taken up by the chromaffin vesicles dependent on hydrolysis of ATP with a Km value of 22 microM and maximum velocity of 2.9 nmol/min/mg protein. The uptake was sensitive to reserpine (1 microM) and bafilomycin (50 nM) (inhibitors of the vesicular monoamine transporter and vacuolar-type H(+)-ATPase, respectively) and substrates for monoamine transporters, but insensitive to imipramine (an inhibitor of the monoamine transporter present in the plasma membrane). The uptake was greatly reduced upon dissipation of the proton gradient by ammonium ion or nigericin with KCl, but stimulated 1.6-fold by valinomycin plus K+. Dissipation of the proton gradient also induced rapid efflux of MPP+ from the vesicles. The MPP+ (monoamine) transporter was solubilized from chromaffin vesicles and reconstituted into liposomes with purified bacterial F0F1-ATPase. MPP+ was taken up by the liposomes coupled with ATP hydrolysis by F0F1, and the uptake was sensitive to reserpine, dissipation of the proton gradient, and azide. Brain synaptic vesicles also accumulated MPP+, showing similar kinetics, inhibitor sensitivities, and energy coupling to those of chromaffin vesicles. Furthermore, MPP+ inhibited the uptake of dopamine without affecting the uptake of glutamate or gamma-aminobutyrate. These results indicated that MPP+ was taken up through the reserpine-sensitive monoamine transporter into chromaffin vesicles and synaptic vesicles and that the energy for accumulation of MPP+ was supplied as a proton gradient (acidic inside) established by H(+)-ATPase.

  8. Matrix vesicles: structure, composition, formation and function in calcification.

    PubMed

    Wuthier, Roy E; Lipscomb, Guy F

    2011-06-01

    Matrix vesicles (MVs) induce calcification during endochondral bone formation. Experimental methods for structural, compositional, and functional analysis of MVs are reviewed. MV proteins, enzymes, receptors, transporters, regulators, lipids and electrolytes are detailed. MV formation is considered from both structural and biochemical perspectives. Confocal imaging of Ca(2+) and H(+) were used to depict how living chondrocytes form MVs. Biochemical studies revealed that coordinated mitochondrial Ca(2+) and Pi metabolism produce MVs containing a nucleational complex (NC) of amorphous calcium phosphate, phosphatidylserine and annexin A5--all critical to the mechanism of mineral nucleation. Reconstitution of the NC and modeling with unilamellar vesicles reveal how the NC transforms into octacalcium phosphate, regulated by Mg(2+), Zn(2+) and annexin A5. Extravasation of intravesicular mineral is mediated by phospholipases and tissue-nonspecific alkaline phosphatase (TNAP). In the extravesicular matrix, hydroxyapatite crystal propagation is enhanced by cartilage collagens and TNAP, which destroys inhibitory PPi, and by metalloproteases that degrade proteoglycans. Other proteins also modulate mineral formation. Recent findings from single and multiple gene knockouts of TNAP, NPP1, ANK, PHOSPHO1, and Annexin A5 are reviewed.

  9. 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.

  10. Autophagy Modulates Articular Cartilage Vesicle Formation in Primary Articular Chondrocytes*

    PubMed Central

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

    2015-01-01

    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. PMID:25869133

  11. METHOD DEVELOPMENT AND VALIDATION OF AN IN VITRO MODEL OF THE EFFECTS OF METHYLPHENIDATE ON MEMBRANE-ASSOCIATED SYNAPTIC VESICLES

    PubMed Central

    Volz, Trent J.; Farnsworth, Sarah J.; Hanson, Glen R.; Fleckenstein, Annette E.

    2009-01-01

    In vivo methylphenidate (MPD) administration decreases vesicular monoamine transporter-2 (VMAT-2) immunoreactivity in membrane-associated vesicles isolated from the striata of treated rats while concurrently kinetically upregulating VMAT-2-mediated vesicular dopamine (DA) sequestration. The functional consequences of these MPD-induced effects include an increase in both vesicular DA content and exocytotic DA release. This report describes experiments designed to develop and validate an in vitro MPD model to further elucidate the molecular mechanism(s) underlying the effects of MPD on the VMAT-2 in membrane-associated vesicles. Method development experiments revealed that in vitro MPD incubation of striatal homogenates, but not striatal synaptosomes, increased DA transport velocities and decreased VMAT-2 immunoreactivity in membrane-associated vesicles. An incubation time of 30 min with a MPD concentration of 10 mM was optimal. Method validation experiments indicated that in vitro MPD incubation kinetically upregulated VMAT-2 in membrane-associated vesicles, increased vesicular DA content, and increased exocytotic DA release. These results reveal that the in vitro MPD incubation model successfully reproduced the salient features of in vivo MPD administration. This in vitro MPD incubation model may provide novel insights into the receptor-mediated mechanism(s) of action of in vivo MPD in the striatum as well as the physiological regulation of vesicular DA sequestration and synaptic transmission. Accordingly, this in vitro model may help to advance the treatment of disorders involving abnormal DA disposition including Parkinson’s disease, attention-deficit hyperactivity disorder, and substance abuse. PMID:18992277

  12. Unconventional molecular regulation of synaptic vesicle replenishment in cochlear inner hair cells.

    PubMed

    Vogl, Christian; Cooper, Benjamin H; Neef, Jakob; Wojcik, Sonja M; Reim, Kerstin; Reisinger, Ellen; Brose, Nils; Rhee, Jeong-Seop; Moser, Tobias; Wichmann, Carolin

    2015-02-15

    Ribbon synapses of cochlear inner hair cells (IHCs) employ efficient vesicle replenishment to indefatigably encode sound. In neurons, neuroendocrine and immune cells, vesicle replenishment depends on proteins of the mammalian uncoordinated 13 (Munc13, also known as Unc13) and Ca(2+)-dependent activator proteins for secretion (CAPS) families, which prime vesicles for exocytosis. Here, we tested whether Munc13 and CAPS proteins also regulate exocytosis in mouse IHCs by combining immunohistochemistry with auditory systems physiology and IHC patch-clamp recordings of exocytosis in mice lacking Munc13 and CAPS isoforms. Surprisingly, we did not detect Munc13 or CAPS proteins at IHC presynaptic active zones and found normal IHC exocytosis as well as auditory brainstem responses (ABRs) in Munc13 and CAPS deletion mutants. Instead, we show that otoferlin, a C2-domain protein that is crucial for vesicular fusion and replenishment in IHCs, clusters at the plasma membrane of the presynaptic active zone. Electron tomography of otoferlin-deficient IHC synapses revealed a reduction of short tethers holding vesicles at the active zone, which might be a structural correlate of impaired vesicle priming in otoferlin-deficient IHCs. We conclude that IHCs use an unconventional priming machinery that involves otoferlin.

  13. Cholesterol and F-actin are required for clustering of recycling synaptic vesicle proteins in the presynaptic plasma membrane

    PubMed Central

    Dason, Jeffrey S; Smith, Alex J; Marin, Leo; Charlton, Milton P

    2014-01-01

    AbstractSynaptic vesicles (SVs) and their proteins must be recycled for sustained synaptic transmission. We tested the hypothesis that SV cholesterol is required for proper sorting of SV proteins during recycling in live presynaptic terminals. We used the reversible block of endocytosis in the Drosophila temperature-sensitive dynamin mutant shibire-ts1 to trap exocytosed SV proteins, and then examined the effect of experimental treatments on the distribution of these proteins within the presynaptic plasma membrane by confocal microscopy. SV proteins synaptotagmin, vglut and csp were clustered following SV trapping in control experiments but dispersed in samples treated with the cholesterol chelator methyl-β-cyclodextrin to extract SV cholesterol. There was accumulation of phosphatidylinositol (4,5)-bisphosphate (PIP2) in presynaptic terminals following SV trapping and this was reduced following SV cholesterol extraction. Reduced PIP2 accumulation was associated with disrupted accumulation of actin in presynaptic terminals. Similar to vesicular cholesterol extraction, disruption of actin by latrunculin A after SV proteins had been trapped on the plasma membrane resulted in the dispersal of SV proteins and prevented recovery of synaptic transmission due to impaired endocytosis following relief of the endocytic block. Our results demonstrate that vesicular cholesterol is required for aggregation of exocytosed SV proteins in the presynaptic plasma membrane and are consistent with a mechanism involving regulation of PIP2 accumulation and local actin polymerization by cholesterol. Thus, alteration of membrane or SV lipids may affect the ability of synapses to undergo sustained synaptic transmission by compromising the recycling of SV proteins. PMID:24297851

  14. Vesicles

    MedlinePlus

    ... the top of a pin or up to 5 millimeters wide. A larger blister is called a bulla. In many cases, vesicles break easily and release their fluid onto the skin. When this fluid dries, yellow crusts may remain on the skin surface.

  15. 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.

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

    PubMed Central

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

    1996-01-01

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

  17. Glycosylation Is Dispensable for Sorting of Synaptotagmin 1 but Is Critical for Targeting of SV2 and Synaptophysin to Recycling Synaptic Vesicles*

    PubMed Central

    Kwon, Sung E.; Chapman, Edwin R.

    2012-01-01

    Glycosylation is a major form of post-translational modification of synaptic vesicle membrane proteins. For example, the three major synaptic vesicle glycoproteins, synaptotagmin 1, synaptophysin, and SV2, represent ∼30% of the total copy number of vesicle proteins. Previous studies suggested that glycosylation is required for the vesicular targeting of synaptotagmin 1, but the role of glycosylation of synaptophysin and SV2 has not been explored in detail. In this study, we analyzed all glycosylation sites on synaptotagmin 1, synaptophysin, and SV2A via mutagenesis and optical imaging of pHluorin-tagged proteins in cultured neurons from knock-out mice lacking each protein. Surprisingly, these experiments revealed that glycosylation is completely dispensable for the sorting of synaptotagmin 1 to SVs whereas the N-glycans on SV2A are only partially dispensable. In contrast, N-glycan addition is essential for the synaptic localization and function of synaptophysin. Thus, glycosylation plays distinct roles in the trafficking of each of the three major synaptic vesicle glycoproteins. PMID:22908222

  18. Thioredoxin and its reductase are present on synaptic vesicles, and their inhibition prevents the paralysis induced by botulinum neurotoxins.

    PubMed

    Pirazzini, Marco; Azarnia Tehran, Domenico; Zanetti, Giulia; Megighian, Aram; Scorzeto, Michele; Fillo, Silvia; Shone, Clifford C; Binz, Thomas; Rossetto, Ornella; Lista, Florigio; Montecucco, Cesare

    2014-09-25

    Botulinum neurotoxins consist of a metalloprotease linked via a conserved interchain disulfide bond to a heavy chain responsible for neurospecific binding and translocation of the enzymatic domain in the nerve terminal cytosol. The metalloprotease activity is enabled upon disulfide reduction and causes neuroparalysis by cleaving the SNARE proteins. Here, we show that the thioredoxin reductase-thioredoxin protein disulfide-reducing system is present on synaptic vesicles and that it is functional and responsible for the reduction of the interchain disulfide of botulinum neurotoxin serotypes A, C, and E. Specific inhibitors of thioredoxin reductase or thioredoxin prevent intoxication of cultured neurons in a dose-dependent manner and are also very effective inhibitors of the paralysis of the neuromuscular junction. We found that this group of inhibitors of botulinum neurotoxins is very effective in vivo. Most of them are nontoxic and are good candidates as preventive and therapeutic drugs for human botulism.

  19. A role of amphiphysin in synaptic vesicle endocytosis suggested by its binding to dynamin in nerve terminals.

    PubMed Central

    David, C; McPherson, P S; Mundigl, O; de Camilli, P

    1996-01-01

    Amphiphysin, a major autoantigen in paraneoplastic Stiff-Man syndrome, is an SH3 domain-containing neuronal protein, concentrated in nerve terminals. Here, we demonstrate a specific, SH3 domain-mediated, interaction between amphiphysin and dynamin by gel overlay and affinity chromatography. In addition, we show that the two proteins are colocalized in nerve terminals and are coprecipitated from brain extracts consistent with their interactions in situ. We also report that a region of amphiphysin distinct from its SH3 domain mediates its binding to the alpha c subunit of AP2 adaptin, which is also concentrated in nerve terminals. These findings support a role of amphiphysin in synaptic vesicle endocytosis. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 PMID:8552632

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  2. Distinct actions of strontium on mineral formation in matrix vesicles

    SciTech Connect

    Bechkoff, Geraldine; Radisson, Jacqueline; Bessueille, Laurence; Bouchekioua-Bouzaghou, Katia; Buchet, Rene

    2008-08-29

    Matrix vesicles (MVs) are involved in the initial step of mineralization in skeletal tissues and provide an easily model to analyze the hydroxyapatite (HA) formation. Sr stimulates bone formation and its effect was tested on MVs. Sr{sup 2+} (15-50 {mu}M) in the mineralization medium containing MVs, 2 mM Ca{sup 2+} and 3.42 mM P{sub i}, retarded HA formation. Sr{sup 2+} (1-5 mM) in the same medium-induced other types of mineral than HA and cancelled the ATP-, ADP- or PP{sub i}-induced retardation in the mineral formation. Our findings suggest that the beneficial effect of Sr{sup 2+} at a low dose (15-50 {mu}M) is rather an inhibitor of bone resorption than an activator of mineral formation, while at high Sr{sup 2+} concentration (1-5 mM), mineral formation, especially other types of mineral than HA, is favored.

  3. Unique pH dynamics in GABAergic synaptic vesicles illuminates the mechanism and kinetics of GABA loading

    PubMed Central

    Egashira, Yoshihiro; Takase, Miki; Watanabe, Shoji; Ishida, Junji; Fukamizu, Akiyoshi; Kaneko, Ryosuke; Yanagawa, Yuchio; Takamori, Shigeo

    2016-01-01

    GABA acts as the major inhibitory neurotransmitter in the mammalian brain, shaping neuronal and circuit activity. For sustained synaptic transmission, synaptic vesicles (SVs) are required to be recycled and refilled with neurotransmitters using an H+ electrochemical gradient. However, neither the mechanism underlying vesicular GABA uptake nor the kinetics of GABA loading in living neurons have been fully elucidated. To characterize the process of GABA uptake into SVs in functional synapses, we monitored luminal pH of GABAergic SVs separately from that of excitatory glutamatergic SVs in cultured hippocampal neurons. By using a pH sensor optimal for the SV lumen, we found that GABAergic SVs exhibited an unexpectedly higher resting pH (∼6.4) than glutamatergic SVs (pH ∼5.8). Moreover, unlike glutamatergic SVs, GABAergic SVs displayed unique pH dynamics after endocytosis that involved initial overacidification and subsequent alkalization that restored their resting pH. GABAergic SVs that lacked the vesicular GABA transporter (VGAT) did not show the pH overshoot and acidified further to ∼6.0. Comparison of luminal pH dynamics in the presence or absence of VGAT showed that VGAT operates as a GABA/H+ exchanger, which is continuously required to offset GABA leakage. Furthermore, the kinetics of GABA transport was slower (τ > 20 s at physiological temperature) than that of glutamate uptake and may exceed the time required for reuse of exocytosed SVs, allowing reuse of incompletely filled vesicles in the presence of high demand for inhibitory transmission. PMID:27601664

  4. The effects of JM-20 on the glutamatergic system in synaptic vesicles, synaptosomes and neural cells cultured from rat brain.

    PubMed

    Nuñez-Figueredo, Yanier; Pardo Andreu, Gilberto L; Oliveira Loureiro, Samanta; Ganzella, Marcelo; Ramírez-Sánchez, Jeney; Ochoa-Rodríguez, Estael; Verdecia-Reyes, Yamila; Delgado-Hernández, René; Souza, Diogo O

    2015-02-01

    JM-20 (3-ethoxycarbonyl-2-methyl-4-(2-nitrophenyl)-4,11-dihydro-1H-pyrido[2,3-b][1,5]benzodiazepine) is a novel benzodiazepine dihydropyridine hybrid molecule, which has been shown to be a neuroprotective agent in brain disorders involving glutamate receptors. However, the effect of JM-20 on the functionality of the glutamatergic system has not been investigated. In this study, by using different in vitro preparations, we investigated the effects of JM-20 on (i) rat brain synaptic vesicles (L-[(3)H]-glutamate uptake, proton gradient built-up and bafilomycin-sensitive H(+)-ATPase activity), (ii) rat brain synaptosomes (glutamate release) and (iii) primary cultures of rat cortical neurons, astrocytes and astrocyte-neuron co-cultures (L-[(3)H]-glutamate uptake and glutamate release). We observed here that JM-20 impairs H(+)-ATPase activity and consequently reduces vesicular glutamate uptake. This molecule also inhibits glutamate release from brain synaptosomes and markedly increases glutamate uptake in astrocytes alone, and co-cultured neurons and astrocytes. The impairment of vesicular glutamate uptake by inhibition of the H(+)-ATPase caused by JM-20 could decrease the amount of the transmitter stored in synaptic vesicles, increase the cytosolic levels of glutamate, and will thus down-regulate neurotransmitter release. Together, these results contribute to explain the anti-excitotoxic effect of JM-20 and its strong neuroprotective effect observed in different in vitro and in vivo models of brain ischemia. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Synaptic vesicle exocytosis and increased cytosolic calcium are both necessary but not sufficient for activity-dependent bulk endocytosis.

    PubMed

    Morton, Andrew; Marland, Jamie R K; Cousin, Michael A

    2015-08-01

    Activity-dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle (SV) endocytosis mode in central nerve terminals during intense neuronal activity. By definition this mode is triggered by neuronal activity; however, key questions regarding its mechanism of activation remain unaddressed. To determine the basic requirements for ADBE triggering in central nerve terminals, we decoupled SV fusion events from activity-dependent calcium influx using either clostridial neurotoxins or buffering of intracellular calcium. ADBE was monitored both optically and morphologically by observing uptake of the fluid phase markers tetramethylrhodamine-dextran and horse radish peroxidase respectively. Ablation of SV fusion with tetanus toxin resulted in the arrest of ADBE, but had no effect on other calcium-dependent events such as activity-dependent dynamin I dephosphorylation, indicating that SV exocytosis is necessary for triggering. Furthermore, the calcium chelator EGTA abolished ADBE while leaving SV exocytosis intact, demonstrating that ADBE is triggered by intracellular free calcium increases outside the active zone. Activity-dependent dynamin I dephosphorylation was also arrested in EGTA-treated neurons, consistent with its proposed role in triggering ADBE. Thus, SV fusion and increased cytoplasmic free calcium are both necessary but not sufficient individually to trigger ADBE. Activity-dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle (SV) endocytosis mode in central nerve terminals during intense neuronal activity. To determine the minimal requirements for ADBE triggering, we decoupled SV fusion events from activity-dependent calcium influx using either clostridial neurotoxins or buffering of intracellular calcium. We found that SV fusion and increased cytoplasmic free calcium are both necessary but not sufficient to trigger ADBE.

  6. Synaptotagmin I's Intrinsically Disordered Region Interacts with Synaptic Vesicle Lipids and Exerts Allosteric Control over C2A.

    PubMed

    Fealey, Michael E; Mahling, Ryan; Rice, Anne M; Dunleavy, Katie; Kobany, Stephanie E G; Lohese, K Jean; Horn, Benjamin; Hinderliter, Anne

    2016-05-31

    Synaptotagmin I (Syt I) is a vesicle-localized integral membrane protein that senses the calcium ion (Ca(2+)) influx to trigger fast synchronous release of neurotransmitter. How the cytosolic domains of Syt I allosterically communicate to propagate the Ca(2+) binding signal throughout the protein is not well understood. In particular, it is unclear whether the intrinsically disordered region (IDR) between Syt I's transmembrane helix and first C2 domain (C2A) plays an important role in allosteric modulation of Ca(2+) binding. Moreover, the structural propensity of this IDR with respect to membrane lipid composition is unknown. Using differential scanning and isothermal titration calorimetry, we found that inclusion of the IDR does indeed allosterically modulate Ca(2+) binding within the first C2 domain. Additionally through application of nuclear magnetic resonance, we found that Syt I's IDR interacts with membranes whose lipid composition mimics that of a synaptic vesicle. These findings not only indicate that Syt I's IDR plays a role in regulating Syt I's Ca(2+) sensing but also indicate the IDR is exquisitely sensitive to the underlying membrane lipids. The latter observation suggests the IDR is a key route for communication of lipid organization to the adjacent C2 domains.

  7. The Structure of the Synaptic Vesicle-Plasma Membrane Interface Constrains SNARE Models of Rapid, Synchronous Exocytosis at Nerve Terminals

    PubMed Central

    Gundersen, Cameron B.

    2017-01-01

    Contemporary models of neurotransmitter release invoke direct or indirect interactions between the Ca2+ sensor, synaptotagmin and the incompletely zippered soluble, N-ethyl-maleimide-sensitive factor attachment protein receptor (SNARE) complex. However, recent electron microscopic (EM) investigations have raised pragmatic issues concerning the mechanism by which SNAREs trigger membrane fusion at nerve terminals. The first issue is related to the finding that the area of contact between a “fully primed” synaptic vesicle and the plasma membrane can exceed 600 nm2. Approximately four-thousands lipid molecules can inhabit this contact zone. Thus, renewed efforts will be needed to explain how the zippering of as few as two SNARE complexes mobilizes these lipids to achieve membrane fusion. The second issue emerges from the finding that “docking filaments” are sandwiched within the area of vesicle-plasma membrane contact. It is challenging to reconcile the location of these filaments with SNARE models of exocytosis. Instead, this commentary outlines how these data are more compatible with a model in which a cluster of synaptotagmins catalyzes exocytotic membrane fusion. PMID:28280457

  8. Calmodulin binding proteins of the cholinergic electromotor synapse: synaptosomes, synaptic vesicles, receptor-enriched membranes, and cytoskeleton.

    PubMed

    Walker, J H; Stadler, H; Witzemann, V

    1984-02-01

    Calmodulin binding proteins (CBPs) have been identified using a gel overlay technique for fractions isolated from Torpedo electromotor nerve endings. Different fractions possessed characteristic patterns of CBPs. Synaptosomes showed five major CBPs--Mr 220,000, 160,000, 125,000, 55,000, and 51,000. Polypeptides of Mr 55,000 and 51,000 were found in the cytoplasm and the others are membrane-associated. The Triton X-100-insoluble cytoskeleton of synaptosomes was isolated in the presence or absence of calcium. The major CBPs had Mr of 19,000, 18,000, and 16,000. In the presence of calcium, no other CBPs were seen. In the absence of calcium, an Mr 160,000 polypeptide was present in the Triton cytoskeleton. Synaptic vesicles showed CBPs of Mr 160,000, 25,000, and 20,000. Membrane fragments enriched in acetylcholine receptors contained two major CBPs, Mr 160,000 and 125,000, together with a less prominent protein at Mr 26,000. A protein of Mr similar to that of fodrin was present in synaptosomes and acetylcholine receptor membrane fragments, but only in small amounts relative to the other polypeptides observed. The heavy and light chains of clathrin-coated vesicles from pig brain did not bind calmodulin, although strong labelling of an Mr 47,000 polypeptide was found. Results showed that calelectrin does not bind calmodulin. The possible identity of the calmodulin binding proteins is discussed.

  9. 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

  10. Structural and Genetic Studies Demonstrate Neurologic Dysfunction in Triosephosphate Isomerase Deficiency Is Associated with Impaired Synaptic Vesicle Dynamics

    SciTech Connect

    Roland, Bartholomew P.; Zeccola, Alison M.; Larsen, Samantha B.; Amrich, Christopher G.; Talsma, Aaron D.; Stuchul, Kimberly A.; Heroux, Annie; Levitan, Edwin S.; VanDemark, Andrew P.; Palladino, Michael J.; Pallanck, Leo J.

    2016-03-31

    Triosephosphate isomerase (TPI) deficiency is a poorly understood disease characterized by hemolytic anemia, cardiomyopathy, neurologic dysfunction, and early death. TPI deficiency is one of a group of diseases known as glycolytic enzymopathies, but is unique for its severe patient neuropathology and early mortality. The disease is caused by missense mutations and dysfunction in the glycolytic enzyme, TPI. Previous studies have detailed structural and catalytic changes elicited by disease-associated TPI substitutions, and samples of patient erythrocytes have yielded insight into patient hemolytic anemia; however, the neuropathophysiology of this disease remains a mystery. This study combines structural, biochemical, and genetic approaches to demonstrate that perturbations of the TPI dimer interface are sufficient to elicit TPI deficiency neuropathogenesis. The present study demonstrates that neurologic dysfunction resulting from TPI deficiency is characterized by synaptic vesicle dysfunction, and can be attenuated with catalytically inactive TPI. Collectively, our findings are the first to identify, to our knowledge, a functional synaptic defect in TPI deficiency derived from molecular changes in the TPI dimer interface.

  11. Structural and Genetic Studies Demonstrate Neurologic Dysfunction in Triosephosphate Isomerase Deficiency Is Associated with Impaired Synaptic Vesicle Dynamics

    SciTech Connect

    Roland, Bartholomew P.; Zeccola, Alison M.; Larsen, Samantha B.; Amrich, Christopher G.; Talsma, Aaron D.; Stuchul, Kimberly A.; Heroux, Annie; VanDemark, Andrew P.; Palladino, Michael J.

    2016-03-31

    Triosephosphate isomerase (TPI) deficiency is a poorly understood disease characterized by hemolytic anemia, cardiomyopathy, neurologic dysfunction, and early death. TPI deficiency is one of a group of diseases known as glycolytic enzymopathies, but is unique for its severe patient neuropathology and early mortality. The disease is caused by missense mutations and dysfunction in the glycolytic enzyme, TPI. Previous studies have detailed structural and catalytic changes elicited by disease-associated TPI substitutions, and samples of patient erythrocytes have yielded insight into patient hemolytic anemia; however, the neuropathophysiology of this disease remains a mystery. This study combines structural, biochemical, and genetic approaches to demonstrate that perturbations of the TPI dimer interface are sufficient to elicit TPI deficiency neuropathogenesis. Also, the present study demonstrates that neurologic dysfunction resulting from TPI deficiency is characterized by synaptic vesicle dysfunction, and can be attenuated with catalytically inactive TPI. Collectively, our findings are the first to identify, to our knowledge, a functional synaptic defect in TPI deficiency derived from molecular changes in the TPI dimer interface.

  12. 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.

  13. 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.

  14. Structural and Genetic Studies Demonstrate Neurologic Dysfunction in Triosephosphate Isomerase Deficiency Is Associated with Impaired Synaptic Vesicle Dynamics

    DOE PAGES

    Roland, Bartholomew P.; Zeccola, Alison M.; Larsen, Samantha B.; ...

    2016-03-31

    Triosephosphate isomerase (TPI) deficiency is a poorly understood disease characterized by hemolytic anemia, cardiomyopathy, neurologic dysfunction, and early death. TPI deficiency is one of a group of diseases known as glycolytic enzymopathies, but is unique for its severe patient neuropathology and early mortality. The disease is caused by missense mutations and dysfunction in the glycolytic enzyme, TPI. Previous studies have detailed structural and catalytic changes elicited by disease-associated TPI substitutions, and samples of patient erythrocytes have yielded insight into patient hemolytic anemia; however, the neuropathophysiology of this disease remains a mystery. This study combines structural, biochemical, and genetic approaches tomore » demonstrate that perturbations of the TPI dimer interface are sufficient to elicit TPI deficiency neuropathogenesis. Also, the present study demonstrates that neurologic dysfunction resulting from TPI deficiency is characterized by synaptic vesicle dysfunction, and can be attenuated with catalytically inactive TPI. Collectively, our findings are the first to identify, to our knowledge, a functional synaptic defect in TPI deficiency derived from molecular changes in the TPI dimer interface.« less

  15. The Human Synaptic Vesicle Protein, SV2A, Functions as a Galactose Transporter in Saccharomyces cerevisiae * ♦

    PubMed Central

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

    2014-01-01

    SV2A is a synaptic vesicle membrane protein expressed in neurons and endocrine cells and involved in the regulation of neurotransmitter release. Although the exact function of SV2A still remains elusive, it was identified as the specific binding site for levetiracetam, a second generation antiepileptic drug. Our sequence analysis demonstrates that SV2A has significant homology with several yeast transport proteins belonging to the major facilitator superfamily (MFS). Many of these transporters are involved in sugar transport into yeast cells. Here we present evidence showing, for the first time, that SV2A is a galactose transporter. We expressed human SV2A in hexose transport-deficient EBY.VW4000 yeast cells and demonstrated that these cells are able to grow on galactose-containing medium but not on other fermentable carbon sources. Furthermore, the addition of the SV2A-binding antiepileptic drug levetiracetam to the medium inhibited the galactose-dependent growth of hexose transport-deficient EBY.VW4000 yeast cells expressing human SV2A. Most importantly, direct measurement of galactose uptake in the same strain verified that SV2A is able to transport extracellular galactose inside the cells. The newly identified galactose transport capability of SV2A may have an important role in regulating/modulating synaptic function. PMID:25326386

  16. Structural and Genetic Studies Demonstrate Neurologic Dysfunction in Triosephosphate Isomerase Deficiency Is Associated with Impaired Synaptic Vesicle Dynamics

    PubMed Central

    Roland, Bartholomew P.; Zeccola, Alison M.; Larsen, Samantha B.; Amrich, Christopher G.; Talsma, Aaron D.; Stuchul, Kimberly A.; Heroux, Annie; Levitan, Edwin S.; VanDemark, Andrew P.; Palladino, Michael J.

    2016-01-01

    Triosephosphate isomerase (TPI) deficiency is a poorly understood disease characterized by hemolytic anemia, cardiomyopathy, neurologic dysfunction, and early death. TPI deficiency is one of a group of diseases known as glycolytic enzymopathies, but is unique for its severe patient neuropathology and early mortality. The disease is caused by missense mutations and dysfunction in the glycolytic enzyme, TPI. Previous studies have detailed structural and catalytic changes elicited by disease-associated TPI substitutions, and samples of patient erythrocytes have yielded insight into patient hemolytic anemia; however, the neuropathophysiology of this disease remains a mystery. This study combines structural, biochemical, and genetic approaches to demonstrate that perturbations of the TPI dimer interface are sufficient to elicit TPI deficiency neuropathogenesis. The present study demonstrates that neurologic dysfunction resulting from TPI deficiency is characterized by synaptic vesicle dysfunction, and can be attenuated with catalytically inactive TPI. Collectively, our findings are the first to identify, to our knowledge, a functional synaptic defect in TPI deficiency derived from molecular changes in the TPI dimer interface. PMID:27031109

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

    PubMed

    Elhenawy, Wael; Bording-Jorgensen, Michael; Valguarnera, Ezequiel; Haurat, M Florencia; Wine, Eytan; Feldman, Mario F

    2016-07-12

    Outer membrane vesicles (OMV) are proposed to mediate multiple functions during pathogenesis and symbiosis. However, the mechanisms responsible for OMV formation remain poorly understood. It has been shown in eukaryotic membranes that lipids with an inverted-cone shape favor the formation of positive membrane curvatures. Based on these studies, we formulated the hypothesis that lipid A deacylation might impose shape modifications that result in the curvature of the outer membrane (OM) and subsequent OMV formation. We tested the effect of lipid A remodeling on OMV biogenesis employing Salmonella enterica serovar Typhimurium as a model organism. Expression of the lipid A deacylase PagL resulted in increased vesiculation, without inducing an envelope stress response. Mass spectrometry analysis revealed profound differences in the patterns of lipid A in OM and OMV, with accumulation of deacylated lipid A forms exclusively in OMV. OMV biogenesis by intracellular bacteria upon macrophage infection was drastically reduced in a pagL mutant strain. We propose a novel mechanism for OMV biogenesis requiring lipid A deacylation in the context of a multifactorial process that involves the orchestrated remodeling of the outer membrane. The role of lipid remodeling in vesiculation is well documented in eukaryotes. Similarly, bacteria produce membrane-derived vesicles; however, the molecular mechanisms underlying their production are yet to be determined. In this work, we investigated the role of outer membrane remodeling in OMV biogenesis in S Typhimurium. We showed that the expression of the lipid A deacylase PagL results in overvesiculation with deacylated lipid A accumulation exclusively in OMV. An S Typhimurium ΔpagL strain showed a significant reduction in intracellular OMV secretion relative to the wild-type strain. Our results suggest a novel mechanism for OMV biogenesis that involves outer membrane remodeling through lipid A modification. Understanding how OMV are

  18. 3D analysis of synaptic vesicle density and distribution after acute foot-shock stress by using serial section transmission electron microscopy.

    PubMed

    Khanmohammadi, M; Darkner, S; Nava, N; Nyengaard, J R; Wegener, G; Popoli, M; Sporring, J

    2017-01-01

    Behavioural stress has shown to strongly affect neurotransmission within the neocortex. In this study, we analysed the effect of an acute stress model on density and distribution of neurotransmitter-containing vesicles within medial prefrontal cortex. Serial section transmission electron microscopy was employed to compare two groups of male rats: (1) rats subjected to foot-shock stress and (2) rats with sham stress as control group. Two-dimensional (2D) density measures are common in microscopic images and are estimated by following a 2D path in-section. However, this method ignores the slant of the active zone and thickness of the section. In fact, the active zone is a surface in three-dimension (3D) and the 2D measures do not accurately reflect the geometric configuration unless the active zone is perpendicular to the sectioning angle. We investigated synaptic vesicle density as a function of distance from the active zone in 3D. We reconstructed a 3D dataset by estimating the thickness of all sections and by registering all the image sections into a common coordinate system. Finally, we estimated the density as the average number of vesicles per area and volume and modelled the synaptic vesicle distribution by fitting a one-dimensional parametrized distribution that took into account the location uncertainty due to section thickness. Our results showed a clear structural difference in synaptic vesicle density and distribution between stressed and control group with improved separation by 3D measures in comparison to the 2D measures. Our results showed that acute foot-shock stress exposure significantly affected both the spatial distribution and density of the synaptic vesicles within the presynaptic terminal.

  19. Effect of K+ and Na+ on calcium-dependent electron-dense particles in the monoaminergic synaptic vesicles of rat pineal nerves fixed in Ca2+-containing solutions.

    PubMed

    Pellegrino de Iraldi, A; Corazza, J P

    1983-01-01

    The effect of K+ and Na+ on the Ca2+ binding site in the dense core of monoaminergic vesicles of pineal nerves was investigated in the rat. Rat pineal glands, bisected immediately after decapitation, were incubated at room temperature in solutions containing high K+ or high Na+ in the presence or absence of Ca2+. Fixation was performed in glutaraldehyde-osmium tetroxide in collidine buffer, with and without CaCl2. It was confirmed that, after fixation in Ca2+-containing solutions, an electron-dense particle, located in the vesicle core, which can be considered a calcium deposit, appears within the synaptic vesicles. It was observed that this Ca2+ deposit may be modified by incubation in a high K+ or high Na+ milieu before fixation in Ca2+ containing solutions. When the incubation was carried out with high K+ and high Ca2+ simultaneously, Ca2+ deposits were considerably increased. With K+ alone, no Ca2+ deposits were apparent, as when electrical stimulation is applied before fixation. This effect was not observed when the incubation was done in high Na+. Consecutive incubations in high K+ and high Na+, respectively, restored the capability of the vesicle cores to bind Ca2+. Prolonged incubation in high Na+ before fixation increased Ca2+ deposits within the vesicles. These findings are in line with data on the effect of these ions upon the storage and release of biogenic amines and suggest that these ions modify the capability of synaptic vesicles to bind Ca2+.

  20. Myotonic dystrophy CTG expansion affects synaptic vesicle proteins, neurotransmission and mouse behaviour

    PubMed Central

    Hernández-Hernández, Oscar; Guiraud-Dogan, Céline; Sicot, Géraldine; Huguet, Aline; Luilier, Sabrina; Steidl, Esther; Saenger, Stefanie; Marciniak, Elodie; Obriot, Hélène; Chevarin, Caroline; Nicole, Annie; Revillod, Lucile; Charizanis, Konstantinos; Lee, Kuang-Yung; Suzuki, Yasuhiro; Kimura, Takashi; Matsuura, Tohru; Cisneros, Bulmaro; Swanson, Maurice S.; Trovero, Fabrice; Buisson, Bruno; Bizot, Jean-Charles; Hamon, Michel; Humez, Sandrine; Bassez, Guillaume; Metzger, Friedrich; Buée, Luc; Munnich, Arnold; Sergeant, Nicolas; Gourdon, Geneviève

    2013-01-01

    Myotonic dystrophy type 1 is a complex multisystemic inherited disorder, which displays multiple debilitating neurological manifestations. Despite recent progress in the understanding of the molecular pathogenesis of myotonic dystrophy type 1 in skeletal muscle and heart, the pathways affected in the central nervous system are largely unknown. To address this question, we studied the only transgenic mouse line expressing CTG trinucleotide repeats in the central nervous system. These mice recreate molecular features of RNA toxicity, such as RNA foci accumulation and missplicing. They exhibit relevant behavioural and cognitive phenotypes, deficits in short-term synaptic plasticity, as well as changes in neurochemical levels. In the search for disease intermediates affected by disease mutation, a global proteomics approach revealed RAB3A upregulation and synapsin I hyperphosphorylation in the central nervous system of transgenic mice, transfected cells and post-mortem brains of patients with myotonic dystrophy type 1. These protein defects were associated with electrophysiological and behavioural deficits in mice and altered spontaneous neurosecretion in cell culture. Taking advantage of a relevant transgenic mouse of a complex human disease, we found a novel connection between physiological phenotypes and synaptic protein dysregulation, indicative of synaptic dysfunction in myotonic dystrophy type 1 brain pathology. PMID:23404338

  1. Myotonic dystrophy CTG expansion affects synaptic vesicle proteins, neurotransmission and mouse behaviour.

    PubMed

    Hernández-Hernández, Oscar; Guiraud-Dogan, Céline; Sicot, Géraldine; Huguet, Aline; Luilier, Sabrina; Steidl, Esther; Saenger, Stefanie; Marciniak, Elodie; Obriot, Hélène; Chevarin, Caroline; Nicole, Annie; Revillod, Lucile; Charizanis, Konstantinos; Lee, Kuang-Yung; Suzuki, Yasuhiro; Kimura, Takashi; Matsuura, Tohru; Cisneros, Bulmaro; Swanson, Maurice S; Trovero, Fabrice; Buisson, Bruno; Bizot, Jean-Charles; Hamon, Michel; Humez, Sandrine; Bassez, Guillaume; Metzger, Friedrich; Buée, Luc; Munnich, Arnold; Sergeant, Nicolas; Gourdon, Geneviève; Gomes-Pereira, Mário

    2013-03-01

    Myotonic dystrophy type 1 is a complex multisystemic inherited disorder, which displays multiple debilitating neurological manifestations. Despite recent progress in the understanding of the molecular pathogenesis of myotonic dystrophy type 1 in skeletal muscle and heart, the pathways affected in the central nervous system are largely unknown. To address this question, we studied the only transgenic mouse line expressing CTG trinucleotide repeats in the central nervous system. These mice recreate molecular features of RNA toxicity, such as RNA foci accumulation and missplicing. They exhibit relevant behavioural and cognitive phenotypes, deficits in short-term synaptic plasticity, as well as changes in neurochemical levels. In the search for disease intermediates affected by disease mutation, a global proteomics approach revealed RAB3A upregulation and synapsin I hyperphosphorylation in the central nervous system of transgenic mice, transfected cells and post-mortem brains of patients with myotonic dystrophy type 1. These protein defects were associated with electrophysiological and behavioural deficits in mice and altered spontaneous neurosecretion in cell culture. Taking advantage of a relevant transgenic mouse of a complex human disease, we found a novel connection between physiological phenotypes and synaptic protein dysregulation, indicative of synaptic dysfunction in myotonic dystrophy type 1 brain pathology.

  2. Mathematical modeling of vesicle drug delivery systems 1: vesicle formation and stability along with drug loading and release.

    PubMed

    Mosley, Garrett L; Yamanishi, Cameron D; Kamei, Daniel T

    2013-02-01

    Vesicles represent an important class of nanoscale drug delivery vehicles. To significantly reduce the time and resources that are required to optimize these drug carriers, this review article discusses the mathematical models that have been derived for understanding the formation of vesicles and their stability, as well as for predicting drug loading and their release. With regard to vesicle formation and stability, the packing parameter can be used to predict how the solution environment, surfactant composition, and surfactant molecular architecture can influence the supermolecular self-assembled structures that are formed from amphiphiles. In the context of drug delivery, this is useful for facilitating vesicle formation and stability during transit through the body. At the target site, this information can be used to help trigger a rapid release of the drug. With regard to drug loading, kinetic and equilibrium models provide guidelines for appropriate pH conditions and drug incubation times during loading. The diffusivity, partition coefficient, and bilayer thickness also play significant roles during loading and release of the drug. Our hope is that more researchers in this exciting field will complement their experimental approaches with these mathematical models to more efficiently develop vesicle-based drug carriers.

  3. Mutations in the major gas vesicle protein GvpA and impacts on gas vesicle formation in Haloferax volcanii.

    PubMed

    Knitsch, Regine; Schneefeld, Marie; Weitzel, Kerstin; Pfeifer, Felicitas

    2017-09-12

    Gas vesicles are proteinaceous, gas-filled nanostructures produced by some bacteria and archaea. The hydrophobic major structural protein GvpA forms the ribbed gas vesicle wall. An in-silico 3D-model of GvpA of the predicted coil-α1-β1-β2-α2-coil structure is available and implies that the two β-chains constitute the hydrophobic interior surface of the gas vesicle wall. To test the importance of individual amino acids in GvpA we performed 85 single substitutions and analyzed these variants in Haloferax volcanii ΔA + Amut transformants for their ability to form gas vesicles (Vac(+) phenotype). In most cases, an alanine substitution of a non-polar residue did not abolish gas vesicle formation, but the replacement of single non-polar by charged residues in β1 or β2 resulted in Vac(-) transformants. A replacement of residues near the β-turn altered the spindle-shape to a cylindrical morphology of the gas vesicles. Vac(-) transformants were also obtained with alanine substitutions of charged residues of helix α1 suggesting that these amino acids form salt-bridges with another GvpA monomer. In helix α2, only the alanine substitution of His53 or Tyr54, led to Vac(-) transformants, whereas most other substitutions had no effect. We discuss our results in respect to the GvpA structure and data available from solid-state NMR. © 2017 John Wiley & Sons Ltd.

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

    PubMed Central

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

    2016-01-01

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

  5. Inhibition of Calpains Protects Mn-Induced Neurotransmitter release disorders in Synaptosomes from Mice: Involvement of SNARE Complex and Synaptic Vesicle Fusion.

    PubMed

    Wang, Can; Xu, Bin; Ma, Zhuo; Liu, Chang; Deng, Yu; Liu, Wei; Xu, Zhao-Fa

    2017-06-16

    Overexposure to manganese (Mn) could disrupt neurotransmitter release via influencing the formation of SNARE complex, but the underlying mechanisms are still unclear. A previous study demonstrated that SNAP-25 is one of substrate of calpains. The current study investigated whether calpains were involved in Mn-induced disorder of SNARE complex. After mice were treated with Mn for 24 days, Mn deposition increased significantly in basal nuclei in Mn-treated and calpeptin pre-treated groups. Behaviorally, less time spent in the center of the area and decreased average velocity significantly in an open field test after 24 days of Mn exposure. With the increase in MnCl2 dosage, intracellular Ca(2+) increased significantly, but pretreatment with calpeptin caused a dose-dependent decrease in calpains activity. There were fragments of N-terminal of SNAP-25 protein appearance in Mn-treated groups, but it is decreased with pretreatment of calpeptin. FM1-43-labeled synaptic vesicles also provided evidence that the treatment with Mn resulted in increasing first and then decreasing, which was consistent with Glu release and the 80 kDa protein levels of SNARE complexes. In summary, Mn induced the disorder of neurotransmitter release through influencing the formation of SNARE complex via cleaving SNAP-25 by overactivation of calpains in vivo.

  6. Inhibitory synapse dynamics: coordinated presynaptic and postsynaptic mobility and the major contribution of recycled vesicles to new synapse formation.

    PubMed

    Dobie, Frederick A; Craig, Ann Marie

    2011-07-20

    Dynamics of GABAergic synaptic components have been studied previously over milliseconds to minutes, revealing mobility of postsynaptic scaffolds and receptors. Here we image inhibitory synapses containing fluorescently tagged postsynaptic scaffold Gephyrin, together with presynaptic vesicular GABA transporter (VGAT) or postsynaptic GABA(A) receptor γ2 subunit (GABA(A)Rγ2), over seconds to days in cultured rat hippocampal neurons, revealing modes of inhibitory synapse formation and remodeling. Entire synapses were mobile, translocating rapidly within a confined region and exhibiting greater nonstochastic motion over multihour periods. Presynaptic and postsynaptic components moved in unison, maintaining close apposition while translocating distances of several micrometers. An observed flux in the density of synaptic puncta partially resulted from the apparent merging and splitting of preexisting clusters. De novo formation of inhibitory synapses was observed, marked by the appearance of stably apposed Gephyrin and VGAT clusters at sites previously lacking either component. Coclustering of GABA(A)Rγ2 supports the identification of such new clusters as synapses. Nascent synapse formation occurred by gradual accumulation of components over several hours, with VGAT clustering preceding that of Gephyrin and GABA(A)Rγ2. Comparing VGAT labeling by active uptake of a luminal domain antibody with post hoc immunocytochemistry indicated that recycling vesicles from preexisting boutons significantly contribute to vesicle pools at the majority of new inhibitory synapses. Although new synapses formed primarily on dendrite shafts, some also formed on dendritic protrusions, without apparent interconversion. Altogether, the long-term imaging of GABAergic presynaptic and postsynaptic components reveals complex dynamics and perpetual remodeling with implications for mechanisms of assembly and synaptic integration.

  7. Structure formation in binary mixtures of lipids and detergents: self-assembly and vesicle division.

    PubMed

    Noguchi, Hiroshi

    2013-01-14

    Self-assembly dynamics in binary surfactant mixtures and structure changes of lipid vesicles induced by detergent solution are studied using coarse-grained molecular simulations. Disk-shaped micelles, the bicelles, are stabilized by detergents surrounding the rim of a bilayer disk of lipids. The self-assembled bicelles are considerably smaller than bicelles formed from vesicle rupture, and their size is determined by the concentrations of lipids and detergents and the interactions between the two species. The detergent-adsorption induces spontaneous curvature of the vesicle bilayer and results in vesicle division into two vesicles or vesicle rupture into worm-like micelles. The division occurs mainly via the inverse pathway of the modified stalk model. For large spontaneous curvature of the monolayers of the detergents, a pore is often opened, thereby leading to vesicle division or worm-like micelle formation.

  8. Structure formation in binary mixtures of lipids and detergents: Self-assembly and vesicle division

    NASA Astrophysics Data System (ADS)

    Noguchi, Hiroshi

    2013-01-01

    Self-assembly dynamics in binary surfactant mixtures and structure changes of lipid vesicles induced by detergent solution are studied using coarse-grained molecular simulations. Disk-shaped micelles, the bicelles, are stabilized by detergents surrounding the rim of a bilayer disk of lipids. The self-assembled bicelles are considerably smaller than bicelles formed from vesicle rupture, and their size is determined by the concentrations of lipids and detergents and the interactions between the two species. The detergent-adsorption induces spontaneous curvature of the vesicle bilayer and results in vesicle division into two vesicles or vesicle rupture into worm-like micelles. The division occurs mainly via the inverse pathway of the modified stalk model. For large spontaneous curvature of the monolayers of the detergents, a pore is often opened, thereby leading to vesicle division or worm-like micelle formation.

  9. The Effects of Concentration and Temperature on Vesicle Adsorption and Bilayer Formation

    NASA Astrophysics Data System (ADS)

    Weirich, Kimberly; Israelachvili, Jacob; Fygenson, Deborah

    2010-03-01

    Supported lipid bilayers (SLBs) are pursued as thin surface coatings and as model systems in which to study membrane-bound processes. We investigate the adsorption of small unilamellar phospholipid vesicles onto glass and the subsequent formation of planar SLBs using temperature-controlled, time-resolved fluorescence microscopy. We report the effects of vesicle concentration and temperature on the time course of lipid adsorption. Our results suggest that isolated vesicle rupture is a rare event and that bilayer edge plays a key role in SLB formation. It enhances vesicle-surface affinity and promotes further rupture.

  10. Dendritic spine formation and synaptic function require neurobeachin

    PubMed Central

    Niesmann, Katharina; Breuer, Dorothee; Brockhaus, Johannes; Born, Gesche; Wolff, Ilka; Reissner, Carsten; Kilimann, Manfred W.; Rohlmann, Astrid; Missler, Markus

    2011-01-01

    A challenge in neuroscience is to understand the mechanisms underlying synapse formation. Most excitatory synapses in the brain are built on spines, which are actin-rich protrusions from dendrites. Spines are a major substrate of brain plasticity, and spine pathologies are observed in various mental illnesses. Here we investigate the role of neurobeachin (Nbea), a multidomain protein previously linked to cases of autism, in synaptogenesis. We show that deletion of Nbea leads to reduced numbers of spinous synapses in cultured neurons from complete knockouts and in cortical tissue from heterozygous mice, accompanied by altered miniature postsynaptic currents. In addition, excitatory synapses terminate mostly at dendritic shafts instead of spine heads in Nbea mutants, and actin becomes less enriched synaptically. As actin and synaptopodin, a spine-associated protein with actin-bundling activity, accumulate ectopically near the Golgi apparatus of mutant neurons, a role emerges for Nbea in trafficking important cargo to pre- and postsynaptic compartments. PMID:22109531

  11. Prebiotic Vesicle Formation and the Necessity of Salts

    NASA Astrophysics Data System (ADS)

    Maurer, Sarah E.; Nguyen, Gunarso

    2016-06-01

    Self-assembly is considered one of the driving forces behind abiogenesis and would have been affected by the environmental conditions of early Earth. The formation of membranes is a key step in this process, and unlike large dialkyl membranes of modern cells the first membranes were likely formed from small single-chain amphiphiles, which are environment-sensitive. Fatty acids and their derivatives have been previously characterized in this role without concern for the concentrations of ionic solutes in the suspension. We determined the critical vesicle concentration (CVC) for three single-chain amphiphiles with increasing concentrations of NaCl. All amphiphile species had decreasing CVCs correlated to increasing NaCl concentrations. Decanoic acid and oleic acid were impacted more strongly than monoacylglycerol, likely because of electric shielding of the negatively charged headgroups in the presence of salt. There was no impact on the salt species as 100 mM NaBr, NaCl, and KCl all exhibited the same effect on CVC. This research shows the importance of salt in both the formation of life and in experimental design for aggregation experiments.

  12. Milk extracellular vesicles accelerate osteoblastogenesis but impair bone matrix formation.

    PubMed

    Oliveira, Marina C; Arntz, Onno J; Blaney Davidson, Esmeralda N; van Lent, Peter L E M; Koenders, Marije I; van der Kraan, Peter M; van den Berg, Wim B; Ferreira, Adaliene V M; van de Loo, Fons A J

    2016-04-01

    The claimed beneficial effect of milk on bone is still a matter for debate. Recently extracellular vesicles (EVs) that contain proteins and RNA were discovered in milk, but their effect on bone formation has not yet been determined. We demonstrated previously that bovine milk-derived EVs (BMEVs) have immunoregulatory properties. Our aim was to evaluate the effect of BMEVs on osteogenesis by mice and human mesenchymal stem cells (hMSCs). Oral delivery of two concentrations of BMEVs to female DBA/1J mice during 7weeks did not alter the tibia trabecular bone area; however, the osteocytes number increased. In addition, the highest dose of BMEVs markedly increased the woven bone tissue, which is more brittle. The exposure of hMSCs to BMEVs during 21days resulted in less mineralization but higher cell proliferation. Interestingly BMEVs reduced the collagen production, but enhanced the expression of genes characteristic for immature osteoblasts. A kinetic study showed that BMEVs up-regulated many osteogenic genes within the first 4days. However, the production of type I collagen and expression of its genes (COL1A1 and COL1A2) were markedly reduced at days 21 and 28. At day 28, BMEVs again lead to higher proliferation, but mineralization was significantly increased. This was associated with increased expression of sclerostin, a marker for osteocytes, and reduced osteonectin, which is associated to bone matrix formation. Our study adds BMEVs to the list of milk components that can affect bone formation and may shed new light on the contradictory claims of milk on bone formation. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Effect of interlamellar interactions on shear induced multilamellar vesicle formation

    NASA Astrophysics Data System (ADS)

    Kawabata, Y.; Bradbury, R.; Kugizaki, S.; Weigandt, K.; Melnichenko, Y. B.; Sadakane, K.; Yamada, N. L.; Endo, H.; Nagao, M.; Seto, H.

    2017-07-01

    Shear-induced multilamellar vesicle (MLV) formation has been studied by coupling the small-angle neutron scattering (SANS) technique with neutron spin echo (NSE) spectroscopy. A 10% mass fraction of the nonionic surfactant pentaethylene glycol dodecyl ether (C12E5) in water was selected as a model system for studying weak inter-lamellar interactions. These interactions are controlled either by adding an anionic surfactant, sodium dodecyl sulfate, or an antagonistic salt, rubidium tetraphenylborate. Increasing the charge density in the bilayer induces an enhanced ordering of the lamellar structure. The charge density dependence of the membrane bending modulus was determined by NSE and showed an increasing trend with charge. This behavior is well explained by a classical theoretical model. By considering the Caillé parameters calculated from the SANS data, the layer compressibility modulus B ¯ is estimated and the nature of the dominant inter-lamellar interaction is determined. Shear flow induces MLV formation around a shear rate of 10 s-1, when a small amount of charge is included in the membrane. The flow-induced layer undulations are in-phase between neighboring layers when the inter-lamellar interaction is sufficiently strong. Under these conditions, MLV formation can occur without significantly changing the inter-lamellar spacing. On the other hand, in the case of weak inter-lamellar interactions, the flow-induced undulations are not in-phase, and greater steric repulsion leads to an increase in the inter-lamellar spacing with shear rate. In this case, MLV formation occurs as the amplitude of the undulations gets larger and the steric interaction leads to in-phase undulations between neighboring membranes.

  14. Glomerular Podocytes Possess the Synaptic Vesicle Molecule Rab3A and Its Specific Effector Rabphilin-3a

    PubMed Central

    Rastaldi, Maria Pia; Armelloni, Silvia; Berra, Silvia; Li, Min; Pesaresi, Marzia; Poczewski, Helga; Langer, Brigitte; Kerjaschki, Dontscho; Henger, Anna; Blattner, Simone Monika; Kretzler, Matthias; Wanke, Rudiger; D’Amico, Giuseppe

    2003-01-01

    Several recent studies have focused on similarities between glomerular podocytes and neurons because the two cells share a specialized cytoskeletal organization and several expression-restricted proteins, such as nephrin and synaptopodin. In neurons, the small guanosine triphosphatase Rab3A and its effector rabphilin-3A form a complex required for the correct docking of synaptic vesicles to their target membrane. Because rabphilin-3A binds in neurons to cytoskeletal proteins also important for podocyte homeostasis, and the complex rabphilin-3A-Rab3A has been demonstrated in neurons and neuroendocrine cells, the aim of our work was to investigate their possible expression and regulation in podocytes. Normal kidneys from mouse, rat, and human were studied by immunohistochemistry, Western blotting, and reverse transcriptase-polymerase chain reaction to evaluate the expression of Rab3A and rabphilin-3A. Double-staining immunohistochemistry and immunogold electron microscopy were then used to precisely localize the two proteins at the cellular and subcellular levels. Rab-3A and rabphilin-3A regulations in disease were then analyzed in growth hormone-transgenic mice, a well established model of focal and segmental glomerulosclerosis, and in human biopsies from proteinuric patients. Our results demonstrated that rabphilin-3A and Rab3A are present in normal mouse, rat, and human kidneys, with an exclusively glomerular expression and a comma-like pattern of positivity along the glomerular capillary wall, suggestive for podocyte staining. Co-localization of both molecules with synaptopodin confirmed their presence in podocytes. By immunogold electron microscopy both proteins were found around vesicles contained in podocyte foot processes. Their expression was increased in growth hormone-transgenic mice compared to their wild-type counterpart, and in a subset of biopsies from proteinuric patients. Our data, demonstrating the presence of two synaptic proteins in podocytes

  15. Formation of Kinetically Trapped Nanoscopic Unilamellar Vesicles from Metastable Nanodiscs

    SciTech Connect

    Nieh, Mu-Ping; Dolinar, Paul; Kucerka, Norbert; Kline, Steven R.; Debeer-Schmitt, Lisa M.; Littrell, Kenneth C.; Katsaras, John

    2011-09-27

    Zwitterionic long-chain lipids (e.g., dimyristoyl phosphatidylcholine, DMPC) spontaneously form onion-like, thermodynamically stable structures in aqueous solutions (commonly known as multilamellar vesicles, or MLVs). It has also been reported that the addition of zwitterionic short-chain (i.e., dihexanoyl phosphatidylcholine, DHPC) and charged long-chain (i.e., dimyristoyl phosphatidylglycerol, DMPG) lipids to zwitterionic long-chain lipid solutions results in the formation of unilamellar vesicles (ULVs). Here, we report a kinetic study on lipid mixtures composed of DMPC, DHPC, and DMPG. Two membrane charge densities (i.e., [DMPG]/[DMPC] = 0.01 and 0.001) and two solution salinities (i.e., [NaCl] = 0 and 0.2 M) are investigated. Upon dilution of the high-concentration samples at 50 °C, thermodynamically stable MLVs are formed, in the case of both weakly charged and high salinity solution mixtures, implying that the electrostatic interactions between bilayers are insufficient to cause MLVs to unbind. Importantly, in the case of these samples small angle neutron scattering (SANS) data show that, initially, nanodiscs (also known as bicelles) or bilayered ribbons form at low temperatures (i.e., 10 °C), but transform into uniform size, nanoscopic ULVs after incubation at 10 °C for 20 h, indicating that the nanodisc is a metastable structure. The instability of nanodiscs may be attributed to low membrane rigidity due to a reduced charge density and high salinity. Moreover, the uniform-sized ULVs persist even after being heated to 50 °C, where thermodynamically stable MLVs are observed. This result clearly demonstrates that these ULVs are kinetically trapped, and that the mechanical properties (e.g., bending rigidity) of 10 C nanodiscs favor the formation of nanoscopic ULVs over that of MLVs. From a practical point of view, this method of forming uniform-sized ULVs may lend itself to their mass production, thus making them economically feasible for medical

  16. Binding characteristics of levetiracetam to synaptic vesicle protein 2A (SV2A) in human brain and in CHO cells expressing the human recombinant protein.

    PubMed

    Gillard, Michel; Chatelain, Pierre; Fuks, Bruno

    2006-04-24

    A specific binding site for the antiepileptic drug levetiracetam (2S-(oxo-1-pyrrolidinyl)butanamide, Keppra) in rat brain, referred to as the levetiracetam binding site, was discovered several years ago. More recently, this binding site has been identified as the synaptic vesicle protein 2A (SV2A), a protein present in synaptic vesicles [Lynch, B., Lambeng, N., Nocka, K., Kensel-Hammes, P., Bajjalieh, S.M., Matagne, A., Fuks, B., 2004. The synaptic vesicle protein SV2A is the binding site for the antiepileptic drug levetiracetam. Proc. Natl. Acad. Sci. USA, 101, 9861-9866.]. In this study, we characterized the binding properties of levetiracetam in post-mortem human brain and compared them to human SV2A expressed in Chinese hamster ovary (CHO) cells. The results showed that the binding properties of levetiracetam and [3H]ucb 30889, an analogue that was previously characterized as a suitable ligand for levetiracetam binding site/SV2A in rat brain [Gillard, M., Fuks, B., Michel, P., Vertongen, P., Massingham, R. Chatelain, P., 2003. Binding characteristics of [3H]ucb 30889 to levetiracetam binding sites in rat brain. Eur. J. Pharmacol. 478, 1-9.], are almost identical in human brain samples (cerebral cortex, hippocampus and cerebellum) and in CHO cell membranes expressing the human SV2A protein. Moreover, the results are also similar to those previously obtained in rat brain. [3H]ucb 30889 binding in human brain and to SV2A was saturable and reversible. At 4 degrees C, its binding kinetics were best fitted assuming a two-phase model in all tissues. The half-times of association for the fast component ranged between 1 to 2 min and represent 30% to 36% of the sites whereas the half-times for the slow component ranged from 20 to 29 min. In dissociation experiments, the half-times were from 2 to 4 min for the fast component (33% to 49% of the sites) and 20 to 41 min for the slow component. Saturation binding curves led to Kd values for [3H]ucb 30889 of 53+/-7, 55+/-9, 70

  17. Synaptic efficacy cluster formation across the dendrite via STDP.

    PubMed

    Iannella, Nicolangelo; Tanaka, Shigeru

    2006-07-31

    The role of spike-timing-dependent plasticity (STDP) in shaping the strength of a synapse located on the dendritic tree has gained recent interest. Previous theoretical studies using STDP have mostly used simplified integrate-and-fire models to investigate the evolution of synaptic efficacy with time. Such studies usually show that the final weight distribution is unimodal or bimodal resulting from a multiplicative or additive STDP rule, respectively. However, very little is known about how STDP shapes the spatial organization of synaptic efficacies. Here, for the first time, we demonstrate that spatial clustering of synaptic efficacies can occur on the dendrite via STDP, where changes in synaptic efficacy are driven by timing differences between synaptic inputs and the generation of local dendritic spikes. Specifically, when the model neuron is stimulated by two independent groups of correlated afferent inputs, the synaptic efficacies from each group, are not only spatially clustered on the dendrite but also spatially complementary to each other.

  18. Identification of the antiepileptic racetam binding site in the synaptic vesicle protein 2A by molecular dynamics and docking simulations.

    PubMed

    Correa-Basurto, José; Cuevas-Hernández, Roberto I; Phillips-Farfán, Bryan V; Martínez-Archundia, Marlet; Romo-Mancillas, Antonio; Ramírez-Salinas, Gema L; Pérez-González, Óscar A; Trujillo-Ferrara, José; Mendoza-Torreblanca, Julieta G

    2015-01-01

    Synaptic vesicle protein 2A (SV2A) is an integral membrane protein necessary for the proper function of the central nervous system and is associated to the physiopathology of epilepsy. SV2A is the molecular target of the anti-epileptic drug levetiracetam and its racetam analogs. The racetam binding site in SV2A and the non-covalent interactions between racetams and SV2A are currently unknown; therefore, an in silico study was performed to explore these issues. Since SV2A has not been structurally characterized with X-ray crystallography or nuclear magnetic resonance, a three-dimensional (3D) model was built. The model was refined by performing a molecular dynamics simulation (MDS) and the interactions of SV2A with the racetams were determined by docking studies. A reliable 3D model of SV2A was obtained; it reached structural equilibrium during the last 15 ns of the MDS (50 ns) with remaining structural motions in the N-terminus and long cytoplasmic loop. The docking studies revealed that hydrophobic interactions and hydrogen bonds participate importantly in ligand recognition within the binding site. Residues T456, S665, W666, D670 and L689 were important for racetam binding within the trans-membrane hydrophilic core of SV2A. Identifying the racetam binding site within SV2A should facilitate the synthesis of suitable radio-ligands to study treatment response and possibly epilepsy progression.

  19. Identification of the antiepileptic racetam binding site in the synaptic vesicle protein 2A by molecular dynamics and docking simulations

    PubMed Central

    Correa-Basurto, José; Cuevas-Hernández, Roberto I.; Phillips-Farfán, Bryan V.; Martínez-Archundia, Marlet; Romo-Mancillas, Antonio; Ramírez-Salinas, Gema L.; Pérez-González, Óscar A.; Trujillo-Ferrara, José; Mendoza-Torreblanca, Julieta G.

    2015-01-01

    Synaptic vesicle protein 2A (SV2A) is an integral membrane protein necessary for the proper function of the central nervous system and is associated to the physiopathology of epilepsy. SV2A is the molecular target of the anti-epileptic drug levetiracetam and its racetam analogs. The racetam binding site in SV2A and the non-covalent interactions between racetams and SV2A are currently unknown; therefore, an in silico study was performed to explore these issues. Since SV2A has not been structurally characterized with X-ray crystallography or nuclear magnetic resonance, a three-dimensional (3D) model was built. The model was refined by performing a molecular dynamics simulation (MDS) and the interactions of SV2A with the racetams were determined by docking studies. A reliable 3D model of SV2A was obtained; it reached structural equilibrium during the last 15 ns of the MDS (50 ns) with remaining structural motions in the N-terminus and long cytoplasmic loop. The docking studies revealed that hydrophobic interactions and hydrogen bonds participate importantly in ligand recognition within the binding site. Residues T456, S665, W666, D670 and L689 were important for racetam binding within the trans-membrane hydrophilic core of SV2A. Identifying the racetam binding site within SV2A should facilitate the synthesis of suitable radio-ligands to study treatment response and possibly epilepsy progression. PMID:25914622

  20. Stable Positioning of Unc13 Restricts Synaptic Vesicle Fusion to Defined Release Sites to Promote Synchronous Neurotransmission.

    PubMed

    Reddy-Alla, Suneel; Böhme, Mathias A; Reynolds, Eric; Beis, Christina; Grasskamp, Andreas T; Mampell, Malou M; Maglione, Marta; Jusyte, Meida; Rey, Ulises; Babikir, Husam; McCarthy, Anthony W; Quentin, Christine; Matkovic, Tanja; Bergeron, Dominique Dufour; Mushtaq, Zeeshan; Göttfert, Fabian; Owald, David; Mielke, Thorsten; Hell, Stefan W; Sigrist, Stephan J; Walter, Alexander M

    2017-09-13

    Neural information processing depends on precisely timed, Ca(2+)-activated synaptic vesicle exocytosis from release sites within active zones (AZs), but molecular details are unknown. Here, we identify that the (M)Unc13-family member Unc13A generates release sites and show the physiological relevance of their restrictive AZ targeting. Super-resolution and intravital imaging of Drosophila neuromuscular junctions revealed that (unlike the other release factors Unc18 and Syntaxin-1A) Unc13A was stably and precisely positioned at AZs. Local Unc13A levels predicted single AZ activity. Different Unc13A portions selectively affected release site number, position, and functionality. An N-terminal fragment stably localized to AZs, displaced endogenous Unc13A, and reduced the number of release sites, while a C-terminal fragment generated excessive sites at atypical locations, resulting in reduced and delayed evoked transmission that displayed excessive facilitation. Thus, release site generation by the Unc13A C terminus and their specific AZ localization via the N terminus ensure efficient transmission and prevent ectopic, temporally imprecise release. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Bottlebrush additives drive formation of vesicle chains in polymer blends

    NASA Astrophysics Data System (ADS)

    Mah, Hui Zhen; Afzali, Pantea; Verduzco, Rafeal; Stein, Gila

    2015-03-01

    The effects of bottlebrush polymer additive with poly (styrene-r-methyl methacrylate) side-chains on the thin film morphology of polystyrene (PS) and poly (methyl methacrylate) (PMMA) blends were studied. Results were compared to PS/PMMA blends with diblock copolymer PS-b-PMMA compatibilizer and without any additive. Thin films were spin casted from toluene onto a ``neutral'' silicon surface and then annealed at a fixed temperature of 150ºC for a range of times (up to 85 mins). The morphology of the films was characterized using optical microscopy and atomic force microscopy. In the absence of any additive, the PS/PMMA blend rapidly de-mixes to form macroscale domains, while high loadings of the PS-b-PMMA additive can compatibilize the blend and suppress phase separation. However, the bottlebrush polymer additive drives the formation of well-organized vesicle chains in the PS/PMMA blend films. This morphology is favored by entropic considerations as the bottlebrush polymers are more stable than linear chains at the PS/PMMA interface and the brush like surface attracts.

  2. Phosphorylation-dependent interaction of the synaptic vesicle proteins cysteine string protein and synaptotagmin I.

    PubMed Central

    Evans, Gareth J O; Morgan, Alan

    2002-01-01

    The secretory vesicle cysteine string proteins (CSPs) are members of the DnaJ family of chaperones, and function at late stages of Ca2+-regulated exocytosis by an unknown mechanism. To determine novel binding partners of CSPs, we employed a pull-down strategy from purified rat brain membrane or cytosolic proteins using recombinant hexahistidine-tagged (His(6)-)CSP. Western blotting of the CSP-binding proteins identified synaptotagmin I to be a putative binding partner. Furthermore, pull-down assays using cAMP-dependent protein kinase (PKA)-phosphorylated CSP recovered significantly less synaptotagmin. Complexes containing CSP and synaptotagmin were immunoprecipitated from rat brain membranes, further suggesting that these proteins interact in vivo. Binding assays in vitro using recombinant proteins confirmed a direct interaction between the two proteins and demonstrated that the PKA-phosphorylated form of CSP binds synaptotagmin with approximately an order of magnitude lower affinity than the non-phosphorylated form. Genetic studies have implicated each of these proteins in the Ca2+-dependency of exocytosis and, since CSP does not bind Ca2+, this novel interaction might explain the Ca2+-dependent actions of CSP. PMID:11931641

  3. The yin and yang of calcium effects on synaptic vesicle endocytosis.

    PubMed

    Wu, Xin-Sheng; Wu, Ling-Gang

    2014-02-12

    A large number of studies suggest that calcium triggers and accelerates vesicle endocytosis at many synapses and non-neuronal secretory cells. However, many studies show that prolonging the duration of the stimulation train, which induces more calcium influx, slows down endocytosis; and several studies suggest that instead of triggering endocytosis, calcium actually inhibits endocytosis. Here we addressed this apparent conflict at a large nerve terminal, the calyx of Held in rat brainstem, in which recent studies suggest that transient calcium increase up to tens of micromolar concentration at the micro/nano domain triggers endocytosis. By dialyzing 0-1 μM calcium into the calyx via a whole-cell pipette, we found that slow endocytosis was inhibited by calcium dialysis in a concentration-dependent manner. Thus, prolonged, small, and global calcium increase inhibits endocytosis, whereas transient and large calcium increase at the micro/nano domain triggers endocytosis and facilitates endocytosis. This yin and yang effect of calcium may reconcile apparent conflicts regarding whether calcium accelerates or inhibits endocytosis. Whether endocytosis is fast or slow depends on the net outcome between the yin and yang effect of calcium.

  4. β-Amyloid Causes Depletion of Synaptic Vesicles Leading to Neurotransmission Failure*

    PubMed Central

    Parodi, Jorge; Sepúlveda, Fernando J.; Roa, Jorge; Opazo, Carlos; Inestrosa, Nibaldo C.; Aguayo, Luis G.

    2010-01-01

    Alzheimer disease is a progressive neurodegenerative brain disorder that leads to major debilitating cognitive deficits. It is believed that the alterations capable of causing brain circuitry dysfunctions have a slow onset and that the full blown disease may take several years to develop. Therefore, it is important to understand the early, asymptomatic, and possible reversible states of the disease with the aim of proposing preventive and disease-modifying therapeutic strategies. It is largely unknown how amyloid β-peptide (Aβ), a principal agent in Alzheimer disease, affects synapses in brain neurons. In this study, we found that similar to other pore-forming neurotoxins, Aβ induced a rapid increase in intracellular calcium and miniature currents, indicating an enhancement in vesicular transmitter release. Significantly, blockade of these effects by low extracellular calcium and a peptide known to act as an inhibitor of the Aβ-induced pore prevented the delayed failure, indicating that Aβ blocks neurotransmission by causing vesicular depletion. This new mechanism for Aβ synaptic toxicity should provide an alternative pathway to search for small molecules that can antagonize these effects of Aβ. PMID:19915004

  5. 1-O-Alkylglycerol vesicles (Algosomes): their formation and characterization.

    PubMed

    Gopinath, D; Ravi, D; Rao, B R; Apte, S S; Rambhau, D

    2002-10-10

    1-O-alkylglycerols (ALKG) have exhibited several biological activities and a prominent effect on blood-brain barrier permeability. They have markedly improved brain uptake of cancerostatic agents. Since ALKG are amphiphilic, we explored their tendency to assemble into bilayer vesicles, which can be applied as carriers for drugs. Vesicles (Algosomes) were formed by film hydration method using ALKG (tetra-, penta-, hexa-, hepta-, octa- or nona-decylglycerols) in combination with cholesterol (CHOL) and dicetyl phosphate (DCP) (1-O-alkylglycerol:CHOL:DCP in 45:45:10 molar ratio). On microscopic examination, the algosomes were found to be conspicuously spherical and the dispersion was a mixture of multi-lamellar and small-unilamellar vesicles. Phase transition temperatures of 1-O-hexadecylglycerol (HXDG) and CHOL mixtures were tested by differential scanning calorimetry (DSC). The changes in phase transition temperatures indicate the vesicle forming tendency of ALKG in presence of CHOL. Alkyl chain length dependent variations in vesicle size, zeta-potential (ZP) and capture volume (CV) could not be observed. Vesicles of 1-O-tetradecylglycerol (TTDG) showed improvement in CV with increase in CHOL content from 15 to 55 mol%. However the vesicle size decreased. On challenging algosomes with hypertonic salt solution [potassium iodide (KI) in water], vesicle size decreased and thus algosomes were found to be osmotically sensitive. Algosome dispersions on addition of higher concentrations of KI (40-100 mM) brought about increases in vesicle size and at concentrations 60 mM and above showed aggregation. All vesicular dispersions were stable for only a few days.

  6. 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

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

    PubMed Central

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

    1999-01-01

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

  8. Formation of simple single-tailed vesicles mediated by lipophilic solid surfaces.

    PubMed

    Du, Na; Zhu, Xiaoyu; Song, Ruiying; Song, Shue; Hou, Wanguo

    2016-10-19

    Adsorption and aggregation of surfactants at solid-liquid interfaces were fairly well understood, but there was limited knowledge regarding the effect of the presence of a solid surface on aggregate structures in bulk solution. Except for the fatty acid system, most simple single-tailed surfactants (STSs) are well known to form micelles but not vesicles in aqueous solution. Herein, we report a novel phenomenon: with the mediation of lipophilic solid surfaces (LSSs), the zwitterionic STS lauryl sulfobetaine (LSB) formed vesicles from its micellar solution without any additives, producing a mixed solution of vesicles and micelles. More interestingly, the STS vesicles coexisted stably with micelles in the solution and were thermally insensitive even after the removal of LSSs. The quantity of LSB vesicles decreases with the addition of ethanol. The pH effects (4.0-9.0) did not have an obvious influence on the formation and stability of the LSB vesicles. Similar results were obtained from the other STSs, suggesting that the LSS-mediated micelle-to-vesicle transition may be a general phenomenon. We proposed a possible mechanism that adsorption, the matrix effect, and interdigitated bilayer structures were probably crucial for the formation and stability of STS vesicles. We expect this work to provide important insights into the effect of the solid/liquid interface on the self-assembly chemistry of surfactants in bulk solution.

  9. Glutamatergic modulation of synaptic-like vesicle recycling in mechanosensory lanceolate nerve terminals of mammalian hair follicles.

    PubMed

    Banks, Robert W; Cahusac, Peter M B; Graca, Anna; Kain, Nakul; Shenton, Fiona; Singh, Paramjeet; Njå, Arild; Simon, Anna; Watson, Sonia; Slater, Clarke R; Bewick, Guy S

    2013-05-15

    Our aim in the present study was to determine whether a glutamatergic modulatory system involving synaptic-like vesicles (SLVs) is present in the lanceolate ending of the mouse and rat hair follicle and, if so, to assess its similarity to that of the rat muscle spindle annulospiral ending we have described previously. Both types of endings are formed by the peripheral sensory terminals of primary mechanosensory dorsal root ganglion cells, so the presence of such a system in the lanceolate ending would provide support for our hypothesis that it is a general property of fundamental importance to the regulation of the responsiveness of the broad class of primary mechanosensory endings. We show not only that an SLV-based system is present in lanceolate endings, but also that there are clear parallels between its operation in the two types of mechanosensory endings. In particular, we demonstrate that, as in the muscle spindle: (i) FM1-43 labels the sensory terminals of the lanceolate ending, rather than the closely associated accessory (glial) cells; (ii) the dye enters and leaves the terminals primarily by SLV recycling; (iii) the dye does not block the electrical response to mechanical stimulation, in contrast to its effect on the hair cell and dorsal root ganglion cells in culture; (iv) SLV recycling is Ca(2+) sensitive; and (v) the sensory terminals are enriched in glutamate. Thus, in the lanceolate sensory ending SLV recycling is itself regulated, at least in part, by glutamate acting through a phospholipase D-coupled metabotropic glutamate receptor.

  10. Human R1441C LRRK2 regulates the synaptic vesicle proteome and phosphoproteome in a Drosophila model of Parkinson's disease.

    PubMed

    Islam, Md Shariful; Nolte, Hendrik; Jacob, Wright; Ziegler, Anna B; Pütz, Stefanie; Grosjean, Yael; Szczepanowska, Karolina; Trifunovic, Aleksandra; Braun, Thomas; Heumann, Hermann; Heumann, Rolf; Hovemann, Bernhard; Moore, Darren J; Krüger, Marcus

    2016-10-29

    Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset, autosomal dominant familial Parkinson's disease (PD) and variation at the LRRK2 locus contributes to the risk for idiopathic PD. LRRK2 can function as a protein kinase and mutations lead to increased kinase activity. To elucidate the pathophysiological mechanism of the R1441C mutation in the GTPase domain of LRRK2, we expressed human wild-type or R1441C LRRK2 in dopaminergic neurons of Drosophila and observe reduced locomotor activity, impaired survival and an age-dependent degeneration of dopaminergic neurons thereby creating a new PD-like model. To explore the function of LRRK2 variants in vivo, we performed mass spectrometry and quantified 3,616 proteins in the fly brain. We identify several differentially-expressed cytoskeletal, mitochondrial and synaptic vesicle proteins (SV), including synaptotagmin-1, syntaxin-1A and Rab3, in the brain of this LRRK2 fly model. In addition, a global phosphoproteome analysis reveals the enhanced phosphorylation of several SV proteins, including synaptojanin-1 (pThr1131) and the microtubule-associated protein futsch (pSer4106) in the brain of R1441C hLRRK2 flies. The direct phosphorylation of human synaptojanin-1 by R1441C hLRRK2 could further be confirmed by in vitro kinase assays. A protein-protein interaction screen in the fly brain confirms that LRRK2 robustly interacts with numerous SV proteins, including synaptojanin-1 and EndophilinA. Our proteomic, phosphoproteomic and interactome study in the Drosophila brain provides a systematic analyses of R1441C hLRRK2-induced pathobiological mechanisms in this model. We demonstrate for the first time that the R1441C mutation located within the LRRK2 GTPase domain induces the enhanced phosphorylation of SV proteins in the brain.

  11. Effects of rates of spontaneous synaptic vesicle secretions in inner hair cells on information transmission in an auditory nerve fiber model.

    PubMed

    Kumsa, Parichat; Mino, Hiroyuki

    2012-01-01

    In this article, we investigate how the rates of spontaneous synaptic vesicle secretions affect information transmission of the spike trains in response to the inner hair cell (IHC) synaptic currents in an auditory nerve fiber (ANF) model through computer simulations. The IHC synaptic currents were modeled by a filtered inhomogeneous Poisson process modulated with sinusoidal functions, while the stochastic ion channel model was incorporated into each node of Ranvier in the ANF model with spiral ganglion. The information rates were estimated from the entropies of the inter-spike intervals of the spike trains to evaluate information transmission in the ANF model. The results show that the information rates increased, reached a maximum, and then decreased as the rate of spontaneous secretion increased, implying a resonance phenomenon dependent on the rate of spontaneous IHC synaptic secretions. In conclusion, this phenomenon similar to the regular stochastic resonance may be observed due to that spontaneous IHC synaptic secretions may act as an origin of fluctuation or noise, and these findings may play a key role in the design of better auditory prostheses.

  12. The use of highly ordered vesicle gels as template for the formation of silica gels.

    PubMed

    Oppel, Claudia; Prévost, Sylvain; Noirez, Laurence; Gradzielski, Michael

    2011-07-19

    A spontaneously forming gel of unilamellar vesicles based on sodium oleate (Na oleate) and 1-octanol as amphiphiles has been employed as a template in the formation of a silica gel formed by the hydrolysis of the inorganic precursor tetraethyl orthosilicate (TEOS). Up to about 10 wt % TEOS can be incorporated into this vesicle gel without phase separation and in a fully homogeneous formation process by simple mixing of the components. The process itself relies solely upon the self-organizing properties of this amphiphilic template system. The formation process was followed by means of time-resolved turbidity, rheology, and small-angle neutron scattering (SANS) experiments. It can be concluded that the presence of the precursor TEOS affects the kinetics of the process but the original vesicle gel structure is retained even up to highest TEOS content. The kinetic studies confirm that under the chosen conditions the vesicle formation proceeds much faster than the hydrolysis of TEOS and the subsequent formation of the silica gel. SANS displays in the low q-range an additional scattering due to the silica gel network, i.e., a hybrid material of an amphiphilic vesicle gel and an inorganic oxide gel is formed. Thus, this method is a very facile novel route of forming a highly ordered silica/vesicle gel by employing a self-organizing amphiphilic system as template and the formation of the silica network proceeds in a fully homogeneous fashion under kinetic control.

  13. A novel region in the CaV2.1 α1 subunit C-terminus regulates fast synaptic vesicle fusion and vesicle docking at the mammalian presynaptic active zone

    PubMed Central

    Lübbert, Matthias; Goral, R Oliver; Satterfield, Rachel; Putzke, Travis; van den Maagdenberg, Arn MJM; Kamasawa, Naomi; Young, Samuel M

    2017-01-01

    In central nervous system (CNS) synapses, action potential-evoked neurotransmitter release is principally mediated by CaV2.1 calcium channels (CaV2.1) and is highly dependent on the physical distance between CaV2.1 and synaptic vesicles (coupling). Although various active zone proteins are proposed to control coupling and abundance of CaV2.1 through direct interactions with the CaV2.1 α1 subunit C-terminus at the active zone, the role of these interaction partners is controversial. To define the intrinsic motifs that regulate coupling, we expressed mutant CaV2.1 α1 subunits on a CaV2.1 null background at the calyx of Held presynaptic terminal. Our results identified a region that directly controlled fast synaptic vesicle release and vesicle docking at the active zone independent of CaV2.1 abundance. In addition, proposed individual direct interactions with active zone proteins are insufficient for CaV2.1 abundance and coupling. Therefore, our work advances our molecular understanding of CaV2.1 regulation of neurotransmitter release in mammalian CNS synapses. DOI: http://dx.doi.org/10.7554/eLife.28412.001 PMID:28786379

  14. Molecular mechanism underlying RAG1/RAG2 synaptic complex formation.

    PubMed

    Shlyakhtenko, Luda S; Gilmore, Jamie; Kriatchko, Aleksei N; Kumar, Sushil; Swanson, Patrick C; Lyubchenko, Yuri L

    2009-07-31

    Two lymphoid cell-specific proteins, RAG1 and RAG2 (RAG), initiate V(D)J recombination by assembling a synaptic complex with recombination signal sequences (RSSs) abutting two different antigen receptor gene coding segments, and then introducing a DNA double strand break at the end of each RSS. Despite the biological importance of this system, the structure of the synaptic complex, and the RAG protein stoichiometry and arrangement of DNA within the synaptosome, remains poorly understood. Here we applied atomic force microscopy to directly visualize and characterize RAG synaptic complexes. We report that the pre-cleavage RAG synaptic complex contains about twice the protein content as a RAG complex bound to a single RSS, with a calculated mass consistent with a pair of RAG heterotetramers. In the synaptic complex, the RSSs are predominantly oriented in a side-by-side configuration with no DNA strand crossover. The mass of the synaptic complex, and the conditions under which it is formed in vitro, favors an association model of assembly in which isolated RAG-RSS complexes undergo synapsis mediated by RAG protein-protein interactions. The replacement of Mg2+ cations with Ca2+ leads to a dramatic change in protein stoichiometry for all RAG-RSS complexes, suggesting that the cation composition profoundly influences the type of complex assembled.

  15. Molecular Mechanism Underlying RAG1/RAG2 Synaptic Complex Formation*

    PubMed Central

    Shlyakhtenko, Luda S.; Gilmore, Jamie; Kriatchko, Aleksei N.; Kumar, Sushil; Swanson, Patrick C.; Lyubchenko, Yuri L.

    2009-01-01

    Two lymphoid cell-specific proteins, RAG1 and RAG2 (RAG), initiate V(D)J recombination by assembling a synaptic complex with recombination signal sequences (RSSs) abutting two different antigen receptor gene coding segments, and then introducing a DNA double strand break at the end of each RSS. Despite the biological importance of this system, the structure of the synaptic complex, and the RAG protein stoichiometry and arrangement of DNA within the synaptosome, remains poorly understood. Here we applied atomic force microscopy to directly visualize and characterize RAG synaptic complexes. We report that the pre-cleavage RAG synaptic complex contains about twice the protein content as a RAG complex bound to a single RSS, with a calculated mass consistent with a pair of RAG heterotetramers. In the synaptic complex, the RSSs are predominantly oriented in a side-by-side configuration with no DNA strand crossover. The mass of the synaptic complex, and the conditions under which it is formed in vitro, favors an association model of assembly in which isolated RAG-RSS complexes undergo synapsis mediated by RAG protein-protein interactions. The replacement of Mg2+ cations with Ca2+ leads to a dramatic change in protein stoichiometry for all RAG-RSS complexes, suggesting that the cation composition profoundly influences the type of complex assembled. PMID:19502597

  16. Glutamate transport into synaptic vesicles. Roles of membrane potential, pH gradient, and intravesicular pH.

    PubMed

    Tabb, J S; Kish, P E; Van Dyke, R; Ueda, T

    1992-08-05

    Glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, is transported into bovine synaptic vesicles in a manner that is ATP dependent and requires a vesicular electrochemical proton gradient. We studied the electrical and chemical elements of this driving force and evaluated the effects of chloride on transport. Increasing concentrations of Cl- were found to increase the steady-state ATP-dependent vesicular pH gradient (delta pH) and were found to concomitantly decrease the vesicular membrane potential (delta psi). Low millimolar chloride concentrations, which cause 3-6-fold stimulation of vesicular glutamate uptake, caused small but measurable increases in delta pH and decreases in delta psi, when compared to control vesicles in the absence of chloride. Nigericin in potassium buffers was used to alter the relative proportions of delta pH and delta psi. Compared to controls, at all chloride concentrations tested, nigericin virtually abolished delta pH and increased the vesicle interior positive delta psi. Concomitantly, nigericin increased ATP-dependent glutamate uptake in 0-1 mM chloride but decreased glutamate uptake in 4 mM (45%), 20 mM (80%), and 140 mM (75%) Cl- (where delta pH in the absence of nigericin was large). These findings suggest that either delta psi, delta pH, or a combination can drive glutamate uptake, but to different degrees. In the presence of 4 mM Cl-, where uptake is optimal, both delta psi and delta pH contribute to the driving force for uptake. When the extravesicular pH was increased from 7.4 to 8.0, more Cl- was required to stimulate vesicular glutamate uptake. In the absence of Cl-, as extravesicular pH was lowered to 6.8, uptake was over 3-fold greater than it was at pH 7.4. As extravesicular pH was reduced from 8.0 toward 6.8, less Cl- was required for maximal stimulation. Decreasing the extravesicular pH from 8.0 to 6.8 in the absence of Cl- significantly increased glutamate uptake activity, even

  17. Characterization of a Human Point Mutation of VGLUT3 (p.A211V) in the Rodent Brain Suggests a Nonuniform Distribution of the Transporter in Synaptic Vesicles.

    PubMed

    Ramet, Lauriane; Zimmermann, Johannes; Bersot, Tiphaine; Poirel, Odile; De Gois, Stéphanie; Silm, Katlin; Sakae, Diana Yae; Mansouri-Guilani, Nina; Bourque, Marie-Josée; Trudeau, Louis-Eric; Pietrancosta, Nicolas; Daumas, Stéphanie; Bernard, Véronique; Rosenmund, Christian; El Mestikawy, Salah

    2017-04-12

    The atypical vesicular glutamate transporter type 3 (VGLUT3) is expressed by subpopulations of neurons using acetylcholine, GABA, or serotonin as neurotransmitters. In addition, VGLUT3 is expressed in the inner hair cells of the auditory system. A mutation (p.A211V) in the gene that encodes VGLUT3 is responsible for progressive deafness in two unrelated families. In this study, we investigated the consequences of the p.A211V mutation in cell cultures and in the CNS of a mutant mouse. The mutation substantially decreased VGLUT3 expression (-70%). We measured VGLUT3-p.A211V activity by vesicular uptake in BON cells, electrophysiological recording of isolated neurons, and its ability to stimulate serotonergic accumulation in cortical synaptic vesicles. Despite a marked loss of expression, the activity of the mutated isoform was only minimally altered. Furthermore, mutant mice displayed none of the behavioral alterations that have previously been reported in VGLUT3 knock-out mice. Finally, we used stimulated emission depletion microscopy to analyze how the mutation altered VGLUT3 distribution within the terminals of mice expressing the mutated isoform. The mutation appeared to reduce the expression of the VGLUT3 transporter by simultaneously decreasing the number of VGLUT3-positive synaptic vesicles and the amount of VGLUT3 per synapses. These observations suggested that VGLUT3 global activity is not linearly correlated with VGLUT3 expression. Furthermore, our data unraveled a nonuniform distribution of VGLUT3 in synaptic vesicles. Identifying the mechanisms responsible for this complex vesicular sorting will be critical to understand VGLUT's involvement in normal and pathological conditions.SIGNIFICANCE STATEMENT VGLUT3 is an atypical member of the vesicular glutamate transporter family. A point mutation of VGLUT3 (VGLUT3-p.A211V) responsible for a progressive loss of hearing has been identified in humans. We observed that this mutation dramatically reduces VGLUT3

  18. 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

  19. Synaptic vesicles contain small ribonucleic acids (sRNAs) including transfer RNA fragments (trfRNA) and microRNAs (miRNA)

    PubMed Central

    Li, Huinan; Wu, Cheng; Aramayo, Rodolfo; Sachs, Matthew S.; Harlow, Mark L.

    2015-01-01

    Synaptic vesicles (SVs) are neuronal presynaptic organelles that load and release neurotransmitter at chemical synapses. In addition to classic neurotransmitters, we have found that synaptic vesicles isolated from the electric organ of Torpedo californica, a model cholinergic synapse, contain small ribonucleic acids (sRNAs), primarily the 5′ ends of transfer RNAs (tRNAs) termed tRNA fragments (trfRNAs). To test the evolutionary conservation of SV sRNAs we examined isolated SVs from the mouse central nervous system (CNS). We found abundant levels of sRNAs in mouse SVs, including trfRNAs and micro RNAs (miRNAs) known to be involved in transcriptional and translational regulation. This discovery suggests that, in addition to inducing changes in local dendritic excitability through the release of neurotransmitters, SVs may, through the release of specific trfRNAs and miRNAs, directly regulate local protein synthesis. We believe these findings have broad implications for the study of chemical synaptic transmission. PMID:26446566

  20. Synaptic vesicles contain small ribonucleic acids (sRNAs) including transfer RNA fragments (trfRNA) and microRNAs (miRNA).

    PubMed

    Li, Huinan; Wu, Cheng; Aramayo, Rodolfo; Sachs, Matthew S; Harlow, Mark L

    2015-10-08

    Synaptic vesicles (SVs) are neuronal presynaptic organelles that load and release neurotransmitter at chemical synapses. In addition to classic neurotransmitters, we have found that synaptic vesicles isolated from the electric organ of Torpedo californica, a model cholinergic synapse, contain small ribonucleic acids (sRNAs), primarily the 5' ends of transfer RNAs (tRNAs) termed tRNA fragments (trfRNAs). To test the evolutionary conservation of SV sRNAs we examined isolated SVs from the mouse central nervous system (CNS). We found abundant levels of sRNAs in mouse SVs, including trfRNAs and micro RNAs (miRNAs) known to be involved in transcriptional and translational regulation. This discovery suggests that, in addition to inducing changes in local dendritic excitability through the release of neurotransmitters, SVs may, through the release of specific trfRNAs and miRNAs, directly regulate local protein synthesis. We believe these findings have broad implications for the study of chemical synaptic transmission.

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

    PubMed Central

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

    2015-01-01

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

  2. The 4p16.3 Parkinson Disease Risk Locus Is Associated with GAK Expression and Genes Involved with the Synaptic Vesicle Membrane

    PubMed Central

    Nagle, Michael W.; Latourelle, Jeanne C.; Labadorf, Adam; Dumitriu, Alexandra; Hadzi, Tiffany C.; Beach, Thomas G.; Myers, Richard H.

    2016-01-01

    Genome-wide association studies (GWAS) have identified the GAK/DGKQ/IDUA region on 4p16.3 among the top three risk loci for Parkinson’s disease (PD), but the specific gene and risk mechanism are unclear. Here, we report transcripts containing the 3’ clathrin-binding domain of GAK identified by RNA deep-sequencing in post-mortem human brain tissue as having increased expression in PD. Furthermore, carriers of 4p16.3 PD GWAS risk SNPs show decreased expression of one of these transcripts, GAK25 (Gencode Transcript 009), which correlates with the expression of genes functioning in the synaptic vesicle membrane. Together, these findings provide strong evidence for GAK clathrin-binding- and J-domain transcripts’ influence on PD pathogenicity, and for a role for GAK in regulating synaptic function in PD. PMID:27508417

  3. The 4p16.3 Parkinson Disease Risk Locus Is Associated with GAK Expression and Genes Involved with the Synaptic Vesicle Membrane.

    PubMed

    Nagle, Michael W; Latourelle, Jeanne C; Labadorf, Adam; Dumitriu, Alexandra; Hadzi, Tiffany C; Beach, Thomas G; Myers, Richard H

    2016-01-01

    Genome-wide association studies (GWAS) have identified the GAK/DGKQ/IDUA region on 4p16.3 among the top three risk loci for Parkinson's disease (PD), but the specific gene and risk mechanism are unclear. Here, we report transcripts containing the 3' clathrin-binding domain of GAK identified by RNA deep-sequencing in post-mortem human brain tissue as having increased expression in PD. Furthermore, carriers of 4p16.3 PD GWAS risk SNPs show decreased expression of one of these transcripts, GAK25 (Gencode Transcript 009), which correlates with the expression of genes functioning in the synaptic vesicle membrane. Together, these findings provide strong evidence for GAK clathrin-binding- and J-domain transcripts' influence on PD pathogenicity, and for a role for GAK in regulating synaptic function in PD.

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

    PubMed Central

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

    2010-01-01

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

  5. Evidence that phospholipase D mediates ADP ribosylation factor- dependent formation of Golgi coated vesicles

    PubMed Central

    1996-01-01

    Formation of coatomer-coated vesicles from Golgi-enriched membranes requires the activation of a small GTP-binding protein, ADP ribosylation factor (ARF). ARF is also an efficacious activator of phospholipase D (PLD), an activity that is relatively abundant on Golgi- enriched membranes. It has been proposed that ARF, which is recruited onto membranes from cytosolic pools, acts directly to promote coatomer binding and is in a 3:1 stoichiometry with coatomer on coated vesicles. We present evidence that cytosolic ARF is not necessary for initiating coat assembly on Golgi membranes from cell lines with high constitutive PLD activity. Conditions are also described under which ARF is at most a minor component relative to coatomer in coated vesicles from all cell lines tested, including Chinese hamster ovary cells. Formation of coated vesicles was sensitive to ethanol at concentrations that inhibit the production of phosphatidic acid (PA) by PLD. When PA was produced in Golgi membranes by an exogenous bacterial PLD, rather than with ARF and endogenous PLD, coatomer bound to Golgi membranes. Purified coatomer also bound selectively to artificial lipid vesicles that contained PA and phosphatidylinositol (4,5)-bisphosphate (PIP2). We propose that activation of PLD and the subsequent production of PA are key early events for the formation of coatomer-coated vesicles. PMID:8707816

  6. MYOSIN IIB REGULATES ACTIN DYNAMICS DURING SYNAPTIC PLASTICITY AND MEMORY FORMATION

    PubMed Central

    Rex, Christopher S.; Gavin, Cristin F.; Rubio, Maria D.; Kramar, Eniko A.; Chen, Lulu Y.; Jia, Yousheng; Huganir, Richard L.; Muzyczka, Nicholas; Gall, Christine M.; Miller, Courtney A.; Lynch, Gary; Rumbaugh, Gavin

    2010-01-01

    Reorganization of the actin cytoskeleton is essential for synaptic plasticity and memory formation. Presently, the mechanisms that trigger actin dynamics during these brain processes are poorly understood. In this study, we show that myosin II motor activity is downstream of LTP induction and is necessary for the emergence of specialized actin structures that stabilize an early phase of LTP. We also demonstrate that myosin II activity contributes importantly to an actin-dependent process that underlies memory consolidation. Pharmacological treatments that promote actin polymerization reversed the effects of a myosin II inhibitor on LTP and memory. We conclude that myosin II motors regulate plasticity by imparting mechanical forces onto the spine actin cytoskeleton in response to synaptic stimulation. These cytoskeletal forces trigger the emergence of actin structures that stabilize synaptic plasticity. Our studies provide a novel mechanical framework for understanding cytoskeletal dynamics associated with synaptic plasticity and memory formation. PMID:20797537

  7. Gel formation in systems composed of drug containing catanionic vesicles and oppositely charged hydrophobically modified polymer.

    PubMed

    Dew, Noel; Edwards, Katarina; Edsman, Katarina

    2009-05-01

    The aim of this study was to explore if mixtures of drug containing catanionic vesicles and polymers give rise to gel formation, and if so, if drug release from these gels could be prolonged. Catanionic vesicles formed from the drug substances alprenolol or tetracaine, and the oppositely charged surfactant sodium dodecyl sulphate were mixed with polymers. Three polymers with different properties were employed: one bearing hydrophobic modifications, one positively charged and one positively charged polymer bearing hydrophobic modifications. The structure of the vesicles before and after addition of polymer was investigated by using cryo-TEM. Gel formation was confirmed by using rheological measurements. Drug release was studied using a modified USP paddle method. Gels were observed to form only in the case when catanionic vesicles, most likely with a net negative charge, were mixed with positively charged polymer bearing lipophilic modifications. The release of drug substance from these systems, where the vesicles are not trapped within the gel but constitute a founding part of it, could be significantly prolonged. The drug release rate was found to depend on vesicle concentration to a higher extent than on polymer concentration.

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

    PubMed

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

    2015-12-17

    Vesicular nucleo-cytoplasmic transport is becoming recognized as a general cellular mechanism for translocation of large cargoes across the nuclear envelope. Cargo is recruited, enveloped at the inner nuclear membrane (INM), and delivered by membrane fusion at the outer nuclear membrane. To understand the structural underpinning for this trafficking, we investigated nuclear egress of progeny herpesvirus capsids where capsid envelopment is mediated by two viral proteins, forming the nuclear egress complex (NEC). Using a multi-modal imaging approach, we visualized the NEC in situ forming coated vesicles of defined size. Cellular electron cryo-tomography revealed a protein layer showing two distinct hexagonal lattices at its membrane-proximal and membrane-distant faces, respectively. NEC coat architecture was determined by combining this information with integrative modeling using small-angle X-ray scattering data. The molecular arrangement of the NEC establishes the basic mechanism for budding and scission of tailored vesicles at the INM. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Phosphorylation of Synaptic Vesicle Protein 2A at Thr84 by Casein Kinase 1 Family Kinases Controls the Specific Retrieval of Synaptotagmin-1

    PubMed Central

    Zhang, Ning; Gordon, Sarah L.; Fritsch, Maximilian J.; Esoof, Noor; Campbell, David G.; Gourlay, Robert; Velupillai, Srikannathasan; Macartney, Thomas; Peggie, Mark; van Aalten, Daan M.F.

    2015-01-01

    Synaptic vesicle protein 2A (SV2A) is a ubiquitous component of synaptic vesicles (SVs). It has roles in both SV trafficking and neurotransmitter release. We demonstrate that Casein kinase 1 family members, including isoforms of Tau–tubulin protein kinases (TTBK1 and TTBK2), phosphorylate human SV2A at two constellations of residues, namely Cluster-1 (Ser42, Ser45, and Ser47) and Cluster-2 (Ser80, Ser81, and Thr84). These residues are also phosphorylated in vivo, and the phosphorylation of Thr84 within Cluster-2 is essential for triggering binding to the C2B domain of human synaptotagmin-1. We show by crystallographic and other analyses that the phosphorylated Thr84 residue binds to a pocket formed by three conserved Lys residues (Lys314, Lys326, and Lys328) on the surface of the synaptotagmin-1 C2B domain. Finally, we observed dysfunctional synaptotagmin-1 retrieval during SV endocytosis by ablating its phospho-dependent interaction with SV2A, knockdown of SV2A, or rescue with a phosphorylation-null Thr84 SV2A mutant in primary cultures of mouse neurons. This study reveals fundamental details of how phosphorylation of Thr84 on SV2A controls its interaction with synaptotagmin-1 and implicates SV2A as a phospho-dependent chaperone required for the specific retrieval of synaptotagmin-1 during SV endocytosis. PMID:25673844

  10. Phosphorylation of synaptic vesicle protein 2A at Thr84 by casein kinase 1 family kinases controls the specific retrieval of synaptotagmin-1.

    PubMed

    Zhang, Ning; Gordon, Sarah L; Fritsch, Maximilian J; Esoof, Noor; Campbell, David G; Gourlay, Robert; Velupillai, Srikannathasan; Macartney, Thomas; Peggie, Mark; van Aalten, Daan M F; Cousin, Michael A; Alessi, Dario R

    2015-02-11

    Synaptic vesicle protein 2A (SV2A) is a ubiquitous component of synaptic vesicles (SVs). It has roles in both SV trafficking and neurotransmitter release. We demonstrate that Casein kinase 1 family members, including isoforms of Tau-tubulin protein kinases (TTBK1 and TTBK2), phosphorylate human SV2A at two constellations of residues, namely Cluster-1 (Ser42, Ser45, and Ser47) and Cluster-2 (Ser80, Ser81, and Thr84). These residues are also phosphorylated in vivo, and the phosphorylation of Thr84 within Cluster-2 is essential for triggering binding to the C2B domain of human synaptotagmin-1. We show by crystallographic and other analyses that the phosphorylated Thr84 residue binds to a pocket formed by three conserved Lys residues (Lys314, Lys326, and Lys328) on the surface of the synaptotagmin-1 C2B domain. Finally, we observed dysfunctional synaptotagmin-1 retrieval during SV endocytosis by ablating its phospho-dependent interaction with SV2A, knockdown of SV2A, or rescue with a phosphorylation-null Thr84 SV2A mutant in primary cultures of mouse neurons. This study reveals fundamental details of how phosphorylation of Thr84 on SV2A controls its interaction with synaptotagmin-1 and implicates SV2A as a phospho-dependent chaperone required for the specific retrieval of synaptotagmin-1 during SV endocytosis. Copyright © 2015 Zhang et al.

  11. The neuronal protein Neurexin directly interacts with the Scribble-Pix complex to stimulate F-actin assembly for synaptic vesicle clustering.

    PubMed

    Rui, Menglong; Qian, Jinjun; Liu, Lijuan; Cai, Yihan; Lv, Huihui; Han, Junhai; Jia, Zhengping; Xie, Wei

    2017-09-01

    Synaptic vesicles (SVs) form distinct pools at synaptic terminals, and this well-regulated separation is necessary for normal neurotransmission. However, how the SV cluster, in particular synaptic compartments, maintains normal neurotransmitter release remains a mystery. The presynaptic protein Neurexin (NRX) plays a significant role in synaptic architecture and function, and some evidence suggests that NRX is associated with neurological disorders, including autism spectrum disorders. However, the role of NRX in SV clustering is unclear. Here, using the neuromuscular junction at the 2-3 instar stages of Drosophila larvae as a model and biochemical imaging and electrophysiology techniques, we demonstrate that Drosophila NRX (DNRX) plays critical roles in regulating synaptic terminal clustering and release of SVs. We found that DNRX controls the terminal clustering and release of SVs by stimulating presynaptic F-actin. Furthermore, our results indicate that DNRX functions through the scaffold protein Scribble and the GEF protein DPix to activate the small GTPase Ras-related C3 Botulinum toxin substrate 1 (Rac1). We observed a direct interaction between the C-terminal PDZ-binding motif of DNRX and the PDZ domains of Scribble and that Scribble bridges DNRX to DPix, forming a DNRX-Scribble-DPix complex that activates Rac1 and subsequently stimulates presynaptic F-actin assembly and SV clustering. Taken together, our work provides important insights into the function of DNRX in regulating SV clustering, which could help inform further research into pathological neurexin-mediated mechanisms in neurological disorders such as autism. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Energy transduction inside vesicles, photocatalysis by titanium dioxide and formation of NADH

    NASA Astrophysics Data System (ADS)

    Summers, David; Noveron, Juan; Rodoni, David; Basa, Ranor

    /protocells suitable either for simple prebiotic systems and/or for more complex "protobiochemical" systems. It could act as a precursor to metabolic systems and provide a model of how metabolism could have developed prebiotically in a vesicle based "protocell origin of life". It can provide a source of prebiotic compounds inside vesicles, an environment considered to be of great importance for the origin of life. An energy transduction system that is simple enough to have formed at an early stage of the origin of life (even before the formation of enzymatic or ribozymal activity) makes an autotrophic origin of life easier to envision.

  13. 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.

  14. Overexpression of synapsin Ia in the rat calyx of Held accelerates short-term plasticity and decreases synaptic vesicle volume and active zone area

    PubMed Central

    Vasileva, Mariya; Renden, Robert; Horstmann, Heinz; Gitler, Daniel; Kuner, Thomas

    2013-01-01

    Synapsins are synaptic vesicle (SV) proteins organizing a component of the reserve pool of vesicles at most central nervous system synapses. Alternative splicing of the three mammalian genes results in multiple isoforms that may differentially contribute to the organization and maintenance of the SV pools. To address this, we first characterized the expression pattern of synapsin isoforms in the rat calyx of Held. At postnatal day 16, synapsins Ia, Ib, IIb and IIIa were present, while IIa—known to sustain repetitive transmission in glutamatergic terminals—was not detectable. To test if the synapsin I isoforms could mediate IIa-like effect, and if this depends on the presence of the E-domain, we overexpressed either synapsin Ia or synapsin Ib in the rat calyx of Held via recombinant adeno-associated virus-mediated gene transfer. Although the size and overall structure of the perturbed calyces remained unchanged, short-term depression and recovery from depression were accelerated upon overexpression of synapsin I isoforms. Using electron microscopic three-dimensional reconstructions we found a redistribution of SV clusters proximal to the active zones (AZ) alongside with a decrease of both AZ area and SV volume. The number of SVs at individual AZs was strongly reduced. Hence, our data indicate that the amount of synapsin Ia expressed in the calyx regulates the rate and extent of short-term synaptic plasticity by affecting vesicle recruitment to the AZ. Finally, our study reveals a novel contribution of synapsin Ia to define the surface area of AZs. PMID:24391547

  15. Stimulation of Synaptic Vesicle Exocytosis by the Mental Disease Gene DISC1 is Mediated by N-Type Voltage-Gated Calcium Channels

    PubMed Central

    Tang, Willcyn; Thevathasan, Jervis Vermal; Lin, Qingshu; Lim, Kim Buay; Kuroda, Keisuke; Kaibuchi, Kozo; Bilger, Marcel; Soong, Tuck Wah; Fivaz, Marc

    2016-01-01

    Lesions and mutations of the DISC1 (Disrupted-in-schizophrenia-1) gene have been linked to major depression, schizophrenia, bipolar disorder and autism, but the influence of DISC1 on synaptic transmission remains poorly understood. Using two independent genetic approaches—RNAi and a DISC1 KO mouse—we examined the impact of DISC1 on the synaptic vesicle (SV) cycle by population imaging of the synaptic tracer vGpH in hippocampal neurons. DISC1 loss-of-function resulted in a marked decrease in SV exocytic rates during neuronal stimulation and was associated with reduced Ca2+ transients at nerve terminals. Impaired SV release was efficiently rescued by elevation of extracellular Ca2+, hinting at a link between DISC1 and voltage-gated Ca2+ channels. Accordingly, blockade of N-type Cav2.2 channels mimics and occludes the effect of DISC1 inactivation on SV exocytosis, and overexpression of DISC1 in a heterologous system increases Cav2.2 currents. Collectively, these results show that DISC1-dependent enhancement of SV exocytosis is mediated by Cav2.2 and point to aberrant glutamate release as a probable endophenotype of major psychiatric disorders. PMID:27378904

  16. Synaptic scaling enables dynamically distinct short- and long-term memory formation.

    PubMed

    Tetzlaff, Christian; Kolodziejski, Christoph; Timme, Marc; Tsodyks, Misha; Wörgötter, Florentin

    2013-10-01

    Memory storage in the brain relies on mechanisms acting on time scales from minutes, for long-term synaptic potentiation, to days, for memory consolidation. During such processes, neural circuits distinguish synapses relevant for forming a long-term storage, which are consolidated, from synapses of short-term storage, which fade. How time scale integration and synaptic differentiation is simultaneously achieved remains unclear. Here we show that synaptic scaling - a slow process usually associated with the maintenance of activity homeostasis - combined with synaptic plasticity may simultaneously achieve both, thereby providing a natural separation of short- from long-term storage. The interaction between plasticity and scaling provides also an explanation for an established paradox where memory consolidation critically depends on the exact order of learning and recall. These results indicate that scaling may be fundamental for stabilizing memories, providing a dynamic link between early and late memory formation processes.

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

    PubMed

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

    2014-10-13

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

  18. 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

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

    PubMed

    Bacaj, Taulant; Wu, Dick; Burré, Jacqueline; Malenka, Robert C; Liu, Xinran; Südhof, Thomas C

    2015-10-01

    In forebrain neurons, Ca(2+) 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 Ca(2+)-independent, mutations in Ca(2+)-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 Ca(2+) triggering of different phases of release.

  20. A role for BARS at the fission step of COPI vesicle formation from Golgi membrane.

    PubMed

    Yang, Jia-Shu; Lee, Stella Y; Spanò, Stefania; Gad, Helge; Zhang, Leiliang; Nie, Zhongzhen; Bonazzi, Matteo; Corda, Daniela; Luini, Alberto; Hsu, Victor W

    2005-12-07

    The core complex of Coat Protein I (COPI), known as coatomer, is sufficient to induce coated vesicular-like structures from liposomal membrane. In the context of biological Golgi membrane, both palmitoyl-coenzyme A (p-coA) and ARFGAP1, a GTPase-activating protein (GAP) for ADP-Ribosylation Factor 1, also participate in vesicle formation, but how their roles may be linked remains unknown. Moreover, whether COPI vesicle formation from Golgi membrane requires additional factors also remains unclear. We now show that Brefeldin-A ADP-Ribosylated Substrate (BARS) plays a critical role in the fission step of COPI vesicle formation from Golgi membrane. This role of BARS requires its interaction with ARFGAP1, which is in turn regulated oppositely by p-coA and nicotinamide adenine dinucleotide, which act as cofactors of BARS. Our findings not only identify a new factor needed for COPI vesicle formation from Golgi membrane but also reveal a surprising mechanism by which the roles of p-coA and GAP are linked in this process.

  1. Mechanism of Lipid Vesicles Spreading and Bilayer Formation on a Au(111) Surface.

    PubMed

    Pawłowski, Jan; Juhaniewicz, Joanna; Güzeloğlu, Alişan; Sęk, Sławomir

    2015-10-13

    Spreading of small unilamellar vesicles on solid surfaces is one of the most common ways to obtain supported lipid bilayers. Although the method has been used successfully for many years, the details of this process are still the subject of intense debate. Particularly controversial is the mechanism of bilayer formation on metallic surfaces like gold. In this work, we have employed scanning probe microscopy techniques to evaluate the details of lipid vesicles spreading and formation of the lipid bilayer on a Au(111) surface in a phosphate-buffered saline solution. Nanoscale imaging revealed that the mechanism of this process differs significantly from that usually assumed for hydrophilic surfaces such as mica, glass, and silicon oxide. Formation of the bilayer on gold involves several steps. Initially, the vesicles accumulate on a gold surface and release lipid molecules that adsorb on a Au(111) surface, giving rise to the appearance of highly ordered stripelike domains. The latter serve as a template for the buildup of a hemimicellar film, which contributes to the increased hydrophilicity of the external surface and facilitates further adsorption and rupture of the vesicles. As a result, the bilayer is spread over a hemimicellar film, and then it is followed by slow fusion between coupled layers leading to formation of a single bilayer supported on a gold surface. We believe that the results presented in this work may provide some new insights into the area of research related to supported lipid bilayers.

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

    PubMed Central

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

    2009-01-01

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

  3. Protein mutated in paroxysmal dyskinesia interacts with the active zone protein RIM and suppresses synaptic vesicle exocytosis

    PubMed Central

    Shen, Yiguo; Ge, Woo-Ping; Li, Yulong; Hirano, Arisa; Lee, Hsien-Yang; Rohlmann, Astrid; Missler, Markus; Tsien, Richard W.; Jan, Lily Yeh; Fu, Ying-Hui; Ptáček, Louis J.

    2015-01-01

    Paroxysmal nonkinesigenic dyskinesia (PNKD) is an autosomal dominant episodic movement disorder precipitated by coffee, alcohol, and stress. We previously identified the causative gene but the function of the encoded protein remains unknown. We also generated a PNKD mouse model that revealed dysregulated dopamine signaling in vivo. Here, we show that PNKD interacts with synaptic active zone proteins Rab3-interacting molecule (RIM)1 and RIM2, localizes to synapses, and modulates neurotransmitter release. Overexpressed PNKD protein suppresses release, and mutant PNKD protein is less effective than wild-type at inhibiting exocytosis. In PNKD KO mice, RIM1/2 protein levels are reduced and synaptic strength is impaired. Thus, PNKD is a novel synaptic protein with a regulatory role in neurotransmitter release. PMID:25730884

  4. Periodic Vesicle Formation in Tectonic Fault Zones--an Ideal Scenario for Molecular Evolution.

    PubMed

    Mayer, Christian; Schreiber, Ulrich; Dávila, María J

    2015-06-01

    Tectonic fault systems in the continental crust offer huge networks of interconnected channels and cavities. Filled mainly with water and carbon dioxide (CO2), containing a wide variety of hydrothermal chemistry and numerous catalytic surfaces, they may offer ideal reaction conditions for prebiotic chemistry. In these systems, an accumulation zone for organic compounds will develop at a depth of approximately 1 km where CO2 turns sub-critical and dissolved components precipitate. At this point, periodic pressure changes caused for example by tidal influences or geyser activity may generate a cyclic process involving repeated phase transitions of carbon dioxide. In the presence of amphiphilic compounds, this will necessarily lead to the transient formation of coated water droplets in the gas phase and corresponding vesicular structures in the aqueous environment. During this process, the concentration of organic components inside the droplets and vesicles would be drastically increased, allowing for favorable reaction conditions and, in case of the vesicles generated, large trans-membrane concentration gradients. Altogether, the process of periodic formation and destruction of vesicles could offer a perfect environment for molecular evolution in small compartments and for the generation of protocells. The basic process of vesicle formation is reproduced experimentally with a lipid in a water/CO2 system.

  5. Periodic Vesicle Formation in Tectonic Fault Zones—an Ideal Scenario for Molecular Evolution

    NASA Astrophysics Data System (ADS)

    Mayer, Christian; Schreiber, Ulrich; Dávila, María J.

    2015-06-01

    Tectonic fault systems in the continental crust offer huge networks of interconnected channels and cavities. Filled mainly with water and carbon dioxide (CO2), containing a wide variety of hydrothermal chemistry and numerous catalytic surfaces, they may offer ideal reaction conditions for prebiotic chemistry. In these systems, an accumulation zone for organic compounds will develop at a depth of approximately 1 km where CO2 turns sub-critical and dissolved components precipitate. At this point, periodic pressure changes caused for example by tidal influences or geyser activity may generate a cyclic process involving repeated phase transitions of carbon dioxide. In the presence of amphiphilic compounds, this will necessarily lead to the transient formation of coated water droplets in the gas phase and corresponding vesicular structures in the aqueous environment. During this process, the concentration of organic components inside the droplets and vesicles would be drastically increased, allowing for favorable reaction conditions and, in case of the vesicles generated, large trans-membrane concentration gradients. Altogether, the process of periodic formation and destruction of vesicles could offer a perfect environment for molecular evolution in small compartments and for the generation of protocells. The basic process of vesicle formation is reproduced experimentally with a lipid in a water/CO2 system.

  6. A Single Herpesvirus Protein Can Mediate Vesicle Formation in the Nuclear Envelope*

    PubMed Central

    Lorenz, Michael; Vollmer, Benjamin; Unsay, Joseph D.; Klupp, Barbara G.; García-Sáez, Ana J.; Mettenleiter, Thomas C.; Antonin, Wolfram

    2015-01-01

    Herpesviruses assemble capsids in the nucleus and egress by unconventional vesicle-mediated trafficking through the nuclear envelope. Capsids bud at the inner nuclear membrane into the nuclear envelope lumen. The resulting intralumenal vesicles fuse with the outer nuclear membrane, delivering the capsids to the cytoplasm. Two viral proteins are required for vesicle formation, the tail-anchored pUL34 and its soluble interactor, pUL31. Whether cellular proteins are involved is unclear. Using giant unilamellar vesicles, we show that pUL31 and pUL34 are sufficient for membrane budding and scission. pUL34 function can be bypassed by membrane tethering of pUL31, demonstrating that pUL34 is required for pUL31 membrane recruitment but not for membrane remodeling. pUL31 can inwardly deform membranes by oligomerizing on their inner surface to form buds that constrict to vesicles. Therefore, a single viral protein can mediate all events necessary for membrane budding and abscission. PMID:25605719

  7. Fragile X mental retardation protein controls synaptic vesicle exocytosis by modulating N-type calcium channel density.

    PubMed

    Ferron, Laurent; Nieto-Rostro, Manuela; Cassidy, John S; Dolphin, Annette C

    2014-04-07

    Fragile X syndrome (FXS), the most common heritable form of mental retardation, is characterized by synaptic dysfunction. Synaptic transmission depends critically on presynaptic calcium entry via voltage-gated calcium (Ca(V)) channels. Here we show that the functional expression of neuronal N-type Ca(V) channels (Ca(V)2.2) is regulated by fragile X mental retardation protein (FMRP). We find that FMRP knockdown in dorsal root ganglion neurons increases Ca(V) channel density in somata and in presynaptic terminals. We then show that FMRP controls Ca(V)2.2 surface expression by targeting the channels to the proteasome for degradation. The interaction between FMRP and Ca(V)2.2 occurs between the carboxy-terminal domain of FMRP and domains of Ca(V)2.2 known to interact with the neurotransmitter release machinery. Finally, we show that FMRP controls synaptic exocytosis via Ca(V)2.2 channels. Our data indicate that FMRP is a potent regulator of presynaptic activity, and its loss is likely to contribute to synaptic dysfunction in FXS.

  8. Fragile X mental retardation protein controls synaptic vesicle exocytosis by modulating N-type calcium channel density

    NASA Astrophysics Data System (ADS)

    Ferron, Laurent; Nieto-Rostro, Manuela; Cassidy, John S.; Dolphin, Annette C.

    2014-04-01

    Fragile X syndrome (FXS), the most common heritable form of mental retardation, is characterized by synaptic dysfunction. Synaptic transmission depends critically on presynaptic calcium entry via voltage-gated calcium (CaV) channels. Here we show that the functional expression of neuronal N-type CaV channels (CaV2.2) is regulated by fragile X mental retardation protein (FMRP). We find that FMRP knockdown in dorsal root ganglion neurons increases CaV channel density in somata and in presynaptic terminals. We then show that FMRP controls CaV2.2 surface expression by targeting the channels to the proteasome for degradation. The interaction between FMRP and CaV2.2 occurs between the carboxy-terminal domain of FMRP and domains of CaV2.2 known to interact with the neurotransmitter release machinery. Finally, we show that FMRP controls synaptic exocytosis via CaV2.2 channels. Our data indicate that FMRP is a potent regulator of presynaptic activity, and its loss is likely to contribute to synaptic dysfunction in FXS.

  9. Fragile X mental retardation protein controls synaptic vesicle exocytosis by modulating N-type calcium channel density

    PubMed Central

    Ferron, Laurent; Nieto-Rostro, Manuela; Cassidy, John S.; Dolphin, Annette C.

    2014-01-01

    Fragile X syndrome (FXS), the most common heritable form of mental retardation, is characterized by synaptic dysfunction. Synaptic transmission depends critically on presynaptic calcium entry via voltage-gated calcium (CaV) channels. Here we show that the functional expression of neuronal N-type CaV channels (CaV2.2) is regulated by fragile X mental retardation protein (FMRP). We find that FMRP knockdown in dorsal root ganglion neurons increases CaV channel density in somata and in presynaptic terminals. We then show that FMRP controls CaV2.2 surface expression by targeting the channels to the proteasome for degradation. The interaction between FMRP and CaV2.2 occurs between the carboxy-terminal domain of FMRP and domains of CaV2.2 known to interact with the neurotransmitter release machinery. Finally, we show that FMRP controls synaptic exocytosis via CaV2.2 channels. Our data indicate that FMRP is a potent regulator of presynaptic activity, and its loss is likely to contribute to synaptic dysfunction in FXS. PMID:24709664

  10. 24S-Hydroxycholesterol enhances synaptic vesicle cycling in the mouse neuromuscular junction: Implication of glutamate NMDA receptors and nitric oxide.

    PubMed

    Kasimov, M R; Fatkhrakhmanova, M R; Mukhutdinova, K A; Petrov, A M

    2017-01-31

    24S-hydroxycholesterol (24S-HC) is a brain-derived product of lipid metabolism present in the systemic circulation, where its level can change significantly in response to physiological and pathophysiological conditions. Here, using electrophysiological and optical approaches, we have found a high sensitivity to 24S-HC of the synaptic vesicle cycle at the mouse neuromuscular junctions. Treatment with 24S-HC increased the end plate potential amplitude (EPP) in response to a single stimulus and attenuated the EPP amplitude rundown during high frequency (HF) activity but had no influence on miniature EPP amplitude or frequency. The effects on evoked responses were associated with enhanced FM1-43 dye loading and unloading by endo- and exocytosis. Comparison of electrophysiological and optical data revealed an increase in the rate of vesicular cycling. The impact of 24S-HC was abolished or potentiated by stimulation or inhibition of NMDA-receptors respectively. Moreover, 24S-HC, acting in the same manner as the endothelial NO synthase (eNOS) inhibitor cavtratin, suppressed an increase in NO-sensitive dye fluorescence during HF stimulation, while l-glutamate had the opposite effect. Inhibitors of NOS (l-NAME and cavtratin, but not the neuronal NOS inhibitor TRIM), a scavenger of extracellular NO and a protein kinase G blocker all had stimulatory effects, similar to those of 24S-HC, on exocytosis induced by HF activity and completely masked the effect of 24S-HC. The data suggest that 24S-HC enhances synaptic vesicle cycling due to an attenuation of retrograde NO signaling that depends on eNOS. In this regard, 24S-HC counteracts the effects of NMDA-receptor stimulation at mouse neuromuscular junctions.

  11. Instability of a Lamellar Phase under Shear Flow: Formation of Multilamellar Vesicles

    NASA Astrophysics Data System (ADS)

    Courbin, L.; Delville, J. P.; Rouch, J.; Panizza, P.

    2002-09-01

    The formation of closed-compact multilamellar vesicles (referred to in the literature as the ``onion texture'') obtained upon shearing lamellar phases is studied using small-angle light scattering and cross-polarized microscopy. By varying the shear rate γ ˙, the gap cell D, and the smectic distance d, we show that: (i)the formation of this structure occurs homogeneously in the cell at a well-defined wave vector qi, via a strain-controlled process, and (ii)the value of qi varies as (dγ ˙/D)1/3. These results strongly suggest that formation of multilamellar vesicles may be monitored by an undulation (buckling) instability of the membranes, as expected from theory.

  12. Small-Angle Neutron Scattering and Spontaneous Formation of Unilamellar Vesicles: Potential Vehicles for Drug Delivery

    NASA Astrophysics Data System (ADS)

    Katsaras, John

    2004-03-01

    Unilamellar vesicles (ULVs) are single-bilayer shells with radii commonly between 10 and 100 nm, and are widely used as model membranes, drug delivery systems, microreactors and substrates for a variety of enzymes and proteins. A common method of making ULVs is the extrusion of multilamellar vesicles (MLVs) through synthetic membranes of known pore size. These extruded ULVs are invariably unstable and in due time, revert back to MLVs. Over the years there have been reports of the spontaneous formation of stable ULVs in surfactant, lipid, and lipid/detergent mixtures. These ULVs have sometimes been shown to be monodisperse and their radii were found, almost without exception, to vary with concentration. We have carried-out small-angle neutron scattering (SANS) experiments on 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 monodisperse unilamellar vesicles whose radii are concentration independent, in contrast to previous experimental observations.

  13. Trans-Synaptic Transfer of Wnt Signals Through Release of Evi/Wntless Vesicles and Trafficking of Postsynaptic Frizzled-2 Receptors

    PubMed Central

    Korkut, Ceren; Ataman, Bulent; Ramachandran, Preethi; Ashley, James; Barria, Romina; Gherbesi, Norberto; Budnik, Vivian

    2009-01-01

    Wnts play pivotal roles during development and in the mature nervous system. However, the mechanism by which Wnts traffic between cells has remained elusive. Here we demonstrate a novel mechanism of Wnt transmission through release of exosome-like vesicles containing the Wnt-binding protein Evenness Interrupted/Wntless/Sprinter (Evi/Wls/Srt). We show that at the Drosophila larval neuromuscular junction (NMJ), presynaptic vesicular release of Evi is required for the secretion of the Wnt, Wingless (Wg). We also show that Evi acts cell-autonomously in the postsynaptic Wnt-receiving cell to target dGRIP, a Wg-receptor-interacting protein, to postsynaptic sites. Upon Evi loss of function, dGRIP is not properly targeted to synaptic sites, interfering with postsynaptic Wnt signal transduction. These findings uncover a previously unknown cellular mechanism by which a secreted Wnt is transported across synapses by Evi-containing vesicles, and reveal novel trafficking functions of Evi in both the Wnt-producing and the Wnt-receiving cell. PMID:19837038

  14. Zebrafish Cacna1fa is required for cone photoreceptor function and synaptic ribbon formation

    PubMed Central

    Jia, Sujuan; Muto, Akira; Orisme, Wilda; Henson, Hannah E.; Parupalli, Chaithanyarani; Ju, Bensheng; Baier, Herwig; Taylor, Michael R.

    2014-01-01

    Mutations in the human CACNA1F gene cause incomplete congenital stationary night blindness type 2 (CSNB2), a non-progressive, clinically heterogeneous retinal disorder. However, the molecular mechanisms underlying CSNB2 have not been fully explored. Here, we describe the positional cloning of a blind zebrafish mutant, wait until dark (wud), which encodes a zebrafish homolog of human CACNA1F. We identified two zebrafish cacna1f paralogs and showed that the cacna1fa transcript (the gene mutated in wud) is expressed exclusively in the photoreceptor layer. We demonstrated that Cacna1fa localizes at the photoreceptor synapse and is absent from wud mutants. Electroretinograms revealed abnormal cone photoreceptor responses from wud mutants, indicating a defect in synaptic transmission. Although there are no obvious morphological differences, we found that wud mutants lacked synaptic ribbons and that wud is essential for the development of synaptic ribbons. We found that Ribeye, the most prominent synaptic ribbon protein, was less abundant and mislocalized in adult wud mutants. In addition to cloning wud, we identified synaptojanin 1 (synj1) as the defective gene in slacker (slak), a blind mutant with floating synaptic ribbons. We determined that Cacna1fa was expressed in slak photoreceptors and that Synj1 was initially expressed wud photoreceptors, but was absent by 5 days postfertilization. Collectively, our data demonstrate that Cacna1fa is essential for cone photoreceptor function and synaptic ribbon formation and reveal a previously unknown yet critical role of L-type voltage-dependent calcium channels in the expression and/or distribution of synaptic ribbon proteins, providing a new model to study the clinical variability in human CSNB2 patients. PMID:24419318

  15. A Preferable Method for the Formation of Vesicles from Lamellar Liquid Crystals Using Chemical Additives.

    PubMed

    Enomoto, Yasutaka; Imai, Yoko; Tajima, Kazuo

    2017-01-01

    We present a method for vesicle formation from lamellar liquid crystals (LCs) using a cationic amphiphilic substance, namely 2-hydroxyethyl di(alkanol)oxyethyl methylammonium methylsulfate (DEAE). Vesicle formation from the DEAE lamellar dispersion occurred via a two-step chemical addition. This method required neither additional mechanical energy nor the use of special solvents. The transition was solubilized using an organic substance (e.g., limonene) in the lamellar DEAE LC, after which, a small amount of inorganic salt was added to the solubilized lamellar LC dispersion with gentle stirring. The viscosity of the DEAE dispersion following salt addition decreased sharply from 10(5) mPa·s to 10(2) mPa·s, and the DEAE dispersion was converted into a high fluidity liquid. Several organic substances were examined as potential solubilizates to initiate the lamellar-vesicle transition. Inorganic salts were also examined as transition triggers using various types of electrolytes; only neutral salts were effective as trigger additives. Dissociation of inorganic salts yielded anions, which inserted between the DEAE bilayer membranes and induced OH(-) ion exchange. In addition, a number of cations simultaneously formed ion pairs with the DEAE counter ions (CH3SO4(-) ions). However, as the amount of solubilized organic substances in the DEAE bilayer membrane decreased over time, the vesicles were transformed into lamellar LCs once again. The DEAE states in each step were measured by monitoring the zeta potential, pH, viscosity, and by examination of scanning electron microscopy and atomic force microscopy images. A possible molecular mechanism for the lamellar-vesicle transition of DEAE was proposed.

  16. Early steps in primary cilium assembly require EHD1/EHD3-dependent ciliary vesicle formation.

    PubMed

    Lu, Quanlong; Insinna, Christine; Ott, Carolyn; Stauffer, Jimmy; Pintado, Petra A; Rahajeng, Juliati; Baxa, Ulrich; Walia, Vijay; Cuenca, Adrian; Hwang, Yoo-Seok; Daar, Ira O; Lopes, Susana; Lippincott-Schwartz, Jennifer; Jackson, Peter K; Caplan, Steve; Westlake, Christopher J

    2015-03-01

    Membrane association with mother centriole (M-centriole) distal appendages is critical for ciliogenesis initiation. How the Rab GTPase Rab11-Rab8 cascade functions in early ciliary membrane assembly is unknown. Here, we show that the membrane shaping proteins EHD1 and EHD3, in association with the Rab11-Rab8 cascade, function in early ciliogenesis. EHD1 and EHD3 localize to preciliary membranes and the ciliary pocket. EHD-dependent membrane tubulation is essential for ciliary vesicle formation from smaller distal appendage vesicles (DAVs). Importantly, this step functions in M-centriole to basal body transformation and recruitment of transition zone proteins and IFT20. SNAP29, a SNARE membrane fusion regulator and EHD1-binding protein, is also required for DAV-mediated ciliary vesicle assembly. Interestingly, only after ciliary vesicle assembly is Rab8 activated for ciliary growth. Our studies uncover molecular mechanisms informing a previously uncharacterized ciliogenesis step, whereby EHD1 and EHD3 reorganize the M-centriole and associated DAVs before coordinated ciliary membrane and axoneme growth.

  17. Time Resolved Neutron Reflectivity During Supported Membrane Formation by Vesicle Fusion.

    PubMed

    Koutsioubas, Alexandros; Appavou, Marie-Sousai; Lairez, Didier

    2017-09-05

    The formation of supported lipid bilayers (SLB) on hydrophilic substrates through the method of unilamelar vesicle fusion is used routinely in a wide range of biophysical studies. In an effort to control and better understand the fusion process on the substrate, many experimental studies employing different techniques have been devoted to the elucidation of the fusion mechanism. In the present work we follow the kinetics of membrane formation using time-resolved (TR) neutron reflectivity, focussing at the structural changes near the solid/liquid interface. A clear indication of stacked bilayer structure is observed during the intermediate phase of SLB formation. Adsorbed lipid mass decrease is also measured at the final stage of the process. We have found that it is essential for the analysis of the experimental results to treat theoretically the shape of adsorbed lipid vesicles on an attractive substrate. The overall findings are discussed in relation to proposed fusion mechanisms from previous literature, while we argue that our observations favour a model involving enhanced adhesion of incoming vesicles on the edges of already formed bilayer patches.

  18. Rupture of Stochastically Occurring Vesicle Clusters Limits Bilayer Formation on Alkane-PEG-Type Supports: Uncoupling Clustering from Surface Coverage.

    PubMed

    Peel, Matthew J; Cross, Stephen J; Birkholz, Oliver; Aladağ, Amine; Piehler, Jacob; Peel, Suman

    2015-08-18

    Polymer-supported bilayers (PSBs) are a recognized tool for drug discovery through function-interaction analysis of membrane proteins. While silica-supported bilayers (SSBs) spontaneously form from surface-adsorbed vesicles, successful PSB formation via a similar method has thus far been limited by an insufficient understanding of the underlying vesicle-remodelling processes. Here, we generated a polymer support through the incubation of poly-L-lysine conjugated to alkyl-chain-terminated poly(ethylene)glycol on silica. This polymer-coated silica substrate yielded efficient vesicle adsorption and spontaneous bilayer formation, thereby providing a rare opportunity to address the mechanism of PSB formation and compare it to that of SSB. The combined use of super-resolution imaging, kinetics, and simulations indicates that the rupture of stochastically formed vesicle clusters is the rate-limiting step, which is an order of magnitude higher for silica than for polymer-coated silica. This was confirmed by directly demonstrating increased rupture rates for surface adsorbed multivesicle assemblies formed by vesicle cross-linking in solution. On the basis of this key insight we surmised that a low propensity of cluster rupture can be compensated for by an increase in the number density of clusters: the deposition of a mixture of oppositely charged vesicles resulted in bilayer formation on another alkane-PEG type of interface, which despite efficient vesicle adsorption otherwise fails to support spontaneous bilayer formation. This potentially provides a universal strategy for promoting bilayer formation on resistant surfaces without resorting to modifying the surface itself. Therefore, multivesicle assemblies with tailored geometries not only could facilitate bilayer formation on polymers with interesting functional properties but also could instigate the exploration of vesicle architecture for other processes involving vesicle remodelling such as drug delivery.

  19. Kinetics of laterally nanostructured vesicle formation by self-assembly of miktoarm star terpolymers in aqueous solution.

    PubMed

    Guo, Yingying; Ma, Zengwei; Ding, Zejun; Li, Robert K Y

    2013-10-15

    Dissipative particle dynamics (DPD) simulation was used to study the self-assembly of laterally nanostructured vesicles in aqueous solution from μ-[poly(ethylethylene)]-[poly(ethylene oxide)][poly(perfluoropropylene oxide)] (μ-EOF) star terpolymers. The simulated results show that the laterally nanostructured vesicle forms when the length of the hydrophilic O blocks are relatively short. In the lateral nanostructure, the hexagonally packed domains formed by the hydrophobic F blocks are immersed in a two-dimensional hydrophobic E block matrix. The formation conditions and microstructure of the vesicles in our simulation agree with the reported experimental results from the literature. The complicated formation pathway of laterally nanostructured vesicles follows three stages: (1) combination of spherical and short cylindrical raspberry-like micelles into an intermediate polygonal sheet; (2) the intermediate polygonal sheet grows to form a larger polygonal sheet with a tail; (3) the large polygonal sheet with a tail eventually folds and forms a vesicle.

  20. Induced rupture of vesicles adsorbed on glass by pore formation at the surface-bilayer interface.

    PubMed

    Kataoka-Hamai, Chiho; Yamazaki, Tomohiko

    2015-02-03

    Supported lipid bilayers (SLBs) are often formed by spontaneous vesicle rupture and fusion on a solid surface. A well-characterized rupture mechanism for isolated vesicles is pore nucleation and expansion in the solution-exposed nonadsorbed area. In contrast, pore formation in the adsorbed bilayer region has not been investigated to date. In this work, we studied the detailed mechanisms of asymmetric rupture of giant unilamellar vesicles (GUVs) adsorbed on glass using fluorescence microscopy. Asymmetric rupture is the pathway where a rupture pore forms in a GUV near the edge of the glass-bilayer interface with high curvature and then expansion of the pore yields a planar bilayer patch. We show that asymmetric rupture occasionally resulted in SLB patches bearing a defect pore. The defect formation probability depended on lipid composition, salt concentration, and pH. Approximately 40% of negatively charged GUVs under physiological conditions formed pore-containing SLB patches, while negatively charged GUVs at low salt concentration or pH 4.0 and positively charged GUVs exhibited a low probability of defect inclusion. The edge of the defect pore was either in contact with (on-edge) or away from (off-edge) the edge of the planar bilayer. On-edge pores were predominantly formed over off-edge defects. Pores initially formed in the glass-adsorbed region before rupture, most frequently in close contact with the edge of the adsorbed region. When a pore formed near the edge of the adsorbed area or when the edge of a pore reached that of the adsorbed area by pore expansion, asymmetric rupture was induced from the defect site. These induced rupture mechanisms yielded SLB patches with an on-edge pore. In contrast, off-edge pores were produced when defect pore generation and subsequent vesicle rupture were uncoupled. The current results demonstrate that pore formation in the surface-adsorbed region of GUVs is not a negligible event.

  1. Coupling field theory with continuum mechanics: a simulation of domain formation in giant unilamellar vesicles.

    PubMed

    Ayton, Gary S; McWhirter, J Liam; McMurtry, Patrick; Voth, Gregory A

    2005-06-01

    Domain formation is modeled on the surface of giant unilamellar vesicles using a Landau field theory model for phase coexistence coupled to elastic deformation mechanics (e.g., membrane curvature). Smooth particle applied mechanics, a form of smoothed particle continuum mechanics, is used to solve either the time-dependent Landau-Ginzburg or Cahn-Hilliard free-energy models for the composition dynamics. At the same time, the underlying elastic membrane is modeled using smooth particle applied mechanics, resulting in a unified computational scheme capable of treating the response of the composition fields to arbitrary deformations of the vesicle and vice versa. The results indicate that curvature coupling, along with the field theory model for composition free energy, gives domain formations that are correlated with surface defects on the vesicle. In the case that external deformations are included, the domain structures are seen to respond to such deformations. The present simulation capability provides a significant step forward toward the simulation of realistic cellular membrane processes.

  2. Coupling Field Theory with Continuum Mechanics: A Simulation of Domain Formation in Giant Unilamellar Vesicles

    PubMed Central

    Ayton, Gary S.; McWhirter, J. Liam; McMurtry, Patrick; Voth, Gregory A.

    2005-01-01

    Domain formation is modeled on the surface of giant unilamellar vesicles using a Landau field theory model for phase coexistence coupled to elastic deformation mechanics (e.g., membrane curvature). Smooth particle applied mechanics, a form of smoothed particle continuum mechanics, is used to solve either the time-dependent Landau-Ginzburg or Cahn-Hilliard free-energy models for the composition dynamics. At the same time, the underlying elastic membrane is modeled using smooth particle applied mechanics, resulting in a unified computational scheme capable of treating the response of the composition fields to arbitrary deformations of the vesicle and vice versa. The results indicate that curvature coupling, along with the field theory model for composition free energy, gives domain formations that are correlated with surface defects on the vesicle. In the case that external deformations are included, the domain structures are seen to respond to such deformations. The present simulation capability provides a significant step forward toward the simulation of realistic cellular membrane processes. PMID:15792968

  3. Ghrelin stimulates synaptic formation in cultured cortical networks in a dose-dependent manner.

    PubMed

    Stoyanova, Irina I; le Feber, Joost; Rutten, Wim L C

    2013-09-10

    Ghrelin was initially related to appetite stimulation and growth hormone secretion. However, it also has a neuroprotective effect in neurodegenerative diseases and regulates cognitive function. The cellular basis of these processes is related to synaptic efficacy and plasticity. Previous studies indicated that ghrelin has an excitatory effect on neuronal activity, and stimulates synaptic plasticity in vivo. Plasticity in the adult brain occurs in many different ways, including changes in synapse morphology and number. Therefore, we used in vitro neuronal cultures to investigate how ghrelin affects synaptogenesis. We used dissociated cortical cultures of newborn rats, chronically treated with different doses of ghrelin (0.5, 1, 1.5 and 2μM). After one-, two-, three- or four weeks cultures were immunostained for the presynaptic marker synaptophysin. In parallel, additional groups of non-treated cultures were immunostained for detection of ghrelin receptor (GHSR1). During development, GHSR1was increasingly expressed in all type of neurons, as well as the synaptophysin. Synaptic density depended on ghrelin concentration, and was much higher than in controls in all age groups. In conclusion, ghrelin leads to earlier network formation in dissociated cortical networks and an increase in number of synapses. The effect is probably mediated by GHSR1. These findings suggest that ghrelin may provide a novel therapeutic strategy for the treatment of disorders related to synaptic impairment.

  4. Modulation of Neuronal Signal Transduction and Memory Formation by Synaptic Zinc

    PubMed Central

    Sindreu, Carlos; Storm, Daniel R.

    2011-01-01

    The physiological role of synaptic zinc has remained largely enigmatic since its initial detection in hippocampal mossy fibers over 50 years ago. The past few years have witnessed a number of studies highlighting the ability of zinc ions to regulate ion channels and intracellular signaling pathways implicated in neuroplasticity, and others that shed some light on the elusive role of synaptic zinc in learning and memory. Recent behavioral studies using knock-out mice for the synapse-specific zinc transporter ZnT-3 indicate that vesicular zinc is required for the formation of memories dependent on the hippocampus and the amygdala, two brain centers that are prominently innervated by zinc-rich fibers. A common theme emerging from this research is the activity-dependent regulation of the Erk1/2 mitogen-activated-protein kinase pathway by synaptic zinc through diverse mechanisms in neurons. Here we discuss current knowledge on how synaptic zinc may play a role in cognition through its impact on neuronal signaling. PMID:22084630

  5. Phosphorylation of synapsin I by cyclin-dependent kinase-5 sets the ratio between the resting and recycling pools of synaptic vesicles at hippocampal synapses.

    PubMed

    Verstegen, Anne M J; Tagliatti, Erica; Lignani, Gabriele; Marte, Antonella; Stolero, Tamar; Atias, Merav; Corradi, Anna; Valtorta, Flavia; Gitler, Daniel; Onofri, Franco; Fassio, Anna; Benfenati, Fabio

    2014-05-21

    Cyclin-dependent kinase-5 (Cdk5) was reported to downscale neurotransmission by sequestering synaptic vesicles (SVs) in the release-reluctant resting pool, but the molecular targets mediating this activity remain unknown. Synapsin I (SynI), a major SV phosphoprotein involved in the regulation of SV trafficking and neurotransmitter release, is one of the presynaptic substrates of Cdk5, which phosphorylates it in its C-terminal region at Ser(549) (site 6) and Ser(551) (site 7). Here we demonstrate that Cdk5 phosphorylation of SynI fine tunes the recruitment of SVs to the active recycling pool and contributes to the Cdk5-mediated homeostatic responses. Phosphorylation of SynI by Cdk5 is physiologically regulated and enhances its binding to F-actin. The effects of Cdk5 inhibition on the size and depletion kinetics of the recycling pool, as well as on SV distribution within the nerve terminal, are virtually abolished in mouse SynI knock-out (KO) neurons or in KO neurons expressing the dephosphomimetic SynI mutants at sites 6,7 or site 7 only. The observation that the single site-7 mutant phenocopies the effects of the deletion of SynI identifies this site as the central switch in mediating the synaptic effects of Cdk5 and demonstrates that SynI is necessary and sufficient for achieving the effects of the kinase on SV trafficking. The phosphorylation state of SynI by Cdk5 at site 7 is regulated during chronic modification of neuronal activity and is an essential downstream effector for the Cdk5-mediated homeostatic scaling.

  6. The ATG12-Conjugating Enzyme ATG10 Is Essential for Autophagic Vesicle Formation in Arabidopsis thaliana

    PubMed Central

    Phillips, Allison R.; Suttangkakul, Anongpat; Vierstra, Richard D.

    2008-01-01

    Autophagy is an important intracellular recycling system in eukaryotes that utilizes small vesicles to traffic cytosolic proteins and organelles to the vacuole for breakdown. Vesicle formation requires the conjugation of the two ubiquitin-fold polypeptides ATG8 and ATG12 to phosphatidylethanolamine and the ATG5 protein, respectively. Using Arabidopsis thaliana mutants affecting the ATG5 target or the ATG7 E1 required to initiate ligation of both ATG8 and ATG12, we previously showed that the ATG8/12 conjugation pathways together are important when plants encounter nutrient stress and during senescence. To characterize the ATG12 conjugation pathway specifically, we characterized a null mutant eliminating the E2-conjugating enzyme ATG10 that, similar to plants missing ATG5 or ATG7, cannot form the ATG12-ATG5 conjugate. atg10-1 plants are hypersensitive to nitrogen and carbon starvation and initiate senescence and programmed cell death (PCD) more quickly than wild type, as indicated by elevated levels of senescence- and PCD-related mRNAs and proteins during carbon starvation. As detected with a GFP-ATG8a reporter, atg10-1 and atg5-1 mutant plants fail to accumulate autophagic bodies inside the vacuole. These results indicate that ATG10 is essential for ATG12 conjugation and that the ATG12-ATG5 conjugate is necessary to form autophagic vesicles and for the timely progression of senescence and PCD in plants. PMID:18245858

  7. Structure of yeast Ape1 and its role in autophagic vesicle formation

    PubMed Central

    Su, Ming-Yuan; Peng, Wen-Hsin; Ho, Meng-Ru; Su, Shih-Chieh; Chang, Yuan-Chih; Chen, Guang-Chao; Chang, Chung-I

    2015-01-01

    In Saccharomyces cerevisiae, a constitutive biosynthetic transport pathway, termed the cytoplasm-to-vacuole targeting (Cvt) pathway, sequesters precursor aminopeptidase I (prApe1) dodecamers in the form of a large complex into a Cvt vesicle using autophagic machinery, targeting it into the vacuole (the yeast lysosome) where it is proteolytically processed into its mature form, Ape1, by removal of an amino-terminal 45-amino acid propeptide. prApe1 is thought to serve as a scaffolding cargo critical for the assembly of the Cvt vesicle by presenting the propeptide to mediate higher-ordered complex formation and autophagic receptor recognition. Here we report the X-ray crystal structure of Ape1 at 2.5 Å resolution and reveal its dodecameric architecture consisting of dimeric and trimeric units, which associate to form a large tetrahedron. The propeptide of prApe1 exhibits concentration-dependent oligomerization and forms a stable tetramer. Structure-based mutagenesis demonstrates that disruption of the inter-subunit interface prevents dodecameric assembly and vacuolar targeting in vivo despite the presence of the propeptide. Furthermore, by examining the vacuolar import of propeptide-fused exogenous protein assemblies with different quaternary structures, we found that 3-dimensional spatial distribution of propeptides presented by a scaffolding cargo is essential for the assembly of the Cvt vesicle for vacuolar delivery. This study describes a molecular framework for understanding the mechanism of Cvt or autophagosomal biogenesis in selective macroautophagy. PMID:26208681

  8. Protease-activated receptor-1 modulates hippocampal memory formation and synaptic plasticity.

    PubMed

    Almonte, Antoine G; Qadri, Laura H; Sultan, Faraz A; Watson, Jennifer A; Mount, Daniel J; Rumbaugh, Gavin; Sweatt, J David

    2013-01-01

    Protease-activated receptor-1 (PAR1) is an unusual G-protein coupled receptor (GPCR) that is activated through proteolytic cleavage by extracellular serine proteases. Although previous work has shown that inhibiting PAR1 activation is neuroprotective in models of ischemia, traumatic injury, and neurotoxicity, surprisingly little is known about PAR1's contribution to normal brain function. Here, we used PAR1-/- mice to investigate the contribution of PAR1 function to memory formation and synaptic function. We demonstrate that PAR1-/- mice have deficits in hippocampus-dependent memory. We also show that while PAR1-/- mice have normal baseline synaptic transmission at Schaffer collateral-CA1 synapses, they exhibit severe deficits in N-methyl-d-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP). Mounting evidence indicates that activation of PAR1 leads to potentiation of NMDAR-mediated responses in CA1 pyramidal cells. Taken together, this evidence and our data suggest an important role for PAR1 function in NMDAR-dependent processes subserving memory formation and synaptic plasticity.

  9. Protease-activated receptor-1 modulates hippocampal memory formation and synaptic plasticity

    PubMed Central

    Almonte, Antoine G.; Qadri, Laura H.; Sultan, Faraz A.; Watson, Jennifer A.; Mount, Daniel J.; Rumbaugh, Gavin; Sweatt, J. David

    2012-01-01

    Protease-activated receptor-1 (PAR1) is an unusual G-protein coupled receptor (GPCR) that is activated through proteolytic cleavage by extracellular serine proteases. While previous work has shown that inhibiting PAR1 activation is neuroprotective in models of ischemia, traumatic injury, and neurotoxicity, surprisingly little is known about PAR1’s contribution to normal brain function. Here we used PAR1 −/− mice to investigate the contribution of PAR1 function to memory formation and synaptic function. We demonstrate that PAR1 −/− mice have deficits in hippocampus-dependent memory. We also show that while PAR1 −/− mice have normal baseline synaptic transmission at Schaffer collateral-CA1 synapses, they exhibit severe deficits in N-methyl-D-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP). Mounting evidence indicates that activation of PAR1 leads to potentiation of NMDAR-mediated responses in CA1 pyramidal cells. Taken together, this evidence and our data suggest an important role for PAR1 function in NMDAR-dependent processes subserving memory formation and synaptic plasticity. PMID:23113835

  10. Synaptic vesicles isolated from the electric organ of Torpedo californica and from the central nervous system of Mus musculus contain small ribonucleic acids (sRNAs).

    PubMed

    Li, Huinan; Wu, Cheng; Aramayo, Rodolfo; Sachs, Matthew S; Harlow, Mark L

    2017-06-01

    Synaptic vesicles (SVs) are presynaptic organelles that load and release small molecule neurotransmitters at chemical synapses. In addition to classic neurotransmitters, we have demonstrated that SVs isolated from the Peripheral Nervous Systems (PNS) of the electric organ of Torpedo californica, a model cholinergic synapse, and SVs isolated from the Central Nervous System (CNS) of Mus musculus (mouse) contain small ribonucleic acids (sRNAs; ≤ 50 nucleotides) (Scientific Reports, 5:1-14(14918) Li et al. (2015) [1]). Our previous publication provided the five most abundant sequences associated with the T. californica SVs, and the ten most abundant sequences associated with the mouse SVs, representing 59% and 39% of the total sRNA reads sequenced, respectively). We provide here a full repository of the SV sRNAs sequenced from T. californica and the mouse deposited in the NCBI as biosamples. Three data studies are included: SVs isolated from the electric organ of T. californica using standard techniques, SVs isolated from the electric organ of T. californica using standard techniques with an additional affinity purification step, and finally, SVs isolated from the CNS of mouse. The three biosamples are available at https://www.ncbi.nlm.nih.gov/biosample/ SRS1523467, SRS1523466, and SRS1523472 respectively.

  11. Combining modelling and mutagenesis studies of synaptic vesicle protein 2A to identify a series of residues involved in racetam binding.

    PubMed

    Shi, Jiye; Anderson, Dina; Lynch, Berkley A; Castaigne, Jean-Gabriel; Foerch, Patrik; Lebon, Florence

    2011-10-01

    LEV (levetiracetam), an antiepileptic drug which possesses a unique profile in animal models of seizure and epilepsy, has as its unique binding site in brain, SV2A (synaptic vesicle protein 2A). Previous studies have used a chimaeric and site-specific mutagenesis approach to identify three residues in the putative tenth transmembrane helix of SV2A that, when mutated, alter binding of LEV and related racetam derivatives to SV2A. In the present paper, we report a combined modelling and mutagenesis study that successfully identifies another 11 residues in SV2A that appear to be involved in ligand binding. Sequence analysis and modelling of SV2A suggested residues equivalent to critical functional residues of other MFS (major facilitator superfamily) transporters. Alanine scanning of these and other SV2A residues resulted in the identification of residues affecting racetam binding, including Ile273 which differentiated between racetam analogues, when mutated to alanine. Integrating mutagenesis results with docking analysis led to the construction of a mutant in which six SV2A residues were replaced with corresponding SV2B residues. This mutant showed racetam ligand-binding affinity intermediate to the affinities observed for SV2A and SV2B.

  12. ELKS1 localizes the synaptic vesicle priming protein bMunc13-2 to a specific subset of active zones.

    PubMed

    Kawabe, Hiroshi; Mitkovski, Miso; Kaeser, Pascal S; Hirrlinger, Johannes; Opazo, Felipe; Nestvogel, Dennis; Kalla, Stefan; Fejtova, Anna; Verrier, Sophie E; Bungers, Simon R; Cooper, Benjamin H; Varoqueaux, Frederique; Wang, Yun; Nehring, Ralf B; Gundelfinger, Eckart D; Rosenmund, Christian; Rizzoli, Silvio O; Südhof, Thomas C; Rhee, Jeong-Seop; Brose, Nils

    2017-03-06

    Presynaptic active zones (AZs) are unique subcellular structures at neuronal synapses, which contain a network of specific proteins that control synaptic vesicle (SV) tethering, priming, and fusion. Munc13s are core AZ proteins with an essential function in SV priming. In hippocampal neurons, two different Munc13s-Munc13-1 and bMunc13-2-mediate opposite forms of presynaptic short-term plasticity and thus differentially affect neuronal network characteristics. We found that most presynapses of cortical and hippocampal neurons contain only Munc13-1, whereas ∼10% contain both Munc13-1 and bMunc13-2. Whereas the presynaptic recruitment and activation of Munc13-1 depends on Rab3-interacting proteins (RIMs), we demonstrate here that bMunc13-2 is recruited to synapses by the AZ protein ELKS1, but not ELKS2, and that this recruitment determines basal SV priming and short-term plasticity. Thus, synapse-specific interactions of different Munc13 isoforms with ELKS1 or RIMs are key determinants of the molecular and functional heterogeneity of presynaptic AZs.

  13. Purification from black widow spider venom of a protein factor causing the depletion of synaptic vesicles at neuromuscular junctions

    PubMed Central

    1976-01-01

    The aqueous extract of the venom glands of black widow spiders was fractionated on a column of Sephadex G-200 and then on a column of DEAE- Sephadex A-50 pH 8.2. A protein fraction was obtained that caused a great increase in the frequency of occurrence of miniature end plate potentials at the frog neuromuscular junction, and caused swelling of the nerve terminals and depleted them of their vesicles. The fraction consists of a least four protein components that are similar in their molecular weights (about 130,000) and isoelectric points (ranging from pH 5.2 to 5.5) and are immunologically indistinguishable. It contains no sugar residues and has little or no lipolytic or proteolytic activity. The fraction is toxic to mice and is different from the fractions that act on houseflies, the crayfish stretch receptor and the cockroach heart. It seems pure enough to warrant a detailed study of its site and mode of action. PMID:1030703

  14. Structural Ensembles of Membrane-bound α-Synuclein Reveal the Molecular Determinants of Synaptic Vesicle Affinity

    PubMed Central

    Fusco, Giuliana; De Simone, Alfonso; Arosio, Paolo; Vendruscolo, Michele; Veglia, Gianluigi; Dobson, Christopher M.

    2016-01-01

    A detailed characterisation of the molecular determinants of membrane binding by α-synuclein (αS), a 140-residue protein whose aggregation is associated with Parkinson’s disease, is of fundamental significance to clarify the manner in which the balance between functional and dysfunctional processes are regulated for this protein. Despite its biological relevance, the structural nature of the membrane-bound state αS remains elusive, in part because of the intrinsically dynamic nature of the protein and also because of the difficulties in studying this state in a physiologically relevant environment. In the present study we have used solid-state NMR and restrained MD simulations to refine structure and topology of the N-terminal region of αS bound to the surface of synaptic-like membranes. This region has fundamental importance in the binding mechanism of αS as it acts as to anchor the protein to lipid bilayers. The results enabled the identification of the key elements for the biological properties of αS in its membrane-bound state. PMID:27273030

  15. Formation of Protocell-like Vesicles in a Thermal Diffusion Column

    PubMed Central

    2009-01-01

    Many of the properties of bilayer membranes composed of simple single-chain amphiphiles seem to be well-suited for a potential role as primitive cell membranes. However, the spontaneous formation of membranes from such amphiphiles is a concentration-dependent process in which a significant critical aggregate concentration (cac) must be reached. Since most scenarios for the prebiotic synthesis of fatty acids and related amphiphiles would result in dilute solutions well below the cac, the identification of mechanisms that would lead to increased local amphiphile concentrations is an important aspect of defining reasonable conditions for the origin of cellular life. Narrow, vertically oriented channels within the mineral precipitates of hydrothermal vent towers have previously been proposed to act as natural Clusius−Dickel thermal diffusion columns, in which a strong transverse thermal gradient concentrates dilute molecules through the coupling of thermophoresis and convection. Here we experimentally demonstrate that a microcapillary acting as a thermal diffusion column can concentrate a solution of oleic acid. Upon concentration, self-assembly of large vesicles occurs in regions where the cac is exceeded. We detected vesicle formation by fluorescence microscopy of encapsulated dye cargoes, which simultaneously concentrated in our channels. Our findings suggest a novel means by which simple physical processes could have led to the spontaneous formation of cell-like structures from a dilute prebiotic reservoir. PMID:19601679

  16. Formation of protocell-like vesicles in a thermal diffusion column.

    PubMed

    Budin, Itay; Bruckner, Raphael J; Szostak, Jack W

    2009-07-22

    Many of the properties of bilayer membranes composed of simple single-chain amphiphiles seem to be well-suited for a potential role as primitive cell membranes. However, the spontaneous formation of membranes from such amphiphiles is a concentration-dependent process in which a significant critical aggregate concentration (cac) must be reached. Since most scenarios for the prebiotic synthesis of fatty acids and related amphiphiles would result in dilute solutions well below the cac, the identification of mechanisms that would lead to increased local amphiphile concentrations is an important aspect of defining reasonable conditions for the origin of cellular life. Narrow, vertically oriented channels within the mineral precipitates of hydrothermal vent towers have previously been proposed to act as natural Clusius-Dickel thermal diffusion columns, in which a strong transverse thermal gradient concentrates dilute molecules through the coupling of thermophoresis and convection. Here we experimentally demonstrate that a microcapillary acting as a thermal diffusion column can concentrate a solution of oleic acid. Upon concentration, self-assembly of large vesicles occurs in regions where the cac is exceeded. We detected vesicle formation by fluorescence microscopy of encapsulated dye cargoes, which simultaneously concentrated in our channels. Our findings suggest a novel means by which simple physical processes could have led to the spontaneous formation of cell-like structures from a dilute prebiotic reservoir.

  17. Formation of drug-bearing vesicles in mixed colloids of bile salts and phosphatidylcholine

    SciTech Connect

    Hjelm, R.P.; Mang, J.; Hofmann, A.F.; Schteingart, C.; Alkan-Onyuksel, H.; Ayd, S.

    1997-11-01

    This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The authors used small-angle neutron scattering to study drug interactions with mixed colloids of bile salt and phosphatidylcholine. Because the mixed colloids form liposomes spontaneously, this system is a model for drug-bile interactions that are important in understanding the efficacy of oral drug formulations and in advanced applications for liposome drug delivery systems. The authors studied particle formation in incorporation of enzymatic products formed in the gut and the effects of cholesteric drugs and taxol on vesicle formation. The studies show that particle morphology is not affected by inclusion of most cholesteric drugs and taxol, and is not affected by incorporation of the products of enzymatic action. The findings suggest that particle form is important for the physiological function of bile and they are beginning to show which drugs affect liposome formation.

  18. 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

  19. Kinetics of the enzyme-vesicle interaction including the formation of rafts and membrane strain.

    PubMed

    Zhdanov, Vladimir P; Höök, Fredrik

    2012-01-01

    In cells, an appreciable part of enzymes is associated with lipid membranes. Academic experimental studies of the function of membrane enzymes (e.g., PLA(2) representing a prototype for interfacial enzymology) are often focused on the enzyme-vesicle interaction or, more specifically, on conversion of lipid forming the external leaflet of the vesicle membrane. The corresponding kinetics are complicated by many factors inherent to the interfacial physics and chemistry. The understanding of the relative role of such factors and how they should be quantitatively described is still limited. Here, we present the mean-field kinetic equations, taking the formation of rafts in the membrane and the product-induced membrane strain into account, and analyze various scenarios of lipid conversion. In particular, we scrutinize the conditions when the kinetics may exhibit a transition from a relatively long latency period to a steady-state regime with fast nearly constant reaction rate. Specifically, we discuss the likely role of the pore formation in the external lipid layer in this transition. The latter effect may be caused by the product-induced tensile strain in this layer.

  20. The Formation of Multi-synaptic Connections by the Interaction of Synaptic and Structural Plasticity and Their Functional Consequences

    PubMed Central

    Fauth, Michael; Wörgötter, Florentin; Tetzlaff, Christian

    2015-01-01

    Cortical connectivity emerges from the permanent interaction between neuronal activity and synaptic as well as structural plasticity. An important experimentally observed feature of this connectivity is the distribution of the number of synapses from one neuron to another, which has been measured in several cortical layers. All of these distributions are bimodal with one peak at zero and a second one at a small number (3–8) of synapses. In this study, using a probabilistic model of structural plasticity, which depends on the synaptic weights, we explore how these distributions can emerge and which functional consequences they have. We find that bimodal distributions arise generically from the interaction of structural plasticity with synaptic plasticity rules that fulfill the following biological realistic constraints: First, the synaptic weights have to grow with the postsynaptic activity. Second, this growth curve and/or the input-output relation of the postsynaptic neuron have to change sub-linearly (negative curvature). As most neurons show such input-output-relations, these constraints can be fulfilled by many biological reasonable systems. Given such a system, we show that the different activities, which can explain the layer-specific distributions, correspond to experimentally observed activities. Considering these activities as working point of the system and varying the pre- or postsynaptic stimulation reveals a hysteresis in the number of synapses. As a consequence of this, the connectivity between two neurons can be controlled by activity but is also safeguarded against overly fast changes. These results indicate that the complex dynamics between activity and plasticity will, already between a pair of neurons, induce a variety of possible stable synaptic distributions, which could support memory mechanisms. PMID:25590330

  1. Spontaneous vesicle formation from sodium salt of acidic sophorolipid and its application as a skin penetration enhancer.

    PubMed

    Imura, Tomohiro; Morita, Tomotake; Fukuoka, Tokuma; Ryu, Mizuyuki; Igarashi, Keisuke; Hirata, Yoshihiko; Kitamoto, Dai

    2014-01-01

    In this study, spontaneous vesicle formation from the sodium salt of acidic sophorolipid (SLNa) was observed, and its potential application as a skin penetration enhancer for triterpene glycosides extracted from the fruits of Siraitia grosvenorii Swingle was then investigated. Dynamic light scattering (DLS) measurements of the SLNa assemblies prepared by the gentle mixing of SLNa with water (1%) showed their hydrodynamic radius (Rh) to be 96.2 nm, and their structure was assigned to be vesicles by freeze-fracture electron microscopy (FF-TEM). DLS and FF-TEM also revealed that the size of the vesicles increased with an increase in the concentration of the triterpene glycosides, indicating that the triterpene glycosides were incorporated into the SLNa vesicles. The results of an in vitro skin permeation assay, after loading the SLNa vesicles on a 3D cultured skin model, showed that the amount of SLNa that penetrated though the skin model increased with time. It was also found that the amount of permeated mogroside, which is the main active component of triterpene glycosides, was significantly enhanced by the SLNa vesicle formulation. These results clearly demonstrated that spontaneously formed vesicles composed of the bolaamphiphile SLNa are useful for application as penetration enhancers for active ingredients such as mogroside V.

  2. Synthesis and Preclinical Evaluation of 11C-UCB-J as a PET Tracer for Imaging the Synaptic Vesicle Glycoprotein 2A in the Brain.

    PubMed

    Nabulsi, Nabeel B; Mercier, Joël; Holden, Daniel; Carré, Stephane; Najafzadeh, Soheila; Vandergeten, Marie-Christine; Lin, Shu-Fei; Deo, Anand; Price, Nathalie; Wood, Martyn; Lara-Jaime, Teresa; Montel, Florian; Laruelle, Marc; Carson, Richard E; Hannestad, Jonas; Huang, Yiyun

    2016-05-01

    The synaptic vesicle glycoprotein 2A (SV2A) is found in secretory vesicles in neurons and endocrine cells. PET with a selective SV2A radiotracer will allow characterization of drugs that modulate SV2A (e.g., antiepileptic drugs) and potentially could be a biomarker of synaptic density (e.g., in neurodegenerative disorders). Here we describe the synthesis and characterization of the SV2A PET radiotracer (11)C-UCB-J ((R)-1-((3-((11)C-methyl-(11)C)pyridin-4-yl)methyl)-4-(3,4,5-trifluorophenyl)pyrrolidin-2-one) in nonhuman primates, including whole-body biodistribution. (11)C-UCB-J was prepared by C-(11)C-methylation of the 3-pyridyl trifluoroborate precursor with (11)C-methyl iodide via the Suzuki-Miyaura cross-coupling method. Rhesus macaques underwent multiple scans including coinjection with unlabeled UCB-J (17, 50, and 150 μg/kg) or preblocking with the antiepileptic drug levetiracetam at 10 and 30 mg/kg. Scans were acquired for 2 h with arterial sampling and metabolite analysis to measure the input function. Regional volume of distribution (VT) was estimated using the 1-tissue-compartment model. Target occupancy was assessed using the occupancy plot; the dissociation constant (Kd) was determined by fitting self-blocking occupancies to a 1-site model, and the maximum number of receptor binding sites (Bmax) values were derived from baseline VT and from the estimated Kd and the nondisplaceable distribution volume (VND). (11)C-UCB-J was synthesized with greater than 98% purity. (11)C-UCB-J exhibited high free fraction (0.46 ± 0.02) and metabolized at a moderate rate (39% ± 5% and 24% ± 3% parent remaining at 30 and 90 min) in plasma. In the monkey brain, (11)C-UCB-J displayed high uptake and fast kinetics. VT was high (∼25-55 mL/cm(3)) in all gray matter regions, consistent with the ubiquitous expression of SV2A. Preblocking with 10 and 30 mg/kg of levetiracetam resulted in approximately 60% and 90% occupancy, respectively. Analysis of the self-blocking scans

  3. Vesicle Docking to the Spindle Pole Body Is Necessary to Recruit the Exocyst During Membrane Formation in Saccharomyces cerevisiae

    PubMed Central

    Mathieson, Erin M.; Suda, Yasuyuki; Nickas, Mark; Snydsman, Brian; Davis, Trisha N.; Muller, Eric G. D.

    2010-01-01

    During meiosis II in Saccharomyces cerevisiae, the cytoplasmic face of the spindle pole body, referred to as the meiosis II outer plaque (MOP), is modified in both composition and structure to become the initiation site for de novo formation of a membrane called the prospore membrane. The MOP serves as a docking complex for precursor vesicles that are targeted to its surface. Using fluorescence resonance energy transfer analysis, the orientation of coiled-coil proteins within the MOP has been determined. The N-termini of two proteins, Mpc54p and Spo21p, were oriented toward the outer surface of the structure. Mutations in the N-terminus of Mpc54p resulted in a unique phenotype: precursor vesicles loosely tethered to the MOP but did not contact its surface. Thus, these mpc54 mutants separate the steps of vesicle association and docking. Using these mpc54 mutants, we determined that recruitment of the Rab GTPase Sec4p, as well as the exocyst components Sec3p and Sec8p, to the precursor vesicles requires vesicle docking to the MOP. This suggests that the MOP promotes membrane formation both by localization of precursor vesicles to a particular site and by recruitment of a second tethering complex, the exocyst, that stimulates downstream events of fusion. PMID:20826607

  4. Anti-convulsive and anti-epileptic properties of brivaracetam (ucb 34714), a high-affinity ligand for the synaptic vesicle protein, SV2A

    PubMed Central

    Matagne, A; Margineanu, D-G; Kenda, B; Michel, P; Klitgaard, H

    2008-01-01

    Background and purpose: Screening of 12 000 compounds for binding affinity to the synaptic vesicle protein 2A (SV2A), identified a high-affinity pyrrolidone derivative, brivaracetam (ucb 34714). This study examined its pharmacological profile in various in vitro and in vivo models of seizures and epilepsy, to evaluate its potential as a new antiepileptic drug. Experimental approach: The effects of brivaracetam and levetiracetam on epileptiform activity and seizure expression were examined in rat hippocampal slices, corneally kindled mice, audiogenic seizure–susceptible mice, maximal electroshock and pentylenetetrazol seizures in mice, hippocampal-kindled rats, amygdala-kindled rats and genetic absence epilepsy rats. Key results: Brivaracetam and levetiracetam reduced epileptiform responses in rat hippocampal slices, brivaracetam being most potent. Brivaracetam also differed from levetiracetam by its ability to protect against seizures in normal mice induced by a maximal electroshock or maximal dose of pentylenetetrazol. In corneally kindled mice and hippocampal-kindled rats, brivaracetam induced potent protection against secondarily generalized motor seizures and showed anti-kindling properties superior to levetiracetam. In amygdala-kindled rats, brivaracetam induced a significant suppression in motor-seizure severity and, contrary to levetiracetam, reduced the after-discharge at a higher dose. Audiogenic seizure–susceptible mice were protected more potently against the expression of clonic convulsions by brivaracetam than by levetiracetam. Brivaracetam induced a more complete suppression of spontaneous spike-and-wave discharges in genetic absence epilepsy rats than levetiracetam. Conclusions and implications: Brivaracetam has higher potency and efficacy than levetiracetam as an anti-seizure and anti-epileptogenic agent in various experimental models of epilepsy, and a wide therapeutic index. PMID:18500360

  5. Low distribution of synaptic vesicle protein 2A and synaptotagimin-1 in the cerebral cortex and hippocampus of spontaneously epileptic rats exhibiting both tonic convulsion and absence seizure.

    PubMed

    Hanaya, R; Hosoyama, H; Sugata, S; Tokudome, M; Hirano, H; Tokimura, H; Kurisu, K; Serikawa, T; Sasa, M; Arita, K

    2012-09-27

    The spontaneously epileptic rat (SER) is a double mutant (zi/zi, tm/tm) which begins to exhibit tonic convulsions and absence seizures after 6 weeks of age, and repetitive tonic seizures over time induce sclerosis-like changes in SER hippocampus with high brain-derived neurotrophic factor (BDNF) expression. Levetiracetam, which binds to synaptic vesicle protein 2A (SV2A), inhibited both tonic convulsions and absence seizures in SERs. We studied SER brains histologically and immunohistochemically after verification by electroencephalography (EEG), as SERs exhibit seizure-related alterations in the cerebral cortex and hippocampus. SERs did not show interictal abnormal spikes and slow waves typical of focal epilepsy or symptomatic generalized epilepsy. The difference in neuronal density of the cerebral cortex was insignificant between SER and Wistar rats, and apoptotic neurons did not appear in SERs. BDNF distributions portrayed higher values in the entorhinal and piriform cortices which would relate with hippocampal sclerosis-like changes. Similar synaptophysin expression in the cerebral cortex and hippocampus was found in both animals. Low and diffuse SV2A distribution portrayed in the cerebral cortex and hippocampus of SERs was significantly less than that of all cerebral lobes and inner molecular layer (IML) of the dentate gyrus (DG) of Wistar rats. The extent of low SV2A expression/distribution in SERs was particularly remarkable in the frontal (51% of control) and entorhinal cortices (47%). Lower synaptotagmin-1 expression (vs Wistar rats) was located in the frontal (31%), piriform (13%) and entorhinal (39%) cortices, and IML of the DG (38%) in SER. Focal low distribution of synaptotagmin-1 accompanying low SV2A expression may contribute to epileptogenesis and seizure propagation in SER.

  6. GluRδ2 assembles four neurexins into trans-synaptic triad to trigger synapse formation.

    PubMed

    Lee, Sung-Jin; Uemura, Takeshi; Yoshida, Tomoyuki; Mishina, Masayoshi

    2012-03-28

    Elucidation of molecular mechanisms of synapse formation is a prerequisite for the understanding of neural wiring, higher brain functions, and mental disorders. The trans-synaptic interaction of postsynaptic glutamate receptor δ2 (GluRδ2) and presynaptic neurexins (NRXNs) through cerebellin precursor protein 1 (Cbln1) mediates synapse formation in vivo in the cerebellum. Here, we asked how the trans-synaptic triad induces synapse formation. Native GluRδ2 existed as a tetramer in the membrane, whereas the N-terminal domain (NTD) of GluRδ2 formed a stable homodimer. When incubated with cultured mouse cerebellar granule cells (GCs), dimeric GluRδ2-NTD and Cbln1 exerted little effect on the accumulation of punctate immunostaining signals for Bassoon and vesicular glutamate transporter 1 in GC axons. However, tetramerized GluRδ2-NTD stimulated the accumulation of these presynaptic proteins in the axons. Analysis of Cbln1 mutants suggested that the binding sites of GluRδ2 and NRXN1β on Cbln1 are differential. Furthermore, there was no competition in the binding to Cbln1 between GluRδ2-NTD and the extracellular domain (ECD) of NRXN1β. Thus, GluRδ2 and Cbln1 interacted with each other rather independently of Cbln1-NRXN1β interaction and vice versa. Gel filtration and isothermal titration calorimetry analyses consistently showed that dimeric GluRδ2-NTD and hexameric Cbln1 assembled in the 1:1 ratio, whereas hexameric Cbln1 and the laminin-neurexin-sex hormone-binding globulin domain of NRXN1β-ECD assembled in the 1:2 ratio. Thus, the synaptogenic triad is assembled from tetrameric GluRδ2, hexameric Cbln1, and monomeric NRXN in the ratio of 1:2:4. These results suggest that GluRδ2 triggers synapse formation by clustering four NRXNs through triad formation.

  7. Early steps in primary cilium assembly require EHD1- and EHD3-dependent ciliary vesicle formation

    PubMed Central

    Ott, Carolyn; Stauffer, Jimmy; Pintado, Petra A.; Rahajeng, Juliati; Baxa, Ulrich; Walia, Vijay; Cuenca, Adrian; Hwang, Yoo-Seok; Daar, Ira O.; Lopes, Susana; Lippincott-Schwartz, Jennifer; Jackson, Peter K.; Caplan, Steve; Westlake, Christopher J.

    2015-01-01

    Membrane association with mother centriole (M-centriole) distal appendages is critical for ciliogenesis initiation. How the Rab GTPase Rab11-Rab8 cascade functions in early ciliary membrane assembly is unknown. Here, we show that the membrane shaping proteins EHD1 and EHD3, in association with the Rab11-Rab8 cascade, function in early ciliogenesis. EHD1 and EHD3 localize to pre-ciliary membranes and the ciliary pocket. EHD-dependent membrane tubulation is essential for ciliary vesicle (CV) formation from smaller distal appendage vesicles (DAV). Importantly, this step functions in M-centriole to basal body transformation and recruitment of transition zone proteins and IFT20. SNAP29, a SNARE membrane fusion regulator and EHD1-binding protein, is also required for DAV-mediated CV assembly. Interestingly, only after CV assembly is Rab8 activated for ciliary growth. Our studies uncover molecular mechanisms informing a previously uncharacterized ciliogenesis step whereby EHD1 and EHD3 reorganize the M-centriole and associated DAV prior to coordinated ciliary membrane and axoneme growth. PMID:25686250

  8. Controlling the Formation of Phospholipid Monolayer, Bilayer, and Intact Vesicle Layer on Graphene.

    PubMed

    Tabaei, Seyed R; Ng, Wei Beng; Cho, Sang-Joon; Cho, Nam-Joon

    2016-05-11

    Exciting progress has been made in the use of graphene for bio- and chemical sensing applications. In this regard, interfacing lipid membranes with graphene provides a high-sealing interface that is resistant to nonspecific protein adsorption and suitable for measuring biomembrane-associated interactions. However, a controllable method to form well-defined lipid bilayer coatings remains elusive, and there are varying results in the literature. Herein, we demonstrate how design strategies based on molecular self-assembly and surface chemistry can be employed to coat graphene surface with different classes of lipid membrane architectures. We characterize the self-assembly of lipid membranes on CVD-graphene using quartz crystal microbalance with dissipation, field-effect transistor, and Raman spectroscopy. By employing the solvent-assisted lipid bilayer (SALB) method, a lipid monolayer and bilayer were formed on pristine and oxygen-plasma-treated CVD-graphene, respectively. On these surfaces, vesicle fusion method resulted in formation of a lipid monolayer and intact vesicle layer, respectively. Collectively, these findings provide the basis for improved surface functionalization strategies on graphene toward bioelectronic applications.

  9. Mitochondria-Regulated Formation of Endothelial Extracellular Vesicles Shifts the Mediator of Flow-Induced Vasodilation.

    PubMed

    Freed, Julie K; Durand, Matthew J; Hoffmann, Brian Robert; Densmore, John C; Greene, Andrew S; Gutterman, David D

    2017-02-17

    In order to examine the effect of endothelial-derived extracellular vesicles (eEVs) on the mechanism of flow-induced dilation (FID) composition, formation, and functional effects on the mechanism of FID were examined from two different eEV subtypes, one produced from ceramide, the other from plasminogen-activator inhibitor 1 (PAI-1). Using videomicroscopy, internal diameter changes in response to increases in flow were measured in human adipose resistance arteries acutely exposed (30min) to eEVs derived from cultured endothelial cells exposed to ceramide or PAI-1. FID was significantly impaired following exposure to 500K/mL (K=1000) of ceramide-induced eEVs (Cer-eEVs) but unaffected by 250K/mL. FID was reduced in the presence of PEG-catalase following administration of 250K/mL of Cer-eEVs and PAI-1 eEVs whereas Nω-Nitro-L-arginine methyl ester (ʟ-NAME) had no effect. Pathway analysis following protein composition examination using liquid chromatography tandem mass spectrometry (LC MS/MS) demonstrated that both subtypes were strongly linked to similar biological functions, primarily, mitochondrial dysfunction. Flow cytometry was utilized to quantify eEVs in the presence or absence of PBA and mito PBA, cytosolic and mitochondrial-targeted anti-oxidants, respectively. eEV formation was significantly and dramatically reduced with mito PBA treatment. In conclusion, eEVs have a biphasic effect with higher doses impairing and lower doses shifting the mediator of FID from nitric oxide (NO) to hydrogen peroxide (H2O2). Despite differences in protein content, eEVs may alter vascular function in similar directions regardless of the stimulus used for their formation. Further, mitochondrial ROS production is required for the generation of these vesicles.

  10. Astrocytes optimize synaptic fidelity

    NASA Astrophysics Data System (ADS)

    Nadkarni, Suhita; Jung, Peter; Levine, Herbert

    2007-03-01

    Most neuronal synapses in the central nervous system are enwrapped by an astrocytic process. This relation allows the astrocyte to listen to and feed back to the synapse and to regulate synaptic transmission. We combine a tested mathematical model for the Ca^2+ response of the synaptic astrocyte and presynaptic feedback with a detailed model for vesicle release of neurotransmitter at active zones. The predicted Ca^2+ dependence of the presynaptic synaptic vesicle release compares favorably for several types of synapses, including the Calyx of Held. We hypothesize that the feedback regulation of the astrocyte onto the presynaptic terminal optimizes the fidelity of the synapse in terms of information transmission.

  11. Involvement of ryanodine receptors in neurotrophin-induced hippocampal synaptic plasticity and spatial memory formation

    PubMed Central

    Adasme, Tatiana; Haeger, Paola; Paula-Lima, Andrea C.; Espinoza, Italo; Casas-Alarcón, M. Mercedes; Carrasco, M. Angélica; Hidalgo, Cecilia

    2011-01-01

    Ryanodine receptors (RyR) amplify activity-dependent calcium influx via calcium-induced calcium release. Calcium signals trigger postsynaptic pathways in hippocampal neurons that underlie synaptic plasticity, learning, and memory. Recent evidence supports a role of the RyR2 and RyR3 isoforms in these processes. Along with calcium signals, brain-derived neurotrophic factor (BDNF) is a key signaling molecule for hippocampal synaptic plasticity and spatial memory. Upon binding to specific TrkB receptors, BDNF initiates complex signaling pathways that modify synaptic structure and function. Here, we show that BDNF-induced remodeling of hippocampal dendritic spines required functional RyR. Additionally, incubation with BDNF enhanced the expression of RyR2, RyR3, and PKMζ, an atypical protein kinase C isoform with key roles in hippocampal memory consolidation. Consistent with their increased RyR protein content, BDNF-treated neurons generated larger RyR-mediated calcium signals than controls. Selective inhibition of RyR-mediated calcium release with inhibitory ryanodine concentrations prevented the PKMζ, RyR2, and RyR3 protein content enhancement induced by BDNF. Intrahippocampal injection of BDNF or training rats in a spatial memory task enhanced PKMζ, RyR2, RyR3, and BDNF hippocampal protein content, while injection of ryanodine at concentrations that stimulate RyR-mediated calcium release improved spatial memory learning and enhanced memory consolidation. We propose that RyR-generated calcium signals are key features of the complex neuronal plasticity processes induced by BDNF, which include increased expression of RyR2, RyR3, and PKMζ and the spine remodeling required for spatial memory formation. PMID:21282625

  12. Interaction of quorum signals with outer membrane lipids: insights into prokaryotic membrane vesicle formation.

    PubMed

    Mashburn-Warren, Lauren; Howe, Jörg; Garidel, Patrick; Richter, Walter; Steiniger, Frank; Roessle, Manfred; Brandenburg, Klaus; Whiteley, Marvin

    2008-07-01

    Bacteria have evolved elaborate communication strategies to co-ordinate their group activities, a process termed quorum sensing (QS). Pseudomonas aeruginosa is an opportunistic pathogen that utilizes QS for diverse activities, including disease pathogenesis. P. aeruginosa has evolved a novel communication system in which the signal molecule 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas Quinolone Signal, PQS) is trafficked between cells via membrane vesicles (MVs). Not only is PQS packaged into MVs, it is required for MV formation. Although MVs are involved in important biological processes aside from signalling, the molecular mechanism of MV formation is unknown. To provide insight into the molecular mechanism of MV formation, we examined the interaction of PQS with bacterial lipids. Here, we show that PQS interacts strongly with the acyl chains and 4'-phosphate of bacterial lipopolysaccharide (LPS). Using PQS derivatives, we demonstrate that the alkyl side-chain and third position hydroxyl of PQS are critical for these interactions. Finally, we show that PQS stimulated purified LPS to form liposome-like structures. These studies provide molecular insight into P. aeruginosa MV formation and demonstrate that quorum signals serve important non-signalling functions.

  13. Role of Pseudomonas aeruginosa Peptidoglycan-Associated Outer Membrane Proteins in Vesicle Formation

    PubMed Central

    Wessel, Aimee K.; Liew, Jean; Kwon, Taejoon; Marcotte, Edward M.

    2013-01-01

    Gram-negative bacteria produce outer membrane vesicles (OMVs) that package and deliver proteins, small molecules, and DNA to prokaryotic and eukaryotic cells. The molecular details of OMV biogenesis have not been fully elucidated, but peptidoglycan-associated outer membrane proteins that tether the outer membrane to the underlying peptidoglycan have been shown to be critical for OMV formation in multiple Enterobacteriaceae. In this study, we demonstrate that the peptidoglycan-associated outer membrane proteins OprF and OprI, but not OprL, impact production of OMVs by the opportunistic pathogen Pseudomonas aeruginosa. Interestingly, OprF does not appear to be important for tethering the outer membrane to peptidoglycan but instead impacts OMV formation through modulation of the levels of the Pseudomonas quinolone signal (PQS), a quorum signal previously shown by our laboratory to be critical for OMV formation. Thus, the mechanism by which OprF impacts OMV formation is distinct from that for other peptidoglycan-associated outer membrane proteins, including OprI. PMID:23123904

  14. Role of Pseudomonas aeruginosa peptidoglycan-associated outer membrane proteins in vesicle formation.

    PubMed

    Wessel, Aimee K; Liew, Jean; Kwon, Taejoon; Marcotte, Edward M; Whiteley, Marvin

    2013-01-01

    Gram-negative bacteria produce outer membrane vesicles (OMVs) that package and deliver proteins, small molecules, and DNA to prokaryotic and eukaryotic cells. The molecular details of OMV biogenesis have not been fully elucidated, but peptidoglycan-associated outer membrane proteins that tether the outer membrane to the underlying peptidoglycan have been shown to be critical for OMV formation in multiple Enterobacteriaceae. In this study, we demonstrate that the peptidoglycan-associated outer membrane proteins OprF and OprI, but not OprL, impact production of OMVs by the opportunistic pathogen Pseudomonas aeruginosa. Interestingly, OprF does not appear to be important for tethering the outer membrane to peptidoglycan but instead impacts OMV formation through modulation of the levels of the Pseudomonas quinolone signal (PQS), a quorum signal previously shown by our laboratory to be critical for OMV formation. Thus, the mechanism by which OprF impacts OMV formation is distinct from that for other peptidoglycan-associated outer membrane proteins, including OprI.

  15. Evolution of diverse cell division and vesicle formation systems in Archaea

    PubMed Central

    Makarova, Kira S.; Yutin, Natalya; Bell, Stephen D.; Koonin, Eugene V.

    2012-01-01

    Recently a novel cell division system comprised of homologues of eukaryotic ESCRT-III (endosomal sorting complex required for transport III) proteins was discovered in the hyperthermophilic crenarchaeote Sulfolobus acidocaldarius. On the basis of this discovery, we undertook a comparative genomic analysis of the machineries for cell division and vesicle formation in Archaea. Archaea possess at least three distinct membrane remodelling systems: the FtsZ-based bacterial-type system, the ESCRT-III-based eukaryote-like system and a putative novel system that uses an archaeal actin-related protein. Many archaeal genomes encode assortments of components from different systems. Evolutionary reconstruction from these findings suggests that the last common ancestor of the extant Archaea possessed a complex membrane remodelling apparatus, different components of which were lost during subsequent evolution of archaeal lineages. By contrast, eukaryotes seem to have inherited all three ancestral systems. PMID:20818414

  16. Fast formation of low-defect-density tethered bilayers by fusion of multilamellar vesicles.

    PubMed

    Ragaliauskas, Tadas; Mickevicius, Mindaugas; Rakovska, Bozena; Penkauskas, Tadas; Vanderah, David J; Heinrich, Frank; Valincius, Gintaras

    2017-05-01

    A facile and reproducible preparation of surface-supported lipid bilayers is essential for fundamental membrane research and biotechnological applications. We demonstrate that multilamellar vesicles fuse to molecular-anchor-grafted surfaces yielding low-defect-density, tethered bilayer membranes. Continuous bilayers are formed within 10min, while the electrically insulating bilayers with <0.1μm(-2) defect density can be accomplished within 60min. Surface plasmon resonance spectroscopy indicates that an amount of lipid material transferred from vesicles to a surface is inversely proportional to the density of an anchor, while the total amount of lipid that includes tethered and transferred lipid remains constant within 5% standard error. This attests for the formation of intact bilayers independent of the tethering agent density. Neutron reflectometry (NR) revealed the atomic level structural details of the tethered bilayer showing, among other things, that the total thickness of the hydrophobic slab of the construct was 3.2nm and that the molar fraction of cholesterol in lipid content is essentially the same as the molar fraction of cholesterol in the multilamellar liposomes. NR also indicated the formation of an overlayer with an effective thickness of 1.9nm. These overlayers may be easily removed by a single rinse of the tethered construct with 30% ethanol solution. Fast assembly and low residual defect density achievable within an hour of fusion makes our tethered bilayer methodology an attractive platform for biosensing of membrane damaging agents, such as pore forming toxins. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Auxilin is required for formation of Golgi-derived clathrin-coated vesicles during Drosophila spermatogenesis

    PubMed Central

    Zhou, Xin; Fabian, Lacramioara; Bayraktar, Jennifer L.; Ding, Hong-Mei; Brill, Julie A.; Chang, Henry C.

    2011-01-01

    SUMMARY Clathrin has previously been implicated in Drosophila male fertility and spermatid individualization. To understand further the role of membrane transport in this process, we analyzed the phenotypes of mutations in Drosophila auxilin (aux), a regulator of clathrin function, in spermatogenesis. Like partial loss-of-function Clathrin heavy chain (Chc) mutants, aux mutant males are sterile and produce no mature sperm. The reproductive defects of aux males were rescued by male germ cell-specific expression of aux, indicating that auxilin function is required autonomously in the germ cells. Furthermore, this rescue depends on both the clathrin-binding and J domains, suggesting that the ability of Aux to bind clathrin and the Hsc70 ATPase is essential for sperm formation. aux mutant spermatids show a deficit in formation of the plasma membrane during elongation, which probably disrupts the subsequent coordinated migration of investment cones during individualization. In wild-type germ cells, GFP-tagged clathrin localized to clusters of vesicular structures near the Golgi. These structures also contained the Golgi-associated clathrin adaptor AP-1, suggesting that they were Golgi-derived. By contrast, in aux mutant cells, clathrin localized to abnormal patches surrounding the Golgi and its colocalization with AP-1 was disrupted. Based on these results, we propose that Golgi-derived clathrin-positive vesicles are normally required for sustaining the plasma membrane increase necessary for spermatid differentiation. Our data suggest that Aux participates in forming these Golgi-derived clathrin-positive vesicles and that Aux, therefore, has a role in the secretory pathway. PMID:21343365

  18. Promoter-Specific Effects of DREADD Modulation on Hippocampal Synaptic Plasticity and Memory Formation

    PubMed Central

    López, Alberto J.; Kramár, Enikö; Matheos, Dina P.; White, André O.; Kwapis, Janine; Vogel-Ciernia, Annie; Sakata, Keith; Espinoza, Monica

    2016-01-01

    Designer receptors exclusively activated by designer drug (DREADDs) are a novel tool with the potential to bidirectionally drive cellular, circuit, and ultimately, behavioral changes. We used DREADDs to evaluate memory formation in a hippocampus-dependent task in mice and effects on synaptic physiology in the dorsal hippocampus. We expressed neuron-specific (hSyn promoter) DREADDs that were either excitatory (HM3D) or inhibitory (HM4D) in the dorsal hippocampus. As predicted, hSyn–HM3D was able to transform a subthreshold learning event into long-term memory (LTM), and hSyn–HM4D completely impaired LTM formation. Surprisingly, the opposite was observed during experiments examining the effects on hippocampal long-term potentiation (LTP). hSyn–HM3D impaired LTP and hSyn–HM4D facilitated LTP. Follow-up experiments indicated that the hSyn–HM3D-mediated depression of fEPSP appears to be driven by presynaptic activation of inhibitory currents, whereas the hSyn–HM4D-mediated increase of fEPSP is induced by a reduction in GABAA receptor function. To determine whether these observations were promoter specific, we next examined the effects of using the CaMKIIα promoter that limits expression to forebrain excitatory neurons. CaMKIIα–HM3D in the dorsal hippocampus led to the transformation of a subthreshold learning event into LTM, whereas CaMKIIα–HM4D blocked LTM formation. Consistent with these findings, baseline synaptic transmission and LTP was increased in CaMKIIα–HM3D hippocampal slices, whereas slices from CaMKIIα–HM4D mice produced expected decreases in baseline synaptic transmission and LTP. Together, these experiments further demonstrate DREADDs as being a robust and reliable means of modulating neuronal function to manipulate long-term changes in behavior, while providing evidence for specific dissociations between LTM and LTP. SIGNIFICANCE STATEMENT This study evaluates the efficacy of designer receptors exclusively activated by designer

  19. Promoter-Specific Effects of DREADD Modulation on Hippocampal Synaptic Plasticity and Memory Formation.

    PubMed

    López, Alberto J; Kramár, Enikö; Matheos, Dina P; White, André O; Kwapis, Janine; Vogel-Ciernia, Annie; Sakata, Keith; Espinoza, Monica; Wood, Marcelo A

    2016-03-23

    Designer receptors exclusively activated by designer drug (DREADDs) are a novel tool with the potential to bidirectionally drive cellular, circuit, and ultimately, behavioral changes. We used DREADDs to evaluate memory formation in a hippocampus-dependent task in mice and effects on synaptic physiology in the dorsal hippocampus. We expressed neuron-specific (hSyn promoter) DREADDs that were either excitatory (HM3D) or inhibitory (HM4D) in the dorsal hippocampus. As predicted, hSyn-HM3D was able to transform a subthreshold learning event into long-term memory (LTM), and hSyn-HM4D completely impaired LTM formation. Surprisingly, the opposite was observed during experiments examining the effects on hippocampal long-term potentiation (LTP). hSyn-HM3D impaired LTP and hSyn-HM4D facilitated LTP. Follow-up experiments indicated that the hSyn-HM3D-mediated depression of fEPSP appears to be driven by presynaptic activation of inhibitory currents, whereas the hSyn-HM4D-mediated increase of fEPSP is induced by a reduction in GABAA receptor function. To determine whether these observations were promoter specific, we next examined the effects of using the CaMKIIα promoter that limits expression to forebrain excitatory neurons. CaMKIIα-HM3D in the dorsal hippocampus led to the transformation of a subthreshold learning event into LTM, whereas CaMKIIα-HM4D blocked LTM formation. Consistent with these findings, baseline synaptic transmission and LTP was increased in CaMKIIα-HM3D hippocampal slices, whereas slices from CaMKIIα-HM4D mice produced expected decreases in baseline synaptic transmission and LTP. Together, these experiments further demonstrate DREADDs as being a robust and reliable means of modulating neuronal function to manipulate long-term changes in behavior, while providing evidence for specific dissociations between LTM and LTP. This study evaluates the efficacy of designer receptors exclusively activated by designer drug (DREADDs) as a means of bidirectionally

  20. Synaptic plasticity in the trigeminal principal nucleus during the period of barrelette formation and consolidation

    PubMed Central

    Guido, William; Lo, Fu-Sun; Erzurumlu, Reha S.

    2013-01-01

    We examined whether the postsynaptic responses of cells in the principal sensory nucleus of the trigeminal nerve (PrV) are subject to long-term changes in synaptic strength, and if such changes were correlated the whisker-specific patterning during and just after the critical period for pattern formation. We used an in vitro brainstem preparation in which the trigeminal ganglion (TG) and PrV remained attached. By electrically activating TG afferents, we evoked large-amplitude extracellular field potentials. These responses were postsynaptic in origin and blocked by the glutamate antagonist, DNQX. At P1, a time when barrelettes are consolidating, high frequency stimulation of their afferents led to an immediate (<1 min) and long-lasting (≥90 min) reduction (35%) in the amplitude of the evoked response. At P3–7, when the pattern of barrelettes have stabilized, the same form of tetanus led to an immediate and long-lasting increase (40%) in the amplitude of the response. Both forms of synaptic plasticity were mediated by the activation of L-type Ca2+ channels. Application of the L-type channel blocker, nitrendipine, led to a complete blockade of any the tetanus induced changes. These associative processes may regulate the patterning and maintenance of whisker-specific patterns in the brainstem trigeminal nuclei. PMID:11744112

  1. Regulators of synaptic transmission: roles in the pathogenesis and treatment of epilepsy.

    PubMed

    Casillas-Espinosa, Pablo M; Powell, Kim L; O'Brien, Terence J

    2012-12-01

    Synaptic transmission is the communication between a presynaptic and a postsynaptic neuron, and the subsequent processing of the signal. These processes are complex and highly regulated, reflecting their importance in normal brain functioning and homeostasis. Sustaining synaptic transmission depends on the continuing cycle of synaptic vesicle formation, release, and endocytosis, which requires proteins such as dynamin, syndapin, synapsin, and synaptic vesicle protein 2A. Synaptic transmission is regulated by diverse mechanisms, including presynaptic modulators of synaptic vesicle formation and release, postsynaptic receptors and signaling, and modulators of neurotransmission. Neurotransmitters released presynaptically can bind to their postsynaptic receptors, the inhibitory γ-aminobutyric acid (GABA)ergic receptors or the excitatory glutamate receptors. Once released, glutamate activates a variety of postsynaptic receptors including α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-D-aspartate (NMDA), kainate, and metabotropic receptors. The activation of the receptors triggers downstream signaling cascades generating a vast array of effects, which can be modulated by a numerous auxiliary regulatory subunits. Moreover, different neuropeptides such as neuropeptide Y, brain-derived neurotrophic factor (BDNF), somatostatin, ghrelin, and galanin, act as regulators of diverse synaptic functions and along with the classic neurotransmitters. Abnormalities in the regulation of synaptic transmission play a critical role in the pathogenesis of numerous brain diseases, including epilepsy. This review focuses on the different mechanisms involved in the regulation of synaptic transmission, which may play a role in the pathogenesis of epilepsy: the presynaptic modulators of synaptic vesicle formation and release, postsynaptic receptors, and modulators of neurotransmission, including the mechanism by which drugs can modulate the frequency and severity of

  2. The Neurexin/N-Ethylmaleimide-sensitive Factor (NSF) Interaction Regulates Short Term Synaptic Depression*♦

    PubMed Central

    Li, Tao; Tian, Yao; Li, Qian; Chen, Huiying; Lv, Huihui; Xie, Wei; Han, Junhai

    2015-01-01

    Although Neurexins, which are cell adhesion molecules localized predominantly to the presynaptic terminals, are known to regulate synapse formation and synaptic transmission, their roles in the regulation of synaptic vesicle release during repetitive nerve stimulation are unknown. Here, we show that nrx mutant synapses exhibit rapid short term synaptic depression upon tetanic nerve stimulation. Moreover, we demonstrate that the intracellular region of NRX is essential for synaptic vesicle release upon tetanic nerve stimulation. Using a yeast two-hybrid screen, we find that the intracellular region of NRX interacts with N-ethylmaleimide-sensitive factor (NSF), an enzyme that mediates soluble NSF attachment protein receptor (SNARE) complex disassembly and plays an important role in synaptic vesicle release. We further map the binding sites of each molecule and demonstrate that the NRX/NSF interaction is critical for both the distribution of NSF at the presynaptic terminals and SNARE complex disassembly. Our results reveal a previously unknown role of NRX in the regulation of short term synaptic depression upon tetanic nerve stimulation and provide new mechanistic insights into the role of NRX in synaptic vesicle release. PMID:25953899

  3. Structure formation in binary mixtures of surfactants: vesicle opening-up to bicelles and octopus-like micelles

    NASA Astrophysics Data System (ADS)

    Noguchi, Hiroshi

    Micelle formation in binary mixtures of surfactants is studied using a coarse-grained molecular simulation. When a vesicle composed of lipid and detergent types of molecules is ruptured, a disk-shaped micelle, the bicelle, is typically formed. It is found that cup-shaped vesicles and bicelles connected with worm-like micelles are also formed depending on the surfactant ratio and critical micelle concentration. The obtained octopus shape of micelles agree with those observed in the cryo-TEM images reported in [S. Jain and F. S. Bates, Macromol. 37, 1511 (2004).]. Two types of connection structures between the worm-like micelles and the bicelles are revealed.

  4. Ternary phase behaviour and vesicle formation of a sodium N-lauroylsarcosinate hydrate/1-decanol/water system

    NASA Astrophysics Data System (ADS)

    Akter, Nasima; Radiman, Shahidan; Mohamed, Faizal; Rahman, Irman Abdul; Reza, Mohammad Imam Hasan

    2011-08-01

    The phase behaviour of a system composed of amino acid-based surfactant (sodium N-lauroylsarcosinate hydrate), 1-decanol and deionised water was investigated for vesicle formation. Changing the molar ratio of the amphiphiles, two important aggregate structures were observed in the aqueous corner of the phase diagram. Two different sizes of microemulsions were found at two amphiphile-water boundaries. A stable single vesicle lobe was found for 1∶2 molar ratios in 92 wt% water with vesicles approximately 100 nm in size and with high zeta potential value. Structural variation arises due to the reduction of electrostatic repulsions among the ionic headgroups of the surfactants and the hydration forces due to adsorbed water onto monolayer's. The balance of these two forces determines the aggregate structures. Analysis was followed by the molecular geometrical structure. These findings may have implications for the development of drug delivery systems for cancer treatments, as well as cosmetic and food formulations.

  5. Ternary phase behaviour and vesicle formation of a sodium N-lauroylsarcosinate hydrate/1-decanol/water system.

    PubMed

    Akter, Nasima; Radiman, Shahidan; Mohamed, Faizal; Rahman, Irman Abdul; Reza, Mohammad Imam Hasan

    2011-01-01

    The phase behaviour of a system composed of amino acid-based surfactant (sodium N-lauroylsarcosinate hydrate), 1-decanol and deionised water was investigated for vesicle formation. Changing the molar ratio of the amphiphiles, two important aggregate structures were observed in the aqueous corner of the phase diagram. Two different sizes of microemulsions were found at two amphiphile-water boundaries. A stable single vesicle lobe was found for 1∶2 molar ratios in 92 wt% water with vesicles approximately 100 nm in size and with high zeta potential value. Structural variation arises due to the reduction of electrostatic repulsions among the ionic headgroups of the surfactants and the hydration forces due to adsorbed water onto monolayer's. The balance of these two forces determines the aggregate structures. Analysis was followed by the molecular geometrical structure. These findings may have implications for the development of drug delivery systems for cancer treatments, as well as cosmetic and food formulations.

  6. Multi-step formation of a hemifusion diaphragm for vesicle fusion revealed by all-atom molecular dynamics simulations.

    PubMed

    Tsai, Hui-Hsu Gavin; Chang, Che-Ming; Lee, Jian-Bin

    2014-06-01

    Membrane fusion is essential for intracellular trafficking and virus infection, but the molecular mechanisms underlying the fusion process remain poorly understood. In this study, we employed all-atom molecular dynamics simulations to investigate the membrane fusion mechanism using vesicle models which were pre-bound by inter-vesicle Ca(2+)-lipid clusters to approximate Ca(2+)-catalyzed fusion. Our results show that the formation of the hemifusion diaphragm for vesicle fusion is a multi-step event. This result contrasts with the assumptions made in most continuum models. The neighboring hemifused states are separated by an energy barrier on the energy landscape. The hemifusion diaphragm is much thinner than the planar lipid bilayers. The thinning of the hemifusion diaphragm during its formation results in the opening of a fusion pore for vesicle fusion. This work provides new insights into the formation of the hemifusion diaphragm and thus increases understanding of the molecular mechanism of membrane fusion. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    SciTech Connect

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

    2005-07-01

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

  8. Vectorial budding of vesicles by asymmetrical enzymatic formation of ceramide in giant liposomes.

    PubMed Central

    Holopainen, J M; Angelova, M I; Kinnunen, P K

    2000-01-01

    Sphingomyelin is an abundant component of eukaryotic membranes. A specific enzyme, sphingomyelinase can convert this lipid to ceramide, a central second messenger in cellular signaling for apoptosis (programmed cell death), differentiation, and senescence. We used microinjection and either Hoffman modulation contrast or fluorescence microscopy of giant liposomes composed of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), N-palmitoyl-sphingomyelin (C16:0-SM), and Bodipy-sphingomyelin as a fluorescent tracer (molar ratio 0.75:0.20:0.05, respectively) to observe changes in lipid lateral distribution and membrane morphology upon formation of ceramide. Notably, in addition to rapid domain formation (capping), vectorial budding of vesicles, i.e., endocytosis and shedding, can be induced by the asymmetrical sphingomyelinase-catalyzed generation of ceramide in either the outer or the inner leaflet, respectively, of giant phosphatidylcholine/sphingomyelin liposomes. These results are readily explained by 1) the lateral phase separation of ceramide enriched domains, 2) the area difference between the adjacent monolayers, 3) the negative spontaneous curvature, and 4) the augmented bending rigidity of the ceramide-containing domains, leading to membrane invagination and vesiculation of the bilayer. PMID:10653795

  9. Expression of mammalian protein kinase C in Schizosaccharomyces pombe: isotype-specific induction of growth arrest, vesicle formation, and endocytosis.

    PubMed Central

    Goode, N T; Hajibagheri, M A; Warren, G; Parker, P J

    1994-01-01

    Mammalian protein kinase C (PKC) isotypes elicit a number of effects on expression in Schizosaccharomyces pombe. A small decrease in growth rate results from PKC-gamma expression, and treatment of these cells with phorbol esters leads to marked growth inhibition and vesicle formation. PKC-delta and -eta expression causes growth inhibition and vesiculation, and the magnitude of both of these effects is increased by phorbol esters. In contrast, PKC-epsilon expression produces growth inhibition but no vesicle accumulation, and this effect is not responsive to phorbol ester. Finally, PKC-zeta has no observable effect. Thus, isotype-specific biological effects are observed. The accumulation of vesicles correlates with phorbol ester-dependent growth inhibition and occurs only with expression of those isotypes that down-regulate in response to phorbol esters in these cells. Antibodies against mammalian clathrin light chain 1a identified clathrin-coated vesicles and up-regulation of clathrin expression in those cells where vesicles accumulate; the increased vesicular traffic includes an element of endocytosis. Thus expression of specific mammalian PKC isotypes up-regulates endocytosis in S. pombe, providing a likely explanation for PKC-mediated receptor internalization in higher eukaryotes. Images PMID:7803858

  10. Visualizing the effect of dynamin inhibition on annular gap vesicle formation and fission

    PubMed Central

    Nickel, Beth; Boller, Marie; Schneider, Kimberly; Shakespeare, Teresa; Gay, Vernon; Murray, Sandra A.

    2013-01-01

    Summary Although gap junction plaque assembly has been extensively studied, mechanisms involved in plaque disassembly are not well understood. Disassembly involves an internalization process in which annular gap junction vesicles are formed. These vesicles undergo fission, but the molecular machinery needed for these fissions has not been described. The mechanoenzyme dynamin has been previously demonstrated to play a role in gap junction plaque internalization. To investigate the role of dynamin in annular gap junction vesicle fission, immunocytochemical, time-lapse and transmission electron microscopy were used to analyze SW-13 adrenocortical cells in culture. Dynamin was demonstrated to colocalize with gap junction plaques and vesicles. Dynamin inhibition, by siRNA knockdown or treatment with the dynamin GTPase inhibitor dynasore, increased the number and size of gap junction ‘buds’ suspended from the gap junction plaques. Buds, in control populations, were frequently released to form annular gap junction vesicles. In dynamin-inhibited populations, the buds were larger and infrequently released and thus fewer annular gap junction vesicles were formed. In addition, the number of annular gap junction vesicle fissions per hour was reduced in the dynamin-inhibited populations. We believe this to be the first report addressing the details of annular gap junction vesicle fissions and demonstrating a role of dynamin in this process. This information is crucial for elucidating the relationship between gap junctions, membrane regulation and cell behavior. PMID:23591819

  11. The identification of protein tyrosine phosphatase receptor type O (PTPRO) as a synaptic adhesion molecule that promotes synapse formation.

    PubMed

    Jiang, Wei; Wei, Mengping; Liu, Mengna; Pan, Yunlong; Yang, Xiaofei; Zhang, Chen

    2017-09-04

    The proper formation of synapses-specialized unitary structures formed between two neurons-is critical to mediate the information flow in the brain. Synaptic cell adhesion molecules (CAMs) are thought to participate in the initiation of the synapse formation process. However, in-vivo functional analysis demonstrates that most well-known synaptic CAMs regulate synaptic maturation and plasticity rather than synapse formation, suggesting that either CAMs work synergistically in the process of forming synapses or more CAMs remain to be found. By screening for unknown CAMs using a co-culture system, we revealed protein tyrosine phosphatase receptor type O (PTPRO) is a potent CAM that induces the formation of artificial synapse clusters in co-cultures of human embryonic kidney (HEK) 293 cells and hippocampal neurons cultured from newborn mice irrespective of gender. PTPRO was enriched in the mouse brain and localized to postsynaptic sites at excitatory synapses. The overexpression of PTPRO in cultured hippocampal neurons increased the number of synapses and the frequency of miniature excitatory postsynaptic currents (mEPSCs). The knockdown of PTPRO expression in cultured neurons by short hairpin RNA (shRNA) reduced the number of synapses and the frequencies of the mEPSCs. The effects of shRNA knockdown were rescued by expressing either full-length PTPRO or a truncated PTPRO lacking the cytoplasmic domain. Consistent with these results, the N-terminal extracellular domain of PTPRO was required for its synaptogenic activity in the co-culture assay. Taken together, our data show that PTPRO is a synaptic CAM that serves as a potent initiator of the formation of excitatory synapses.SIGNIFICANCE STATEMENTThe formation of synapses is critical for the brain to execute its function, and synaptic cell adhesion molecules (CAMs) play essential roles in initiating the formation of synapses. By screening for unknown CAMs using a co-culture system, we revealed protein tyrosine

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

    PubMed

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

    2005-04-01

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

  13. Formation of temporal-feature maps by axonal propagation of synaptic learning

    PubMed Central

    Kempter, Richard; Leibold, Christian; Wagner, Hermann; van Hemmen, J. Leo

    2001-01-01

    Computational maps are of central importance to a neuronal representation of the outside world. In a map, neighboring neurons respond to similar sensory features. A well studied example is the computational map of interaural time differences (ITDs), which is essential to sound localization in a variety of species and allows resolution of ITDs of the order of 10 μs. Nevertheless, it is unclear how such an orderly representation of temporal features arises. We address this problem by modeling the ontogenetic development of an ITD map in the laminar nucleus of the barn owl. We show how the owl's ITD map can emerge from a combined action of homosynaptic spike-based Hebbian learning and its propagation along the presynaptic axon. In spike-based Hebbian learning, synaptic strengths are modified according to the timing of pre- and postsynaptic action potentials. In unspecific axonal learning, a synapse's modification gives rise to a factor that propagates along the presynaptic axon and affects the properties of synapses at neighboring neurons. Our results indicate that both Hebbian learning and its presynaptic propagation are necessary for map formation in the laminar nucleus, but the latter can be orders of magnitude weaker than the former. We argue that the algorithm is important for the formation of computational maps, when, in particular, time plays a key role. PMID:11274439

  14. Critical importance of RAB proteins for synaptic function.

    PubMed

    Mignogna, Maria Lidia; D'Adamo, Patrizia

    2017-02-01

    Neurons are highly polarized cells that exhibit one of the more complex morphology and function. Neuronal intracellular trafficking plays a key role in dictating the directionality and specificity of vesicle formation, transport and fusion, allowing the transmission of information in sophisticate cellular network. Thus, the integrity of protein trafficking and spatial organization is especially important in neuronal cells. RAB proteins, small monomeric GTPases belonging to the RAS superfamily, spatially and temporally orchestrate specific vesicular trafficking steps. In this review we summarise the known roles of RAB GTPases involved in the maintenance of neuronal vesicular trafficking in the central nervous system. In particular, we discriminate the axonal pre-synaptic trafficking and dendritic post-synaptic trafficking, to better underlie how a correct orchestration of vesicle movement is necessary to maintain neuronal polarity and then, to permit an accurate architecture and functionality of synaptic activity.

  15. Phospholipase D Is Involved in the Formation of Golgi Associated Clathrin Coated Vesicles in Human Parotid Duct Cells

    PubMed Central

    Brito de Souza, Lorena; Pinto da Silva, Luis Lamberti; Jamur, Maria Célia; Oliver, Constance

    2014-01-01

    Phospholipase D (PLD) has been implicated in many cellular functions, such as vesicle trafficking, exocytosis, differentiation, and proliferation. The aim of this study was to characterize the role of PLD in HSY cells, a human cell line originating from the intercalated duct of the parotid gland. As the function and intracellular localization of PLD varies according to cell type, initially, the intracellular localization of PLD1 and PLD2 was determined. By immunofluorescence, PLD1 and PLD2 both showed a punctate cytoplasmic distribution with extensive co-localization with TGN-46. PLD1 was also found in the nucleus, while PLD2 was associated with the plasma membrane. Treatment of cells with the primary alcohol 1-butanol inhibits the hydrolysis of phosphatidylcoline by PLD thereby suppressing phosphatidic acid (PA) production. In untreated HSY cells, there was only a slight co-localization of PLD with the clathrin coated vesicles. When HSY cells were incubated with 1-butanol the total number of clathrin coated vesicles increased, especially in the juxtanuclear region and the co-localization of PLD with the clathrin coated vesicles was augmented. Transmission electron microscopy confirmed that the number of Golgi-associated coated vesicles was greater. Treatment with 1-butanol also affected the Golgi apparatus, increasing the volume of the Golgi saccules. The decrease in PA levels after treatment with 1-butanol likewise resulted in an accumulation of enlarged lysosomes in the perinuclear region. Therefore, in HSY cells PLD appears to be involved in the formation of Golgi associated clathrin coated vesicles as well as in the structural maintenance of the Golgi apparatus. PMID:24618697

  16. Analysis of outer membrane vesicle protein involved in biofilm formation of Helicobacter pylori.

    PubMed

    Yonezawa, Hideo; Osaki, Takako; Woo, Timothy; Kurata, Satoshi; Zaman, Cynthia; Hojo, Fuhito; Hanawa, Tomoko; Kato, Shuichi; Kamiya, Shigeru

    2011-12-01

    Helicobacter pylori is one of the most common causes of bacterial infection in humans. Infection with H. pylori is closely associated with gastritis and peptic ulcers and is a risk factor for gastric cancer and mucosa-associated lymphoid tissue lymphoma. H. pylori forms biofilms on glass surfaces at the air-liquid interface in in-vitro batch cultures. We previously reported that strain TK1402 showed a strong biofilm-forming ability in vitro. We also suggested the outer membrane vesicles (OMV) produced by strain TK1402 might be related to its biofilm forming ability. In the present study, we analyzed the protein profile of the OMV produced by strain TK1402 and found a unique 22-kDa protein in TK1402 OMV cultured for 2-3 days. In addition, this protein could not be detected in the OMVs produced by other H. pylori strains. These results suggest that the 22-kDa protein is involved in effective biofilm formation by strain TK1402. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. Cell collectivity regulation within migrating cell cluster during Kupffer's vesicle <