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Sample records for synaptic vesicle formation

  1. Synaptic vesicle endocytosis.

    PubMed

    Saheki, Yasunori; De Camilli, Pietro

    2012-09-01

    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.

  2. 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

  3. Proper synaptic vesicle formation and neuronal network activity critically rely on syndapin I

    PubMed Central

    Koch, Dennis; Spiwoks-Becker, Isabella; Sabanov, Victor; Sinning, Anne; Dugladze, Tamar; Stellmacher, Anne; Ahuja, Rashmi; Grimm, Julia; Schüler, Susann; Müller, Anke; Angenstein, Frank; Ahmed, Tariq; Diesler, Alexander; Moser, Markus; tom Dieck, Susanne; Spessert, Rainer; Boeckers, Tobias Maria; Fässler, Reinhard; Hübner, Christian Andreas; Balschun, Detlef; Gloveli, Tengis; Kessels, Michael Manfred; Qualmann, Britta

    2011-01-01

    Synaptic transmission relies on effective and accurate compensatory endocytosis. F-BAR proteins may serve as membrane curvature sensors and/or inducers and thereby support membrane remodelling processes; yet, their in vivo functions urgently await disclosure. We demonstrate that the F-BAR protein syndapin I is crucial for proper brain function. Syndapin I knockout (KO) mice suffer from seizures, a phenotype consistent with excessive hippocampal network activity. Loss of syndapin I causes defects in presynaptic membrane trafficking processes, which are especially evident under high-capacity retrieval conditions, accumulation of endocytic intermediates, loss of synaptic vesicle (SV) size control, impaired activity-dependent SV retrieval and defective synaptic activity. Detailed molecular analyses demonstrate that syndapin I plays an important role in the recruitment of all dynamin isoforms, central players in vesicle fission reactions, to the membrane. Consistently, syndapin I KO mice share phenotypes with dynamin I KO mice, whereas their seizure phenotype is very reminiscent of fitful mice expressing a mutant dynamin. Thus, syndapin I acts as pivotal membrane anchoring factor for dynamins during regeneration of SVs. PMID:21926968

  4. Synaptic vesicle pools: an update.

    PubMed

    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

  5. Molecular underpinnings of synaptic vesicle pool heterogeneity.

    PubMed

    Crawford, Devon C; Kavalali, Ege T

    2015-04-01

    Neuronal communication relies on chemical synaptic transmission for information transfer and processing. Chemical neurotransmission is initiated by synaptic vesicle fusion with the presynaptic active zone resulting in release of neurotransmitters. Classical models have assumed that all synaptic vesicles within a synapse have the same potential to fuse under different functional contexts. In this model, functional differences among synaptic vesicle populations are ascribed to their spatial distribution in the synapse with respect to the active zone. Emerging evidence suggests, however, that synaptic vesicles are not a homogenous population of organelles, and they possess intrinsic molecular differences and differential interaction partners. Recent studies have reported a diverse array of synaptic molecules that selectively regulate synaptic vesicles' ability to fuse synchronously and asynchronously in response to action potentials or spontaneously irrespective of action potentials. Here we discuss these molecular mediators of vesicle pool heterogeneity that are found on the synaptic vesicle membrane, on the presynaptic plasma membrane, or within the cytosol and consider some of the functional consequences of this diversity. This emerging molecular framework presents novel avenues to probe synaptic function and uncover how synaptic vesicle pools impact neuronal signaling.

  6. Spontaneous vesicle recycling in the synaptic bouton.

    PubMed

    Truckenbrodt, Sven; Rizzoli, Silvio O

    2014-01-01

    The trigger for synaptic vesicle exocytosis is Ca(2+), which enters the synaptic bouton following action potential stimulation. However, spontaneous release of neurotransmitter also occurs in the absence of stimulation in virtually all synaptic boutons. It has long been thought that this represents exocytosis driven by fluctuations in local Ca(2+) levels. The vesicles responding to these fluctuations are thought to be the same ones that release upon stimulation, albeit potentially triggered by different Ca(2+) sensors. This view has been challenged by several recent works, which have suggested that spontaneous release is driven by a separate pool of synaptic vesicles. Numerous articles appeared during the last few years in support of each of these hypotheses, and it has been challenging to bring them into accord. We speculate here on the origins of this controversy, and propose a solution that is related to developmental effects. Constitutive membrane traffic, needed for the biogenesis of vesicles and synapses, is responsible for high levels of spontaneous membrane fusion in young neurons, probably independent of Ca(2+). The vesicles releasing spontaneously in such neurons are not related to other synaptic vesicle pools and may represent constitutively releasing vesicles (CRVs) rather than bona fide synaptic vesicles. In mature neurons, constitutive traffic is much dampened, and the few remaining spontaneous release events probably represent bona fide spontaneously releasing synaptic vesicles (SRSVs) responding to Ca(2+) fluctuations, along with a handful of CRVs that participate in synaptic vesicle turnover.

  7. Synaptic Vesicle Proteins and Active Zone Plasticity.

    PubMed

    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.

  8. 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

  9. Synaptic vesicle distribution by conveyor belt.

    PubMed

    Moughamian, Armen J; Holzbaur, Erika L F

    2012-03-01

    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. PMID:22385955

  10. 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

  11. Functionally heterogeneous synaptic vesicle pools support diverse synaptic signalling.

    PubMed

    Chamberland, Simon; Tóth, Katalin

    2016-02-15

    Synaptic communication between neurons is a highly dynamic process involving specialized structures. At the level of the presynaptic terminal, neurotransmission is ensured by fusion of vesicles to the membrane, which releases neurotransmitter in the synaptic cleft. Depending on the level of activity experienced by the terminal, the spatiotemporal properties of calcium invasion will dictate the timing and the number of vesicles that need to be released. Diverse presynaptic firing patterns are translated to neurotransmitter release with a distinct temporal feature. Complex patterns of neurotransmitter release can be achieved when different vesicles respond to distinct calcium dynamics in the presynaptic terminal. Specific vesicles from different pools are recruited during various modes of release as the particular molecular composition of their membrane proteins define their functional properties. Such diversity endows the presynaptic terminal with the ability to respond to distinct physiological signals via the mobilization of specific subpopulation of vesicles. There are several mechanisms by which a diverse vesicle population could be generated in single presynaptic terminals, including distinct recycling pathways that utilize various adaptor proteins. Several additional factors could potentially contribute to the development of a heterogeneous vesicle pool such as specialized release sites, spatial segregation within the terminal and specialized delivery pathways. Among these factors molecular heterogeneity plays a central role in defining the functional properties of different subpopulations of vesicles. PMID:26614712

  12. 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

  13. Probing the interior of synaptic vesicles with internalized nanoparticles

    NASA Astrophysics Data System (ADS)

    Gadd, Jennifer C.; Budzinski, Kristi L.; Chan, Yang-Hsiang; Ye, Fangmao; Chiu, Daniel T.

    2012-03-01

    Synaptic vesicles are subcellular organelles that are found in the synaptic bouton and are responsible for the propagation of signals between neurons. Synaptic vesicles undergo endo- and exocytosis with the neuronal membrane to load and release neurotransmitters. Here we discuss how we utilize this property to load nanoparticles as a means of probing the interior of synaptic vesicles. To probe the intravesicular region of synaptic vesicles, we have developed a highly sensitive pH-sensing polymer dot. We feel the robust nature of the pH-sensing polymer dot will provide insight into the dynamics of proton loading into synaptic vesicles.

  14. 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

  15. 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

  16. Dephosphorylated synapsin I anchors synaptic vesicles to actin cytoskeleton: an analysis by videomicroscopy.

    PubMed

    Ceccaldi, P E; Grohovaz, F; Benfenati, F; Chieregatti, E; Greengard, P; Valtorta, F

    1995-03-01

    Synapsin I is a synaptic vesicle-associated protein which inhibits neurotransmitter release, an effect which is abolished upon its phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). Based on indirect evidence, it was suggested that this effect on neurotransmitter release may be achieved by the reversible anchoring of synaptic vesicles to the actin cytoskeleton of the nerve terminal. Using video-enhanced microscopy, we have now obtained experimental evidence in support of this model: the presence of dephosphorylated synapsin I is necessary for synaptic vesicles to bind actin; synapsin I is able to promote actin polymerization and bundling of actin filaments in the presence of synaptic vesicles; the ability to cross-link synaptic vesicles and actin is specific for synapsin I and is not shared by other basic proteins; the cross-linking between synaptic vesicles and actin is specific for the membrane of synaptic vesicles and does not reflect either a non-specific binding of membranes to the highly surface active synapsin I molecule or trapping of vesicles within the thick bundles of actin filaments; the formation of the ternary complex is virtually abolished when synapsin I is phosphorylated by CaM kinase II. The data indicate that synapsin I markedly affects synaptic vesicle traffic and cytoskeleton assembly in the nerve terminal and provide a molecular basis for the ability of synapsin I to regulate the availability of synaptic vesicles for exocytosis and thereby the efficiency of neurotransmitter release. PMID:7876313

  17. Abnormal Synaptic Vesicle Biogenesis in Drosophila Synaptogyrin Mutants

    PubMed Central

    Stevens, Robin J.; Akbergenova, Yulia; Jorquera, Ramon A.; Littleton, J. Troy

    2012-01-01

    Sustained neuronal communication relies on the coordinated activity of multiple proteins that regulate synaptic vesicle biogenesis and cycling within the presynaptic terminal. Synaptogyrin and synaptophysin are conserved MARVEL domain-containing transmembrane proteins that are among the most abundant synaptic vesicle constituents, although their role in the synaptic vesicle cycle has remained elusive. To further investigate the function of these proteins, we generated and characterized a synaptogyrin (gyr) null mutant in Drosophila, whose genome encodes a single synaptogyrin isoform and lacks a synaptophysin homolog. We demonstrate that Drosophila synaptogyrin plays a modulatory role in synaptic vesicle biogenesis at larval neuromuscular junctions. Drosophila lacking synaptogyrin are viable and fertile and have no overt deficits in motor function. However, ultrastructural analysis of gyr larvae revealed increased synaptic vesicle diameter and enhanced variability in the size of synaptic vesicles. In addition, the resolution of endocytic cisternae into synaptic vesicles in response to strong stimulation is defective in gyr mutants. Electrophysiological analysis demonstrated an increase in quantal size and a concomitant decrease in quantal content, suggesting functional consequences for transmission caused by the loss of synaptogyrin. Furthermore, high-frequency stimulation resulted in increased facilitation and a delay in recovery from synaptic depression, indicating that synaptic vesicle exo-endocytosis is abnormally regulated during intense stimulation conditions. These results suggest that synaptogyrin modulates the synaptic vesicle exo-endocytic cycle and is required for the proper biogenesis of synaptic vesicles at nerve terminals. PMID:23238721

  18. 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

  19. Synaptosomes as a Platform for Loading Nanoparticles into Synaptic Vesicles

    PubMed Central

    2011-01-01

    Synaptosomes are intact, isolated nerve terminals that contain the necessary machinery to recycle synaptic vesicles via endocytosis and exocytosis upon stimulation. Here we use this property of synaptosomes to load quantum dots into synaptic vesicles. Vesicles are then isolated from the synaptosomes, providing a method to probe isolated, individual synaptic vesicles where each vesicle contains a single, encapsulated nanoparticle. This technique provided an encapsulation efficiency of ∼16%; that is, ∼16% of the vesicles contained a single quantum dot while the remaining vesicles were empty. The ability to load single nanoparticles into synaptic vesicles opens new opportunity for employing various nanoparticle-based sensors to study the dynamics of vesicular transporters. PMID:21666849

  20. Prion protein facilitates synaptic vesicle release by enhancing release probability.

    PubMed

    Robinson, Susan W; Nugent, Marie L; Dinsdale, David; Steinert, Joern R

    2014-09-01

    The cellular prion protein (PrP(C)) has been implicated in several neurodegenerative diseases as a result of protein misfolding. In humans, prion disease occurs typically with a sporadic origin where uncharacterized mechanisms induce spontaneous PrP(C) misfolding leading to neurotoxic PrP-scrapie formation (PrP(SC)). The consequences of misfolded PrP(C) signalling are well characterized but little is known about the physiological roles of PrP(C) and its involvement in disease. Here we investigated wild-type PrP(C) signalling in synaptic function as well as the effects of a disease-relevant mutation within PrP(C) (proline-to-leucine mutation at codon 101). Expression of wild-type PrP(C) at the Drosophila neuromuscular junction leads to enhanced synaptic responses as detected in larger miniature synaptic currents which are caused by enlarged presynaptic vesicles. The expression of the mutated PrP(C) leads to reduction of both parameters compared with wild-type PrP(C). Wild-type PrP(C) enhances synaptic release probability and quantal content but reduces the size of the ready-releasable vesicle pool. Partially, these changes are not detectable following expression of the mutant PrP(C). A behavioural test revealed that expression of either protein caused an increase in locomotor activities consistent with enhanced synaptic release and stronger muscle contractions. Both proteins were sensitive to proteinase digestion. These data uncover new functions of wild-type PrP(C) at the synapse with a disease-relevant mutation in PrP(C) leading to diminished functional phenotypes. Thus, our data present essential new information possibly related to prion pathogenesis in which a functional synaptic role of PrP(C) is compromised due to its advanced conversion into PrP(SC) thereby creating a lack-of-function scenario.

  1. Epsin1 modulates synaptic vesicle retrieval capacity at CNS synapses.

    PubMed

    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

  2. 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

  3. Fusion Competent Synaptic Vesicles Persist upon Active Zone Disruption and Loss of Vesicle Docking.

    PubMed

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

    2016-08-17

    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

  4. Fusion Competent Synaptic Vesicles Persist upon Active Zone Disruption and Loss of Vesicle Docking.

    PubMed

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

    2016-08-17

    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.

  5. The role of mitochondrially derived ATP in synaptic vesicle recycling.

    PubMed

    Pathak, Divya; Shields, Lauren Y; Mendelsohn, Bryce A; Haddad, Dominik; Lin, Wei; Gerencser, Akos A; Kim, Hwajin; Brand, Martin D; Edwards, Robert H; Nakamura, Ken

    2015-09-11

    Synaptic mitochondria are thought to be critical in supporting neuronal energy requirements at the synapse, and bioenergetic failure at the synapse may impair neural transmission and contribute to neurodegeneration. However, little is known about the energy requirements of synaptic vesicle release or whether these energy requirements go unmet in disease, primarily due to a lack of appropriate tools and sensitive assays. To determine the dependence of synaptic vesicle cycling on mitochondrially derived ATP levels, we developed two complementary assays sensitive to mitochondrially derived ATP in individual, living hippocampal boutons. The first is a functional assay for mitochondrially derived ATP that uses the extent of synaptic vesicle cycling as a surrogate for ATP level. The second uses ATP FRET sensors to directly measure ATP at the synapse. Using these assays, we show that endocytosis has high ATP requirements and that vesicle reacidification and exocytosis require comparatively little energy. We then show that to meet these energy needs, mitochondrially derived ATP is rapidly dispersed in axons, thereby maintaining near normal levels of ATP even in boutons lacking mitochondria. As a result, the capacity for synaptic vesicle cycling is similar in boutons without mitochondria as in those with mitochondria. Finally, we show that loss of a key respiratory subunit implicated in Leigh disease markedly decreases mitochondrially derived ATP levels in axons, thus inhibiting synaptic vesicle cycling. This proves that mitochondria-based energy failure can occur and be detected in individual neurons that have a genetic mitochondrial defect.

  6. Mobility and Turnover of Vesicles at the Synaptic Ribbon

    PubMed Central

    LoGiudice, Lisamarie; Sterling, Peter; Matthews, Gary

    2008-01-01

    Ribbon synapses release neurotransmitter continuously at high rates, and the ribbons tether a large pool of synaptic vesicles. To determine if the tethered vesicles are actually released, we tracked vesicles labeled with FM4-64 dye in mouse retinal bipolar cell terminals whose ribbons had been labeled with a fluorescent peptide. We photobleached vesicles in regions with ribbons and without them and then followed recovery of fluorescence as bleached regions were repopulated by labeled vesicles. In the resting terminal, fluorescence recovered by ~50% in non-ribbon regions, but by only ~20% at ribbons. Thus, at rest, vesicles associated with ribbons cannot exchange freely with cytoplasmic vesicles. Depolarization stimulated vesicle turnover at ribbons as bleached, immobile vesicles were released by exocytosis and were then replaced by fluorescent vesicles from the cytoplasm, producing a further increase in fluorescence specifically at the ribbon location. We conclude that vesicles immobilized at synaptic ribbons participate in the readily releasable pool that is tapped rapidly during depolarization. PMID:18354018

  7. Comparison of nerve terminal events in vivo effecting retrograde transport of vesicles containing neurotrophins or synaptic vesicle components.

    PubMed

    Weible, M W; Ozsarac, N; Grimes, M L; Hendry, I A

    2004-03-15

    Although vesicular retrograde transport of neurotrophins in vivo is well established, relatively little is known about the mechanisms that underlie vesicle endocytosis and formation before transport. We demonstrate that in vivo not all retrograde transport vesicles are alike, nor are they all formed using identical mechanisms. As characterized by density, there are at least two populations of vesicles present in the synaptic terminal that are retrogradely transported along the axon: those containing neurotrophins (NTs) and those resulting from synaptic vesicle recycling. Vesicles containing nerve growth factor (NGF), NT-3, or NT-4 had similar densities with peak values at about 1.05 g/ml. Synaptic-derived vesicles, labeled with anti-dopamine beta-hydroxylase (DBH), had densities with peak values at about 1.16 g/ml. We assayed the effects of pharmacologic agents in vivo on retrograde transport from the anterior eye chamber to the superior cervical ganglion. Inhibitors of phosphatidylinositol-3-OH (PI-3) kinase and actin function blocked transport of both anti-DBH and NGF, demonstrating an essential role for these molecules in retrograde transport of both vesicle types. Dynamin, a key element in synaptic vesicle recycling, was axonally transported in retrograde and anterograde directions, and compounds able to interfere with dynamin function had a differential effect on retrograde transport of NTs and anti-DBH. Okadaic acid significantly decreased retrograde axonal transport of anti-DBH and increased NGF retrograde transport. We conclude that there are both different and common proteins involved in endocytosis and targeting of retrograde transport of these two populations of vesicles. PMID:14994338

  8. 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.

  9. Examination of synaptic vesicle recycling using FM dyes during evoked, spontaneous, and miniature synaptic activities.

    PubMed

    Iwabuchi, Sadahiro; Kakazu, Yasuhiro; Koh, Jin-Young; Goodman, Kirsty M; Harata, N Charles

    2014-03-31

    Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using styryl FM dyes, which reveal membrane turnover. Conventional protocols for the use of FM dyes were designed for analyzing neurons following stimulated (evoked) synaptic activity. Recently, protocols have become available for analyzing the FM signals that accompany weaker synaptic activities, such as spontaneous or miniature synaptic events. Analysis of these small changes in FM signals requires that the imaging system is sufficiently sensitive to detect small changes in intensity, yet that artifactual changes of large amplitude are suppressed. Here we describe a protocol that can be applied to evoked, spontaneous, and miniature synaptic activities, and use cultured hippocampal neurons as an example. This protocol also incorporates a means of assessing the rate of photobleaching of FM dyes, as this is a significant source of artifacts when imaging small changes in intensity.

  10. Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities

    PubMed Central

    Iwabuchi, Sadahiro; Kakazu, Yasuhiro; Koh, Jin-Young; Goodman, Kirsty M.; Harata, N. Charles

    2014-01-01

    Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using styryl FM dyes, which reveal membrane turnover. Conventional protocols for the use of FM dyes were designed for analyzing neurons following stimulated (evoked) synaptic activity. Recently, protocols have become available for analyzing the FM signals that accompany weaker synaptic activities, such as spontaneous or miniature synaptic events. Analysis of these small changes in FM signals requires that the imaging system is sufficiently sensitive to detect small changes in intensity, yet that artifactual changes of large amplitude are suppressed. Here we describe a protocol that can be applied to evoked, spontaneous, and miniature synaptic activities, and use cultured hippocampal neurons as an example. This protocol also incorporates a means of assessing the rate of photobleaching of FM dyes, as this is a significant source of artifacts when imaging small changes in intensity. PMID:24747983

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

    PubMed Central

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

    1988-01-01

    Several proteins are associated with, or are integral components of, the lipid bilayer that forms the delineating membrane of neuronal synaptic vesicles. To characterize these molecules, we used a polyclonal antiserum raised against purified cholinergic synaptic vesicles from Torpedo to screen a cDNA expression library constructed from mRNA of the electromotor nucleus. One clone encodes VAMP-1 (vesicle-associated membrane protein 1), a nervous-system-specific protein of 120 amino acids whose primary sequence can be divided into three domains: a proline-rich amino terminus, a highly charged internal region, and a hydrophobic carboxyl-terminal domain that is predicted to comprise a membrane anchor. Tryptic digestion of intact and lysed vesicles suggests that the protein faces the cytoplasm, where it may play a role in packaging, transport, or release of neurotransmitters. Images PMID:3380805

  12. 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.

  13. Ion channels in synaptic vesicles from Torpedo electric organ.

    PubMed Central

    Rahamimoff, R; DeRiemer, S A; Sakmann, B; Stadler, H; Yakir, N

    1988-01-01

    A simple method has been developed for fusing synaptic vesicles into spherical structures 20-50 micron in diameter. The method has been applied to purified cholinergic synaptic vesicles from Torpedo electric organ, and the membrane properties of these fused structures have been studied by the "cell"-attached version of the patch clamp technique. A large conductance potassium-preferring channel, termed the P channel, was consistently observed in preparations of fused synaptic vesicles. The selectivity of the channel for potassium over sodium was approximately equal to 2.8-fold. Two major conductance levels were observed during P-channel activity, and their relative proportion was dependent on the voltage applied to the membrane through the patch pipette. P channels were not seen in fused preparations of purified Torpedo lipids, nor was the frequency of their occurrence increased in preparations enriched with plasma membrane or nonvesicular membranes. We suggest, therefore, that the P channels are components of the synaptic vesicle membrane. Their function in synaptic transmission physiology is still unknown. Images PMID:2455900

  14. Sphingosine Facilitates SNARE Complex Assembly and Activates Synaptic Vesicle Exocytosis

    PubMed Central

    Darios, Frédéric; Wasser, Catherine; Shakirzyanova, Anastasia; Giniatullin, Artur; Goodman, Kerry; Munoz-Bravo, Jose L.; Raingo, Jesica; Jorgačevski, Jernej; Kreft, Marko; Zorec, Robert; Rosa, Juliana M.; Gandia, Luis; Gutiérrez, Luis M.; Binz, Thomas; Giniatullin, Rashid; Kavalali, Ege T.; Davletov, Bazbek

    2009-01-01

    Summary Synaptic vesicles loaded with neurotransmitters fuse with the plasma membrane to release their content into the extracellular space, thereby allowing neuronal communication. The membrane fusion process is mediated by a conserved set of SNARE proteins: vesicular synaptobrevin and plasma membrane syntaxin and SNAP-25. Recent data suggest that the fusion process may be subject to regulation by local lipid metabolism. Here, we have performed a screen of lipid compounds to identify positive regulators of vesicular synaptobrevin. We show that sphingosine, a releasable backbone of sphingolipids, activates synaptobrevin in synaptic vesicles to form the SNARE complex implicated in membrane fusion. Consistent with the role of synaptobrevin in vesicle fusion, sphingosine upregulated exocytosis in isolated nerve terminals, neuromuscular junctions, neuroendocrine cells and hippocampal neurons, but not in neurons obtained from synaptobrevin-2 knockout mice. Further mechanistic insights suggest that sphingosine acts on the synaptobrevin/phospholipid interface, defining a novel function for this important lipid regulator. PMID:19524527

  15. Functional Nanoscale Imaging of Synaptic Vesicle Cycling with Superfast Fixation.

    PubMed

    Schikorski, Thomas

    2016-01-01

    Functional imaging is the measurement of structural changes during an ongoing physiological process over time. In many cases, functional imaging has been implemented by tracking a fluorescent signal in live imaging sessions. Electron microscopy, however, excludes live imaging which has hampered functional imaging approaches on the ultrastructural level. This barrier was broken with the introduction of superfast fixation. Superfast fixation is capable of stopping and fixing membrane traffic at sufficient speed to capture a physiological process at a distinct functional state. Applying superfast fixation at sequential time points allows tracking of membrane traffic in a step-by-step fashion.This technique has been applied to track labeled endocytic vesicles at central synapses as they pass through the synaptic vesicle cycle. At synapses, neurotransmitter is released from synaptic vesicles (SVs) via fast activity-dependent exocytosis. Exocytosis is coupled to fast endocytosis that retrieves SVs components from the plasma membrane shortly after release. Fluorescent FM dyes that bind to the outer leaflet of the plasma membrane enter the endocytic vesicle during membrane retrieval and remain trapped in endocytic vesicles have been widely used to study SV exo-endocytic cycling in live imaging sessions. FM dyes can also be photoconverted into an electron-dense diaminobenzidine polymer which allows the investigation of SV cycling in the electron microscope. The combination of FM labeling with superfast fixation made it possible to track the fine structure of endocytic vesicles at 1 s intervals. Because this combination is not specialized to SV cycling, many other cellular processes can be studied. Furthermore, the technique is easy to set up and cost effective.This chapter describes activity-dependent FM dye labeling of SVs in cultured hippocampal neurons, superfast microwave-assisted fixation, photoconversion of the fluorescent endocytic vesicles, and the analysis of

  16. 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

  17. Synaptic vesicle release regulates myelin sheath number of individual oligodendrocytes in vivo.

    PubMed

    Mensch, Sigrid; Baraban, Marion; Almeida, Rafael; Czopka, Tim; Ausborn, Jessica; El Manira, Abdeljabbar; Lyons, David A

    2015-05-01

    The myelination of axons by oligodendrocytes markedly affects CNS function, but how this is regulated by neuronal activity in vivo is not known. We found that blocking synaptic vesicle release impaired CNS myelination by reducing the number of myelin sheaths made by individual oligodendrocytes during their short period of formation. We also found that stimulating neuronal activity increased myelin sheath formation by individual oligodendrocytes. These data indicate that neuronal activity regulates the myelinating capacity of single oligodendrocytes.

  18. Endocytosis of VAMP is facilitated by a synaptic vesicle targeting signal

    PubMed Central

    1996-01-01

    After synaptic vesicles fuse with the plasma membrane and release their contents, vesicle membrane proteins recycle by endocytosis and are targeted to newly formed synaptic vesicles. The membrane traffic of an epitope-tagged form of VAMP-2 (VAMP-TAg) was observed in transfected cells to identify sequence requirements for recycling of a synaptic vesicle membrane protein. In the neuroendocrine PC12 cell line VAMP-TAg is found not only in synaptic vesicles, but also in endosomes and on the plasma membrane. Endocytosis of VAMP-TAg is a rapid and saturable process. At high expression levels VAMP-TAg accumulates at the cell surface. Rapid endocytosis of VAMP-TAg also occurs in transfected CHO cells and is therefore independent of other synaptic proteins. The majority of the measured endocytosis is not directly into synaptic vesicles since mutations in VAMP-TAg that enhance synaptic vesicle targeting did not affect endocytosis. Nonetheless, mutations that inhibited synaptic vesicle targeting, in particular replacement of methionine-46 by alanine, inhibited endocytosis by 85% in PC12 cells and by 35% in CHO cells. These results demonstrate that the synaptic vesicle targeting signal is also used for endocytosis and can be recognized in cells lacking synaptic vesicles. PMID:8647886

  19. Merits and Limitations of Vesicle Pool Models in View of Heterogeneous Populations of Synaptic Vesicles.

    PubMed

    Neher, Erwin

    2015-09-23

    The concept of a readily releasable pool (RRP) of synaptic vesicles has been used extensively for the analysis of neurotransmitter release. Traditionally the properties of vesicles in such a pool have been assumed to be homogeneous, and techniques have been developed to determine pool parameters, such as the size of the pool and the probability with which a vesicle is released during an action potential. Increasing evidence, however, indicates that vesicles may be quite heterogeneous with respect to their release probability. The question, therefore, arises: what do the estimates of pool parameters mean in view of such heterogeneity? Here, four methods for obtaining pool estimates are reviewed, together with their underlying assumptions. The consequences of violation of these assumptions are discussed, and how apparent pool sizes are influenced by stimulation strength is explored by simulations.

  20. Synaptic Mitochondria in Synaptic Transmission and Organization of Vesicle Pools in Health and Disease

    PubMed Central

    Vos, Melissa; Lauwers, Elsa; Verstreken, Patrik

    2010-01-01

    Cell types rich in mitochondria, including neurons, display a high energy demand and a need for calcium buffering. The importance of mitochondria for proper neuronal function is stressed by the occurrence of neurological defects in patients suffering from a great variety of diseases caused by mutations in mitochondrial genes. Genetic and pharmacological evidence also reveal a role of these organelles in various aspects of neuronal physiology and in the pathogenesis of neurodegenerative disorders. Yet the mechanisms by which mitochondria can affect neurotransmission largely remain to be elucidated. In this review we focus on experimental data that suggest a critical function of synaptic mitochondria in the function and organization of synaptic vesicle pools, and in neurotransmitter release during intense neuronal activity. We discuss how calcium handling, ATP production and other mitochondrial mechanisms may influence synaptic vesicle pool organization and synaptic function. Given the link between synaptic mitochondrial function and neuronal communication, efforts toward better understanding mitochondrial biology may lead to novel therapeutic approaches of neurological disorders including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and psychiatric disorders that are at least in part caused by mitochondrial deficits. PMID:21423525

  1. Visualization of synaptic vesicle movement in intact synaptic boutons using fluorescence fluctuation spectroscopy.

    PubMed

    Jordan, Randolf; Lemke, Edward A; Klingauf, Jurgen

    2005-09-01

    Not much is known about the mobility of synaptic vesicles inside small synapses of the central nervous system, reflecting a lack of methods for visualizing these dynamics. We adapted confocal spot detection with fluctuation analysis to monitor the mobility of fluorescently labeled synaptic vesicles inside individual boutons of cultured hippocampal neurons. Using Monte Carlo simulations we were able to propose a simple quantitative model that can describe vesicle mobility in small hippocampal boutons under resting conditions and different pharmacological treatments. We find that vesicle mobility in a time window of 20 s can be well described by caged diffusion (D approximately 5 x 10(-5) microm(2)/s, cage sizes of approximately 50 nm). Mobility can be upregulated by phosphatase blockage and increased further by actin disruption in a dose-dependent manner. Inhibition of the myosin light chain kinase slows down vesicle mobility 10-fold, whereas other kinases like protein kinase C (PKC), A (PKA), and calmodulin kinase II (caMKII) do not affect mobility in unstimulated boutons.

  2. 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

  3. Rabx-5 regulates RAB-5 early endosomal compartments and synaptic vesicles in C. elegans.

    PubMed

    Sann, Sharon B; Crane, Matthew M; Lu, Hang; Jin, Yishi

    2012-01-01

    Early endosomal membrane compartments are required for the formation and recycling of synaptic vesicles, but how these compartments are regulated is incompletely understood. We performed a forward genetic screen in C. elegans for mutations that affect RAB-5 labeled early endosomal compartments in GABAergic motoneurons. Here we report the isolation and characterization of one mutation, rabx-5. The rabx-5 mutation leads to decreased intensity of YFP::RAB-5 in the cell soma but increased intensity in the synaptic and intersynaptic regions of the axon. This effect is due to the bias of the cycling state of RAB-5, and results from a change in the organization of the early endosomal compartment as well as the membrane binding state of RAB-5. Synaptic vesicle accumulation is altered in rabx-5 mutants, and synaptic transmission from cholinergic neurons is decreased. Early endosomal membrane compartments show disorganization with ageing and rabx-5 mutant animals age faster. These results suggest that rabx-5 regulation of RAB-5 compartments is important for maintaining proper synaptic function throughout the lifetime.

  4. 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

  5. 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

  6. The active zone protein CAST regulates synaptic vesicle recycling and quantal size in the mouse hippocampus.

    PubMed

    Kobayashi, Shizuka; Hida, Yamato; Ishizaki, Hiroyoshi; Inoue, Eiji; Tanaka-Okamoto, Miki; Yamasaki, Miwako; Miyazaki, Taisuke; Fukaya, Masahiro; Kitajima, Isao; Takai, Yoshimi; Watanabe, Masahiko; Ohtsuka, Toshihisa; Manabe, Toshiya

    2016-09-01

    Synaptic efficacy is determined by various factors, including the quantal size, which is dependent on the amount of neurotransmitters in synaptic vesicles at the presynaptic terminal. It is essential for stable synaptic transmission that the quantal size is kept within a constant range and that synaptic efficacy during and after repetitive synaptic activation is maintained by replenishing release sites with synaptic vesicles. However, the mechanisms for these fundamental properties have still been undetermined. We found that the active zone protein CAST (cytomatrix at the active zone structural protein) played pivotal roles in both presynaptic regulation of quantal size and recycling of endocytosed synaptic vesicles. In the CA1 region of hippocampal slices of the CAST knockout mice, miniature excitatory synaptic responses were increased in size, and synaptic depression after prolonged synaptic activation was larger, which was attributable to selective impairment of synaptic vesicle trafficking via the endosome in the presynaptic terminal likely mediated by Rab6. Therefore, CAST serves as a key molecule that regulates dynamics and neurotransmitter contents of synaptic vesicles in the excitatory presynaptic terminal in the central nervous system.

  7. The active zone protein CAST regulates synaptic vesicle recycling and quantal size in the mouse hippocampus.

    PubMed

    Kobayashi, Shizuka; Hida, Yamato; Ishizaki, Hiroyoshi; Inoue, Eiji; Tanaka-Okamoto, Miki; Yamasaki, Miwako; Miyazaki, Taisuke; Fukaya, Masahiro; Kitajima, Isao; Takai, Yoshimi; Watanabe, Masahiko; Ohtsuka, Toshihisa; Manabe, Toshiya

    2016-09-01

    Synaptic efficacy is determined by various factors, including the quantal size, which is dependent on the amount of neurotransmitters in synaptic vesicles at the presynaptic terminal. It is essential for stable synaptic transmission that the quantal size is kept within a constant range and that synaptic efficacy during and after repetitive synaptic activation is maintained by replenishing release sites with synaptic vesicles. However, the mechanisms for these fundamental properties have still been undetermined. We found that the active zone protein CAST (cytomatrix at the active zone structural protein) played pivotal roles in both presynaptic regulation of quantal size and recycling of endocytosed synaptic vesicles. In the CA1 region of hippocampal slices of the CAST knockout mice, miniature excitatory synaptic responses were increased in size, and synaptic depression after prolonged synaptic activation was larger, which was attributable to selective impairment of synaptic vesicle trafficking via the endosome in the presynaptic terminal likely mediated by Rab6. Therefore, CAST serves as a key molecule that regulates dynamics and neurotransmitter contents of synaptic vesicles in the excitatory presynaptic terminal in the central nervous system. PMID:27422015

  8. In vivo neuron-wide analysis of synaptic vesicle precursor trafficking.

    PubMed

    Maeder, Celine I; San-Miguel, Adriana; Wu, Emily Ye; Lu, Hang; Shen, Kang

    2014-03-01

    During synapse development, synaptic proteins must be targeted to sites of presynaptic release. Directed transport as well as local sequestration of synaptic vesicle precursors (SVPs), membranous organelles containing many synaptic proteins, might contribute to this process. Using neuron-wide time-lapse microscopy, we studied SVP dynamics in the DA9 motor neuron in Caenorhabditis elegans. SVP transport was highly dynamic and bi-directional throughout the entire neuron, including the dendrite. While SVP trafficking was anterogradely biased in axonal segments prior to the synaptic domain, directionality of SVP movement was stochastic in the dendrite and distal axon. Furthermore, frequency of movement and speed were variable between different compartments. These data provide evidence that SVP transport is differentially regulated in distinct neuronal domains. It also suggests that polarized SVP transport in concert with local vesicle capturing is necessary for accurate presynapse formation and maintenance. SVP trafficking analysis of two hypomorphs for UNC-104/KIF1A in combination with mathematical modeling identified directionality of movement, entry of SVPs into the axon as well as axonal speeds as the important determinants of steady-state SVP distributions. Furthermore, detailed dissection of speed distributions for wild-type and unc-104/kif1a mutant animals revealed an unexpected role for UNC-104/KIF1A in dendritic SVP trafficking.

  9. 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

  10. Piccolo and bassoon maintain synaptic vesicle clustering without directly participating in vesicle exocytosis.

    PubMed

    Mukherjee, Konark; Yang, Xiaofei; Gerber, Stefan H; Kwon, Hyung-Bae; Ho, Angela; Castillo, Pablo E; Liu, Xinran; Südhof, Thomas C

    2010-04-01

    Piccolo and bassoon are highly homologous multidomain proteins of the presynaptic cytomatrix whose function is unclear. Here, we generated piccolo knockin/knockout mice that either contain wild-type levels of mutant piccolo unable to bind Ca(2+) (knockin), approximately 60% decreased levels of piccolo that is C-terminally truncated (partial knockout), or <5% levels of piccolo (knockout). All piccolo mutant mice were viable and fertile, but piccolo knockout mice exhibited increased postnatal mortality. Unexpectedly, electrophysiology and electron microscopy of piccolo-deficient synapses failed to uncover a major phenotype either in acute hippocampal slices or in cultured cortical neurons. To unmask potentially redundant functions of piccolo and bassoon, we thus acutely knocked down expression of bassoon in wild-type and piccolo knockout neurons. Despite a nearly complete loss of piccolo and bassoon, however, we still did not detect an electrophysiological phenotype in cultured piccolo- and bassoon-deficient neurons in either GABAergic or glutamatergic synaptic transmission. In contrast, electron microscopy revealed a significant reduction in synaptic vesicle clustering in double bassoon/piccolo-deficient synapses. Thus, we propose that piccolo and bassoon play a redundant role in synaptic vesicle clustering in nerve terminals without directly participating in neurotransmitter release.

  11. Formation of Golgi-derived active zone precursor vesicles.

    PubMed

    Maas, Christoph; Torres, Viviana I; Altrock, Wilko D; Leal-Ortiz, Sergio; Wagh, Dhananjay; Terry-Lorenzo, Ryan T; Fejtova, Anna; Gundelfinger, Eckart D; Ziv, Noam E; Garner, Craig C

    2012-08-01

    Vesicular trafficking of presynaptic and postsynaptic components is emerging as a general cellular mechanism for the delivery of scaffold proteins, ion channels, and receptors to nascent and mature synapses. However, the molecular mechanisms leading to the selection of cargos and their differential transport to subneuronal compartments are not well understood, in part because of the mixing of cargos at the plasma membrane and/or within endosomal compartments. In the present study, we have explored the cellular mechanisms of active zone precursor vesicle assembly at the Golgi in dissociated hippocampal neurons of Rattus norvegicus. Our studies show that Piccolo, Bassoon, and ELKS2/CAST exit the trans-Golgi network on a common vesicle that requires Piccolo and Bassoon for its proper assembly. In contrast, Munc13 and synaptic vesicle proteins use distinct sets of Golgi-derived transport vesicles, while RIM1α associates with vesicular membranes in a post-Golgi compartment. Furthermore, Piccolo and Bassoon are necessary for ELKS2/CAST to leave the Golgi in association with vesicles, and a core domain of Bassoon is sufficient to facilitate formation of these vesicles. While these findings support emerging principles regarding active zone differentiation, the cellular and molecular analyses reported here also indicate that the Piccolo-Bassoon transport vesicles leaving the Golgi may undergo further changes in protein composition before arriving at synaptic sites.

  12. 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.

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

    PubMed Central

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

    2012-01-01

    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. PMID:23074252

  14. Synaptic vesicle generation from central nerve terminal endosomes.

    PubMed

    Kokotos, Alexandros C; Cousin, Michael A

    2015-03-01

    Central nerve terminals contain a small number of synaptic vesicles (SVs) that must sustain the fidelity of neurotransmission across a wide range of stimulation intensities. For this to be achieved, nerve terminals integrate a number of complementary endocytosis modes whose activation spans the breadth of these neuronal stimulation patterns. Two such modes are ultrafast endocytosis and activity-dependent bulk endocytosis, which are triggered by stimuli at either end of the physiological range. Both endocytosis modes generate endosomes directly from the nerve terminal plasma membrane, before the subsequent production of SVs from these structures. This review will discuss the current knowledge relating to the molecular mechanisms involved in the generation of SVs from nerve terminal endosomes, how this relates to other mechanisms of SV production and the functional role of such SVs.

  15. 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

    2014-07-01

    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.

  16. Synaptic vesicle proteins: targets and routes for botulinum neurotoxins.

    PubMed

    Ahnert-Hilger, Gudrun; Münster-Wandowski, Agnieszka; Höltje, Markus

    2013-01-01

    Synaptic vesicles (SV) are key organelles of neuronal communication. SV are responsible for the storage of neurotransmitters, which are released by Ca(2+)-dependent exocytosis. After release and interaction with postsynaptic receptors, transmitters rapidly diffuse out of the synaptic cleft and are sequestered by plasma membrane transporters (in some cases following enzymatic conversion). SVs undergo endocytosis and are refilled by specific vesicular transmitter transporters different in the various neuronal subtypes. Besides these differences, SVs in general are equipped with a remarkable common set of proteins. Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release from almost all types of neurons by cleaving proteins required for membrane fusion localized either to SVs (synaptobrevin) or to the plasma membrane (SNAP-25 and syntaxin) depending on the BoNT serotype. To enter the neuronal cytoplasm, BoNTs specifically interact with the luminal domain of SV proteins (synaptotagmin or SV2, depending on serotype) transiently exposed during exocytotic membrane fusion and occurring in almost every neuron. Thus, the highly specific interaction with luminal domains of SV proteins commonly expressed on all SV types is one reason why BoNTs exhibit such a high neuronal specificity but attack almost every neuron type.

  17. Ciliary vesicle formation: a prelude to ciliogenesis.

    PubMed

    Yee, Laura E; Reiter, Jeremy F

    2015-03-23

    Reporting recently in Nature Cell Biology, Lu et al. (2015) identify two Eps15-homology-domain-containing proteins as critical effectors of ciliary vesicle formation, an early event in ciliogenesis. Functional dissection reveals that one of them works to convert small vesicles associated with mother centriole distal appendages into a larger ciliary vesicle. PMID:25805133

  18. Synaptic vesicle exocytosis in hippocampal synaptosomes correlates directly with total mitochondrial volume

    PubMed Central

    Ivannikov, Maxim V.; Sugimori, Mutsuyuki; Llinás, Rodolfo R.

    2012-01-01

    Synaptic plasticity in many regions of the central nervous system leads to the continuous adjustment of synaptic strength, which is essential for learning and memory. In this study, we show by visualizing synaptic vesicle release in mouse hippocampal synaptosomes that presynaptic mitochondria and specifically, their capacities for ATP production are essential determinants of synaptic vesicle exocytosis and its magnitude. Total internal reflection microscopy of FM1-43 loaded hippocampal synaptosomes showed that inhibition of mitochondrial oxidative phosphorylation reduces evoked synaptic release. This reduction was accompanied by a substantial drop in synaptosomal ATP levels. However, cytosolic calcium influx was not affected. Structural characterization of stimulated hippocampal synaptosomes revealed that higher total presynaptic mitochondrial volumes were consistently associated with higher levels of exocytosis. Thus, synaptic vesicle release is linked to the presynaptic ability to regenerate ATP, which itself is a utility of mitochondrial density and activity. PMID:22772899

  19. Imaging Exocytosis of Single Synaptic Vesicles at a Fast CNS Presynaptic Terminal.

    PubMed

    Midorikawa, Mitsuharu; Sakaba, Takeshi

    2015-11-01

    Synaptic vesicles are tethered to the active zone where they are docked/primed so that they can fuse rapidly upon Ca(2+) influx. To directly study these steps at a CNS presynaptic terminal, we used total internal reflection fluorescence (TIRF) microscopy at the live isolated calyx of Held terminal and measured the movements of single synaptic vesicle just beneath the plasma membrane. Only a subset of vesicles within the TIRF field underwent exocytosis. Following exocytosis, new vesicles (newcomers) approached the membrane and refilled the release sites slowly with a time constant of several seconds. Uniform elevation of the intracellular Ca(2+) using flash photolysis elicited an exocytotic burst followed by the sustained component, representing release of the readily releasable vesicles and vesicle replenishment, respectively. Surprisingly, newcomers were not released within a second of high Ca(2+). Instead, already-tethered vesicles became release-ready and mediated the replenishment. Our results reveal an important feature of conventional synapses. PMID:26539890

  20. Imaging Exocytosis of Single Synaptic Vesicles at a Fast CNS Presynaptic Terminal.

    PubMed

    Midorikawa, Mitsuharu; Sakaba, Takeshi

    2015-11-01

    Synaptic vesicles are tethered to the active zone where they are docked/primed so that they can fuse rapidly upon Ca(2+) influx. To directly study these steps at a CNS presynaptic terminal, we used total internal reflection fluorescence (TIRF) microscopy at the live isolated calyx of Held terminal and measured the movements of single synaptic vesicle just beneath the plasma membrane. Only a subset of vesicles within the TIRF field underwent exocytosis. Following exocytosis, new vesicles (newcomers) approached the membrane and refilled the release sites slowly with a time constant of several seconds. Uniform elevation of the intracellular Ca(2+) using flash photolysis elicited an exocytotic burst followed by the sustained component, representing release of the readily releasable vesicles and vesicle replenishment, respectively. Surprisingly, newcomers were not released within a second of high Ca(2+). Instead, already-tethered vesicles became release-ready and mediated the replenishment. Our results reveal an important feature of conventional synapses.

  1. Distinct Functions of Endophilin Isoforms in Synaptic Vesicle Endocytosis.

    PubMed

    Zhang, Jifeng; Tan, Minghui; Yin, Yichen; Ren, Bingyu; Jiang, Nannan; Guo, Guoqing; Chen, Yuan

    2015-01-01

    Endophilin isoforms perform distinct characteristics in their interactions with N-type Ca(2+) channels and dynamin. However, precise functional differences for the endophilin isoforms on synaptic vesicle (SV) endocytosis remain unknown. By coupling RNA interference and electrophysiological recording techniques in cultured rat hippocampal neurons, we investigated the functional differences of three isoforms of endophilin in SV endocytosis. The results showed that the amplitude of normalized evoked excitatory postsynaptic currents in endophilin1 knockdown neurons decreased significantly for both single train and multiple train stimulations. Similar results were found using endophilin2 knockdown neurons, whereas endophilin3 siRNA exhibited no change compared with control neurons. Endophilin1 and endophilin2 affected SV endocytosis, but the effect of endophilin1 and endophilin2 double knockdown was not different from that of either knockdown alone. This result suggested that endophilin1 and endophilin2 functioned together but not independently during SV endocytosis. Taken together, our results indicate that SV endocytosis is sustained by endophilin1 and endophilin2 isoforms, but not by endophilin3, in primary cultured hippocampal neurons.

  2. Structure of synaptogyrin (p29) defines novel synaptic vesicle protein

    PubMed Central

    1995-01-01

    Synaptogyrin (p29) is a synaptic vesicle protein that is uniformly distributed in the nervous system (Baumert et al., 1990). We have cloned and sequenced the cDNA encoding synaptogyrin, and the sequence predicts a protein with a molecular mass of 25,900 D with four membrane- spanning domains. The topology of the protein was confirmed by limited proteolysis using domain-specific antibodies. Database searches revealed several cDNA sequences coding polypeptides with sequence identities ranging from 32 to 46%, suggesting that synaptogyrin is a member of a multigene family. When the synaptogyrin cDNA is expressed in COS cells, the generated protein is indistinguishable from native synaptogyrin. To study intracellular sorting, synaptogyrin was expressed in CHO cells that revealed a punctate staining that was very similar to that of synaptophysin and endogenously expressed cellubrevin. Significant overlap with transferrin staining was also observed, suggesting that synaptogyrin is targeted to a recycling compartment involved in membrane traffic to and from the plasma membrane. PMID:8557746

  3. 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

  4. 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

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

    PubMed

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

    2015-09-01

    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.

  6. The Role of Mitochondrially Derived ATP in Synaptic Vesicle Recycling*♦

    PubMed Central

    Pathak, Divya; Shields, Lauren Y.; Mendelsohn, Bryce A.; Haddad, Dominik; Lin, Wei; Gerencser, Akos A.; Kim, Hwajin; Brand, Martin D.; Edwards, Robert H.; Nakamura, Ken

    2015-01-01

    Synaptic mitochondria are thought to be critical in supporting neuronal energy requirements at the synapse, and bioenergetic failure at the synapse may impair neural transmission and contribute to neurodegeneration. However, little is known about the energy requirements of synaptic vesicle release or whether these energy requirements go unmet in disease, primarily due to a lack of appropriate tools and sensitive assays. To determine the dependence of synaptic vesicle cycling on mitochondrially derived ATP levels, we developed two complementary assays sensitive to mitochondrially derived ATP in individual, living hippocampal boutons. The first is a functional assay for mitochondrially derived ATP that uses the extent of synaptic vesicle cycling as a surrogate for ATP level. The second uses ATP FRET sensors to directly measure ATP at the synapse. Using these assays, we show that endocytosis has high ATP requirements and that vesicle reacidification and exocytosis require comparatively little energy. We then show that to meet these energy needs, mitochondrially derived ATP is rapidly dispersed in axons, thereby maintaining near normal levels of ATP even in boutons lacking mitochondria. As a result, the capacity for synaptic vesicle cycling is similar in boutons without mitochondria as in those with mitochondria. Finally, we show that loss of a key respiratory subunit implicated in Leigh disease markedly decreases mitochondrially derived ATP levels in axons, thus inhibiting synaptic vesicle cycling. This proves that mitochondria-based energy failure can occur and be detected in individual neurons that have a genetic mitochondrial defect. PMID:26126824

  7. The organization of synaptic vesicles at tonically transmitting connections of locust visual interneurons.

    PubMed

    Leitinger, Gerd; Simmons, Peter J

    2002-02-01

    Large, second-order neurons of locust ocelli, or L-neurons, make some output connections that transmit small changes in membrane potential and can sustain transmission tonically. The synaptic connections are made from the axons of L-neurons in the lateral ocellar tracts, and are characterized by bar-shaped presynaptic densities and densely packed clouds of vesicles near to the cell membrane. A cloud of vesicles can extend much of the length of this synaptic zone, and there is no border between the vesicles that are associated with neighboring presynaptic densities. In some axons, presynaptic densities are associated with discrete small clusters of vesicles. Up to 6% of the volume of a length of axon in a synaptic zone can be occupied with a vesicle cloud, packed with 4.5 to 5.5 thousand vesicles per microm(3). Presynaptic densities vary in length, from less than 70 nm to 1.5 microm, with shorter presynaptic densities being most frequent. The distribution of vesicles around short presynaptic densities was indistinguishable from that around long presynaptic densities, and vesicles were distributed in a similar way right along the length of a presynaptic density. Within the cytoplasm, vesicles are homogeneously distributed within a cloud. We found no differences in the distribution of vesicles in clouds between locusts that had been dark-adapted and locusts that had been light-adapted before fixation.

  8. 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

    2014-01-01

    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. PMID:24977345

  9. 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

  10. 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.

  11. FM 1–43 Labeling of Synaptic Vesicle Pools at the Drosophila Neuromuscular Junction

    PubMed Central

    Verstreken, Patrik; Ohyama, Tomoko; Bellen, Hugo J.

    2009-01-01

    Summary To maintain transmitter release during intense stimulation, neurons need to efficiently recycle vesicles at the synapse. Following membrane fusion, vesicles are reshaped and formed from the plasma membrane by bulk or clathrin-mediated endocytosis. Most synapses, including the Drosophila neuromuscular junction (NMJ), can also recycle synaptic vesicles directly by closing the fusion pore, a process referred to as “kiss and run.” While the process of clathrin-mediated vesicle retrieval is under intense investigation, the kiss-and-run phenomenon remains much less accepted. To gain better insight into the mechanisms of synaptic vesicle recycling, it is therefore critical not only to identify and characterize novel players involved in the process, but also to develop novel methods to study vesicle recycling. Although in recent years numerous techniques to study vesicle traffic have been developed (see also this volume), in this chapter we outline established procedures that use the fluorescent dye FM 1–43 or related compounds to study vesicle cycling. We describe how FM 1–43 can be used to study and visualize clathrin-mediated or bulk endocytosis from the presynaptic membrane as well as exocytosis of labeled vesicles at the Drosophila NMJ, one of the best-characterized model synapses to study synaptic function in a genetic model system. PMID:18369958

  12. Increased Expression of Alpha-Synuclein Reduces Neurotransmitter Release by Inhibiting Synaptic Vesicle Reclustering After Endocytosis

    PubMed Central

    Nemani, Venu M.; Lu, Wei; Berge, Victoria; Nakamura, Ken; Onoa, Bibiana; Lee, Michael K.; Chaudhry, Farrukh A.; Nicoll, Roger A.; Edwards, Robert H.

    2011-01-01

    Summary The protein α-synuclein accumulates in the brain of patients with sporadic Parkinson’s disease (PD), and increased gene dosage causes a severe, dominantly inherited form of PD, but we know little about the effects of synuclein that precede degeneration. α-Synuclein localizes to the nerve terminal, but the knockout has little if any effect on synaptic transmission. In contrast, we now find that the modest over-expression of α-synuclein, in the range predicted for gene multiplication and in the absence of overt toxicity, markedly inhibits neurotransmitter release. The mechanism, elucidated by direct imaging of the synaptic vesicle cycle, involves a specific reduction in size of the synaptic vesicle recycling pool. Ultrastructural analysis demonstrates reduced synaptic vesicle density at the active zone, and imaging further reveals a defect in the reclustering of synaptic vesicles after endocytosis. Increased levels of α-synuclein thus produce a specific, physiological defect in synaptic vesicle recycling that precedes detectable neuropathology. PMID:20152114

  13. Synaptic vesicle-bound pyruvate kinase can support vesicular glutamate uptake

    PubMed Central

    Ishida, Atsuhiko; Noda, Yasuko; Ueda, Tetsufumi

    2008-01-01

    Glucose metabolism is essential for normal brain function and plays a vital role in synaptic transmission. Recent evidence suggests that ATP synthesized locally by glycolysis, particularly via glyceraldehyde 3-phosphate dehydrogenase/3-phosphoglycerate kinase, is critical for synaptic transmission. We present evidence that ATP generated by synaptic vesicle-associated pyruvate kinase is harnessed to transport glutamate into synaptic vesicles. Isolated synaptic vesicles incorporated [3H]glutamate in the presence of phosphoenolpyruvate (PEP) and ADP. Pyruvate kinase activators and inhibitors stimulated and reduced PEP/ADP-dependent glutamate uptake, respectively. Membrane potential was also formed in the presence of pyruvate kinase activators. “ATP-trapping” experiments using hexokinase and glucose suggest that ATP produced by vesicle-associated pyruvate kinase is more readily used than exogenously added ATP. Other neurotransmitters such as GABA, dopamine, and serotonin were also taken up into crude synaptic vesicles in a PEP/ADP-dependent manner. The possibility that ATP locally generated by glycolysis supports vesicular accumulation of neurotransmitters is discussed. PMID:18751889

  14. 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

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

    NASA Astrophysics Data System (ADS)

    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

    2003-03-01

    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. Abbreviations: ACh, acetylcholine AFM, atomic force microscopy

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

    PubMed Central

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

    2014-01-01

    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. PMID:25422373

  17. 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

  18. Presynaptic Calcium Channel Localization and Calcium Dependent Synaptic Vesicle Exocytosis Regulated by the Fuseless Protein

    PubMed Central

    Long, A. Ashleigh; Kim, Eunju; Leung, Hung-Tat; Woodruff, Elvin; An, Lingling; Doerge, R. W.; Pak, William L.; Broadie, Kendal

    2009-01-01

    Summary A systematic forward genetic Drosophila screen for electroretinogram mutants lacking synaptic transients identified the fuseless (fusl) gene, which encodes a predicted 8-pass transmembrane protein in the presynaptic membrane. Null fusl mutants display >75% reduction in evoked synaptic transmission but, conversely, a ~3-fold increase in the frequency and amplitude of spontaneous synaptic vesicle fusion events. These neurotransmission defects are rescued by a wildtype fusl transgene targeted only to the presynaptic cell, demonstrating a strictly presynaptic requirement for Fusl function. Defects in FM dye turnover at the synapse show a severely impaired exo-endo synaptic vesicle cycling pool. Consistently, ultrastructural analyses reveal accumulated vesicles arrested in clustered and docked pools at presynaptic active zones. In the absence of Fusl, calcium-dependent neurotransmitter release is dramatically compromised and there is little enhancement of synaptic efficacy with elevated external Ca2+ concentrations. These defects are causally linked with severe loss of the Cacophony voltage-gated Ca2+ channels, which fail to localize normally at presynaptic active zone domains in the absence of Fusl. These data indicate that Fusl regulates assembly of the presynaptic active zone Ca2+ channel domains required for efficient coupling of the Ca2+ influx and synaptic vesicle exocytosis during neurotransmission. PMID:18385325

  19. Hydrogen peroxide inhibits the vacuolar H+-ATPase in brain synaptic vesicles at micromolar concentrations.

    PubMed

    Wang, Y; Floor, E

    1998-02-01

    Hydrogen peroxide (H2O2) is produced from several sources in brain and may be involved in neurodegeneration and second messenger signaling. Little is known about the effects of H2O2 on transmitter storage in brain synaptic vesicles. Neurotransmitter uptake into synaptic vesicles is driven by an electrochemical proton gradient generated by the vacuolar H+-ATPase (V-ATPase) in the vesicle membrane. We report here that the VATPase in bovine brain synaptic vesicles is highly sensitive to inhibition by micromolar concentrations of H2O2. Glutamate uptake by the vesicles is also inhibited, very likely as a secondary consequence of ATPase inactivation. Dithiothreitol or reduced glutathione reverse H2O2-induced inhibition of the V-ATPase, and ATP or GTP partially protect the ATPase from inhibition by H2O2. These and other results suggest that the mechanism of inhibition of the V-ATPase by H2O2 involves oxidation of a reactive cysteine sulfhydryl group in the ATP binding site. Inhibition of V-ATPase activity would decrease the amount of transmitter stored in synaptic vesicles and thus down-regulate transmitter release during episodes of oxidative stress or in response to second messenger signaling.

  20. Ultrastructural localization of active zone and synaptic vesicle proteins in a preassembled multi-vesicle transport aggregate.

    PubMed

    Tao-Cheng, J-H

    2007-12-12

    Although it has been suggested that presynaptic active zone (AZ) may be preassembled, it is still unclear which entities carry the various proteins to the AZ during synaptogenesis. Here, I propose that aggregates of dense core vesicles (DCV) and small clear vesicles in the axons of young rat hippocampal cultures are carriers containing preformed AZ and synaptic vesicle (SV) components on their way to developing synapses. The aggregates were positively labeled with antibodies against Bassoon and Piccolo (two AZ cytomatrix proteins), VAMP, SV2, synaptotagmin (three SV membrane proteins), and synapsin I (a SV-associated protein). Bassoon and Piccolo labeling were localized at dense material both in the aggregates and at the AZ. In addition to the SV at the synapses, the SV membrane proteins labeled the clear vesicles in the aggregate as well as many other SV-like and pleiomorphic vesicular structures in the axons, and synapsin I labeling was associated with the vesicles in the aggregates. In single sections, these axonal vesicle aggregates were approximately 0.22 by 0.13 microm in average dimensions and contain one to two DCV and five to six small clear vesicles. Serial sections confirmed that the aggregates were not synaptic junctions sectioned en face. Labeling intensities of Bassoon and Piccolo measured from serially sectioned transport aggregates and AZ were within range of each other, suggesting that one or a few aggregates, but not individual DCV, can carry sufficient Bassoon and Piccolo to form an AZ. The present findings provide the first ultrastructural evidence localizing various AZ and SV proteins in a preassembled multi-vesicle transport aggregate that has the potential to quickly form a functional active zone.

  1. A phosphatidylinositol 4,5-bisphosphate-sensitive casein kinase I alpha associates with synaptic vesicles and phosphorylates a subset of vesicle proteins

    PubMed Central

    1995-01-01

    In interphase cells, alpha-casein kinase I (alpha-CKI) is found associated with cytosolic vesicular structures, the centrosome, and within the nucleus. To identify the specific vesicular structures with which alpha-CKI is associated, established cell lines and primary rat neurons were immunofluorescently labeled with an antibody raised to alpha-CKI. In nonneuronal cells, alpha-CKI colocalizes with vesicular structures which align with microtubules and are partially coincident with both Golgi and endoplasmic reticulum markers. In neurons, alpha- CKI colocalizes with synaptic vesicle markers. When synaptic vesicles were purified from rat brain, they were highly enriched in a CKI, based on activity and immunoreactivity. The synaptic vesicle-associated CKI is an extrinsic kinase and was eluted from synaptic vesicles and purified. This purified CKI has properties most similar to alpha-CKI. When the activities of casein kinase I or II were specifically inhibited on isolated synaptic vesicles, CKI was shown to phosphorylate a specific subset of vesicle proteins, one of which was identified as the synaptic vesicle-specific protein SV2. As with alpha-CKI, the synaptic vesicle CKI is inhibited by phosphatidylinositol 4,5- bisphosphate (PIP2). However, synthesis of PIP2 was detected only in plasma membrane-containing fractions. Therefore, PIP2 may spatially regulate CKI. Since PIP2 synthesis is required for secretion, this inhibition of CKI may be important for the regulation of secretion. PMID:7622570

  2. Endogenous Rho-kinase signaling maintains synaptic strength by stabilizing the size of the readily releasable pool of synaptic vesicles.

    PubMed

    González-Forero, David; Montero, Fernando; García-Morales, Victoria; Domínguez, Germán; Gómez-Pérez, Laura; García-Verdugo, José Manuel; Moreno-López, Bernardo

    2012-01-01

    Rho-associated kinase (ROCK) regulates neural cell migration, proliferation and survival, dendritic spine morphology, and axon guidance and regeneration. There is, however, little information about whether ROCK modulates the electrical activity and information processing of neuronal circuits. At neonatal stage, ROCKα is expressed in hypoglossal motoneurons (HMNs) and in their afferent inputs, whereas ROCKβ is found in synaptic terminals on HMNs, but not in their somata. Inhibition of endogenous ROCK activity in neonatal rat brainstem slices failed to modulate intrinsic excitability of HMNs, but strongly attenuated the strength of their glutamatergic and GABAergic synaptic inputs. The mechanism acts presynaptically to reduce evoked neurotransmitter release. ROCK inhibition increased myosin light chain (MLC) phosphorylation, which is known to trigger actomyosin contraction, and reduced the number of synaptic vesicles docked to active zones in excitatory boutons. Functional and ultrastructural changes induced by ROCK inhibition were fully prevented/reverted by MLC kinase (MLCK) inhibition. Furthermore, ROCK inhibition drastically reduced the phosphorylated form of p21-associated kinase (PAK), which directly inhibits MLCK. We conclude that endogenous ROCK activity is necessary for the normal performance of motor output commands, because it maintains afferent synaptic strength, by stabilizing the size of the readily releasable pool of synaptic vesicles. The mechanism of action involves a tonic inhibition of MLCK, presumably through PAK phosphorylation. This mechanism might be present in adults since unilateral microinjection of ROCK or MLCK inhibitors into the hypoglossal nucleus reduced or increased, respectively, whole XIIth nerve activity.

  3. The GTPase Rab26 links synaptic vesicles to the autophagy pathway.

    PubMed

    Binotti, Beyenech; Pavlos, Nathan J; Riedel, Dietmar; Wenzel, Dirk; Vorbrüggen, Gerd; Schalk, Amanda M; Kühnel, Karin; Boyken, Janina; Erck, Christian; Martens, Henrik; Chua, John J E; Jahn, Reinhard

    2015-01-01

    Small GTPases of the Rab family not only regulate target recognition in membrane traffic but also control other cellular functions such as cytoskeletal transport and autophagy. Here we show that Rab26 is specifically associated with clusters of synaptic vesicles in neurites. Overexpression of active but not of GDP-preferring Rab26 enhances vesicle clustering, which is particularly conspicuous for the EGFP-tagged variant, resulting in a massive accumulation of synaptic vesicles in neuronal somata without altering the distribution of other organelles. Both endogenous and induced clusters co-localize with autophagy-related proteins such as Atg16L1, LC3B and Rab33B but not with other organelles. Furthermore, Atg16L1 appears to be a direct effector of Rab26 and binds Rab26 in its GTP-bound form, albeit only with low affinity. We propose that Rab26 selectively directs synaptic and secretory vesicles into preautophagosomal structures, suggesting the presence of a novel pathway for degradation of synaptic vesicles.

  4. Okadaic acid disrupts synaptic vesicle trafficking in a ribbon-type synapse

    PubMed Central

    Guatimosim, Cristina; Hull, Court; von Gersdorff, Henrique; Prado, Marco A. M.

    2013-01-01

    Protein phosphorylation plays an essential role in regulating synaptic transmission and plasticity. However, regulation of vesicle trafficking towards and away from the plasma membrane is poorly understood. Furthermore, the extent to which phosphorylation modulates ribbon-type synapses is unknown. Using the phosphatase inhibitor okadaic acid (OA), we investigated the influence of persistent phosphorylation on vesicle cycling in goldfish bipolar cells. We followed uptake of FM1-43 during vesicle recycling in control and OA-treated cells. FM1-43 fluorescence spread to the center of control synaptic terminals after depolarization elicited Ca2+ influx. However, OA (1–50 nM) impaired this spatial spread of FM1-43 in a dose-dependent manner. Capacitance measurements revealed that OA (50 nM) did not modify either the amount or kinetics of exocytosis and endocytosis evoked by depolarizing pulses. The extremely low concentrations of OA (1–5 nM) sufficient to observe the inhibition of vesicle mobility implicate phosphatase 2A (PP2A) as a major regulator of vesicle trafficking after endocytosis. These results contrast with those at the neuromuscular junction where OA enhances lateral movement of vesicles between distinct vesicle clusters. Thus, our results suggest that phosphatases regulate vesicle translocation at ribbon synapses in a different manner than conventional active zones. PMID:12358752

  5. 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

  6. A role for vesicular glutamate transporter 1 in synaptic vesicle clustering and mobility.

    PubMed

    Siksou, Léa; Silm, Kätlin; Biesemann, Christoph; Nehring, Ralf B; Wojcik, Sonja M; Triller, Antoine; El Mestikawy, Salah; Marty, Serge; Herzog, Etienne

    2013-05-01

    Synaptic vesicles (SVs) from excitatory synapses carry vesicular glutamate transporters (VGLUTs) that fill the vesicles with neurotransmitter. Although the essential function of VGLUTs as glutamate transporters has been well established, the evidence for additional cell-biological functions is more controversial. Both VGLUT1 and VGLUT2 disruptions in mice result in a reduced number of SVs away from release sites, flattening of SVs, and the appearance of tubular structures. Therefore, we analysed the morphology, biochemical composition and trafficking of SVs at synapses of VGLUT1(-/-) mice in order to test for a function of VGLUTs in the formation or clustering of SVs. Analyses with high-pressure freezing immobilisation and electron tomography pointed to a role of VGLUT1 transport function in the tonicity of excitatory SVs, explaining the aldehyde-induced flattening of SVs observed in VGLUT1(-/-) synapses. We confirmed the steep reduction in the number of SVs previously observed in VGLUT1(-/-) presynaptic terminals, but did not observe accumulation of endocytotic intermediates. Furthermore, SV proteins of adult VGLUT1(-/-) mouse brain tissue were expressed at normal levels in all subcellular fractions, suggesting that they were not displaced to another organelle. We thus assessed the mobility of the recently documented superpool of SVs. Synaptobrevin2-enhanced green fluorescent protein time lapse experiments revealed an oversized superpool of SVs in VGLUT1(-/-) neurons. Our results support the idea that, beyond glutamate loading, VGLUT1 enhances the tonicity of excitatory SVs and stabilises SVs at presynaptic terminals. PMID:23581566

  7. 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

  8. Structural basis of synaptic vesicle assembly promoted by α-synuclein.

    PubMed

    Fusco, Giuliana; Pape, Tillmann; Stephens, Amberley D; Mahou, Pierre; Costa, Ana Rita; Kaminski, Clemens F; Kaminski Schierle, Gabriele S; Vendruscolo, Michele; Veglia, Gianluigi; Dobson, Christopher M; De Simone, Alfonso

    2016-01-01

    α-synuclein (αS) is an intrinsically disordered protein whose fibrillar aggregates are the major constituents of Lewy bodies in Parkinson's disease. Although the specific function of αS is still unclear, a general consensus is forming that it has a key role in regulating the process of neurotransmitter release, which is associated with the mediation of synaptic vesicle interactions and assembly. Here we report the analysis of wild-type αS and two mutational variants linked to familial Parkinson's disease to describe the structural basis of a molecular mechanism enabling αS to induce the clustering of synaptic vesicles. We provide support for this 'double-anchor' mechanism by rationally designing and experimentally testing a further mutational variant of αS engineered to promote stronger interactions between synaptic vesicles. Our results characterize the nature of the active conformations of αS that mediate the clustering of synaptic vesicles, and indicate their relevance in both functional and pathological contexts. PMID:27640673

  9. 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

  10. ATP-dependent directional movement of rat synaptic vesicles injected into the presynaptic terminal of squid giant synapse.

    PubMed Central

    Llinás, R; Sugimori, M; Lin, J W; Leopold, P L; Brady, S T

    1989-01-01

    The question as to whether synaptic vesicles prepared from vertebrate brain can be transported to the active zones of the squid giant synapse was studied by using a combined optical and electrophysiological approach. In order to visualize the behavior of the vertebrate synaptic vesicles in situ, synaptic vesicles isolated from rat brain were labeled with a fluorescent dye (Texas red) and injected into the presynaptic terminal of the squid giant synapse. The pattern of fluorescence that would result from passive diffusion was determined by coinjection of an unconjugated fluorescent dye (fluorescein). The patterns obtained with fluorescent synaptic vesicles were strikingly different from that obtained by simple diffusion of fluorescein. Although the fluorescein diffused freely in both directions, the vesicles moved preferentially into the terminal--i.e., toward the release sites--at a rate of 0.5 microns/sec. The final distribution of the injected fluorescent synaptic vesicles displayed a discrete localization that suggested a distribution coincident with the active zones of the presynaptic terminal. Like fast axonal transport, but unlike fluorescein movements in the terminal, the vesicle movement was energy dependent, since the addition of 2,4-dinitrophenol blocked the redistribution of vesicles completely. In addition, reduction of extracellular calcium concentration reversibly blocked vesicular movement as well. In conclusion, mammalian synaptic vesicles retain the cytoplasmic surface components necessary for translocation, sorting, and targeting to the proper locations by the native machinery of the squid giant synapse. Images PMID:2748609

  11. ATP-dependent directional movement of rat synaptic vesicles injected into the presynaptic terminal of squid giant synapse.

    PubMed

    Llinás, R; Sugimori, M; Lin, J W; Leopold, P L; Brady, S T

    1989-07-01

    The question as to whether synaptic vesicles prepared from vertebrate brain can be transported to the active zones of the squid giant synapse was studied by using a combined optical and electrophysiological approach. In order to visualize the behavior of the vertebrate synaptic vesicles in situ, synaptic vesicles isolated from rat brain were labeled with a fluorescent dye (Texas red) and injected into the presynaptic terminal of the squid giant synapse. The pattern of fluorescence that would result from passive diffusion was determined by coinjection of an unconjugated fluorescent dye (fluorescein). The patterns obtained with fluorescent synaptic vesicles were strikingly different from that obtained by simple diffusion of fluorescein. Although the fluorescein diffused freely in both directions, the vesicles moved preferentially into the terminal--i.e., toward the release sites--at a rate of 0.5 microns/sec. The final distribution of the injected fluorescent synaptic vesicles displayed a discrete localization that suggested a distribution coincident with the active zones of the presynaptic terminal. Like fast axonal transport, but unlike fluorescein movements in the terminal, the vesicle movement was energy dependent, since the addition of 2,4-dinitrophenol blocked the redistribution of vesicles completely. In addition, reduction of extracellular calcium concentration reversibly blocked vesicular movement as well. In conclusion, mammalian synaptic vesicles retain the cytoplasmic surface components necessary for translocation, sorting, and targeting to the proper locations by the native machinery of the squid giant synapse.

  12. 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

  13. 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

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

    PubMed

    Li, Huinan; Harlow, Mark L

    2014-01-01

    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.

  15. Synaptic Vesicle Docking: Sphingosine Regulates Syntaxin1 Interaction with Munc18

    PubMed Central

    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. PMID:19390577

  16. Cholesterol-dependent balance between evoked and spontaneous synaptic vesicle recycling

    PubMed Central

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

    2007-01-01

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

  17. The synaptic vesicle SNARE neuronal Synaptobrevin promotes endolysosomal degradation and prevents neurodegeneration

    PubMed Central

    Haberman, Adam; Williamson, W. Ryan; Epstein, Daniel; Wang, Dong; Rina, Srisha; Meinertzhagen, Ian A.

    2012-01-01

    Soluble NSF attachment protein receptors (SNAREs) are the core proteins in membrane fusion. The neuron-specific synaptic v-SNARE n-syb (neuronal Synaptobrevin) plays a key role during synaptic vesicle exocytosis. In this paper, we report that loss of n-syb caused slow neurodegeneration independent of its role in neurotransmitter release in adult Drosophila melanogaster photoreceptor neurons. In addition to synaptic vesicles, n-Syb localized to endosomal vesicles. Loss of n-syb lead to endosomal accumulations, transmembrane protein degradation defects, and a secondary increase in autophagy. Our evidence suggests a primary defect of impaired delivery of vesicles that contain degradation proteins, including the acidification-activated Cathepsin proteases and the neuron-specific proton pump and V0 adenosine triphosphatase component V100. Overexpressing V100 partially rescued n-syb–dependent degeneration through an acidification-independent endosomal sorting mechanism. Collectively, these findings reveal a role for n-Syb in a neuron-specific sort-and-degrade mechanism that protects neurons from degeneration. Our findings further shed light on which intraneuronal compartments exhibit increased or decreased neurotoxicity. PMID:22270918

  18. Cholinergic synaptic vesicles contain a V-type and a P-type ATPase.

    PubMed

    Yamagata, S K; Parsons, S M

    1989-11-01

    Fifty to eighty-five percent of the ATPase activity in different preparations of cholinergic synaptic vesicles isolated from Torpedo electric organ was half-inhibited by 7 microM vanadate. This activity is due to a recently purified phosphointermediate, or P-type, ATPase, Acetylcholine (ACh) active transport by the vesicles was stimulated about 35% by vanadate, demonstrating that the P-type enzyme is not the proton pump responsible for ACh active transport. Nearly all of the vesicle ATPase activity was inhibited by N-ethylmaleimide. The P-type ATPase could be protected from N-ethylmaleimide inactivation by vanadate, and subsequently reactivated by complexation of vanadate with deferoxamine. The inactivation-protection pattern suggests the presence of a vanadate-insensitive, N-ethylmaleimide-sensitive ATPase consistent with a vacuolar, or V-type, activity expected to drive ACh active transport. ACh active transport was half-inhibited by 5 microM N-ethylmaleimide, even in the presence of vanadate. The presence of a V-type ATPase was confirmed by Western blots using antisera raised against three separate subunits of chromaffin granule vacuolar ATPase I. Both ATPase activities, the P-type polypeptides, and the 38-kilodalton polypeptide of the V-type ATPase precisely copurify with the synaptic vesicles. Solubilization of synaptic vesicles in octaethyleneglycol dodecyl ether detergent results in several-fold stimulation of the P-type activity and inactivation of the V-type activity, thus explaining why the V-type activity was not detected previously during purification of the P-type ATPase. It is concluded that cholinergic vesicles contain a P-type ATPase of unknown function and a V-type ATPase which is the proton pump. PMID:2552014

  19. 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

  20. 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

  1. alpha-Latrotoxin affects mitochondrial potential and synaptic vesicle proton gradient of nerve terminals.

    PubMed

    Tarasenko, A S; Storchak, L G; Himmelreich, N H

    2008-02-01

    Ca(2+)-independent [(3)H]GABA release induced by alpha-latrotoxin was found to consist of two sequential processes: a fast initial release realized via exocytosis and more delayed outflow through the plasma membrane GABA transporters [Linetska, M.V., Storchak, L.G., Tarasenko, A.S., Himmelreich, N.H., 2004. Involvement of membrane GABA transporters in alpha-latrotoxin-stimulated [(3)H]GABA release. Neurochem. Int. 44, 303-312]. To characterize the toxin-stimulated events attributable to the transporter-mediated [(3)H]GABA release from rat brain synaptosomes we studied the effect of alpha-latrotoxin on membrane potentials and generation of the synaptic vesicles proton gradient, using fluorescent dyes: potential-sensitive rhodamine 6G and pH-sensitive acridine orange. We revealed that alpha-latrotoxin induced a progressive dose-dependent depolarization of mitochondrial membrane potential and an irreversible run-down of the synaptic vesicle proton gradient. Both processes were insensitive to the presence of cadmium, a potent blocker of toxin-formed transmembrane pores, indicating that alpha-latrotoxin-induced disturbance of the plasma membrane permeability was not responsible to these effects. A gradual dissipation of the synaptic vesicle proton gradient closely coupled with lowering the vesicular GABA transporter activity results in a leakage of the neurotransmitter from synaptic vesicles to cytoplasm. As a consequence, there is an essential increase in GABA concentration in a soluble cytosolic pool that appears to be critical parameter for altering the mode of the plasma membrane GABA transporter operation from inward to outward. Thus, our data allow clarifying what cell processes underlain a recruitment of the plasma membrane transporter-mediated pathway in alpha-LTX-stimulated secretion.

  2. The Bruchpilot cytomatrix determines the size of the readily releasable pool of synaptic vesicles.

    PubMed

    Matkovic, Tanja; Siebert, Matthias; Knoche, Elena; Depner, Harald; Mertel, Sara; Owald, David; Schmidt, Manuela; Thomas, Ulrich; Sickmann, Albert; Kamin, Dirk; Hell, Stefan W; Bürger, Jörg; Hollmann, Christina; Mielke, Thorsten; Wichmann, Carolin; Sigrist, Stephan J

    2013-08-19

    Synaptic vesicles (SVs) fuse at a specialized membrane domain called the active zone (AZ), covered by a conserved cytomatrix. How exactly cytomatrix components intersect with SV release remains insufficiently understood. We showed previously that loss of the Drosophila melanogaster ELKS family protein Bruchpilot (BRP) eliminates the cytomatrix (T bar) and declusters Ca(2+) channels. In this paper, we explored additional functions of the cytomatrix, starting with the biochemical identification of two BRP isoforms. Both isoforms alternated in a circular array and were important for proper T-bar formation. Basal transmission was decreased in isoform-specific mutants, which we attributed to a reduction in the size of the readily releasable pool (RRP) of SVs. We also found a corresponding reduction in the number of SVs docked close to the remaining cytomatrix. We propose that the macromolecular architecture created by the alternating pattern of the BRP isoforms determines the number of Ca(2+) channel-coupled SV release slots available per AZ and thereby sets the size of the RRP. PMID:23960145

  3. The Bruchpilot cytomatrix determines the size of the readily releasable pool of synaptic vesicles

    PubMed Central

    Matkovic, Tanja; Siebert, Matthias; Knoche, Elena; Depner, Harald; Mertel, Sara; Owald, David; Schmidt, Manuela; Thomas, Ulrich; Sickmann, Albert; Kamin, Dirk; Hell, Stefan W.; Bürger, Jörg; Hollmann, Christina; Mielke, Thorsten

    2013-01-01

    Synaptic vesicles (SVs) fuse at a specialized membrane domain called the active zone (AZ), covered by a conserved cytomatrix. How exactly cytomatrix components intersect with SV release remains insufficiently understood. We showed previously that loss of the Drosophila melanogaster ELKS family protein Bruchpilot (BRP) eliminates the cytomatrix (T bar) and declusters Ca2+ channels. In this paper, we explored additional functions of the cytomatrix, starting with the biochemical identification of two BRP isoforms. Both isoforms alternated in a circular array and were important for proper T-bar formation. Basal transmission was decreased in isoform-specific mutants, which we attributed to a reduction in the size of the readily releasable pool (RRP) of SVs. We also found a corresponding reduction in the number of SVs docked close to the remaining cytomatrix. We propose that the macromolecular architecture created by the alternating pattern of the BRP isoforms determines the number of Ca2+ channel-coupled SV release slots available per AZ and thereby sets the size of the RRP. PMID:23960145

  4. A Stem Cell-Derived Platform for Studying Single Synaptic Vesicles in Dopaminergic Synapses

    PubMed Central

    Gu, Haigang; Lazarenko, Roman M.; Koktysh, Dmitry; Iacovitti, Lorraine

    2015-01-01

    The exocytotic release of dopamine is one of the most characteristic but also one of the least appreciated processes in dopaminergic neurotransmission. Fluorescence imaging has yielded rich information about the properties of synaptic vesicles and the release of neurotransmitters in excitatory and inhibitory neurons. In contrast, imaging-based studies for in-depth understanding of synaptic vesicle behavior in dopamine neurons are lagging largely because of a lack of suitable preparations. Midbrain culture has been one of the most valuable preparations for the subcellular investigation of dopaminergic transmission; however, the paucity and fragility of cultured dopaminergic neurons limits their use for live cell imaging. Recent developments in stem cell technology have led to the successful production of dopamine neurons from embryonic or induced pluripotent stem cells. Although the dopaminergic identity of these stem cell-derived neurons has been characterized in different ways, vesicle-mediated dopamine release from their axonal terminals has been barely assessed. We report a more efficient procedure to reliably generate dopamine neurons from embryonic stem cells, and it yields more dopamine neurons with more dopaminergic axon projections than midbrain culture does. Using a collection of functional measurements, we show that stem cell-derived dopamine neurons are indistinguishable from those in midbrain culture. Taking advantage of this new preparation, we simultaneously tracked the turnover of hundreds of synaptic vesicles individually using pH-sensitive quantum dots. By doing so, we revealed distinct fusion kinetics of the dopamine-secreting vesicles, which is consistent within both preparations. Significance For the use of stem cell-derived neurons in clinical applications, improved differentiation efficiency and more careful characterization of resultant cells are needed. A procedure has been refined for differentiation of mouse embryonic stem cells into

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

    PubMed

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

    2015-04-01

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

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

    PubMed Central

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

    2015-01-01

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

  7. 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

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

    PubMed

    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.

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

    PubMed

    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

  10. Large Structural Change in Isolated Synaptic Vesicles upon Loading with Neurotransmitter

    PubMed Central

    Budzinski, Kristi L.; Allen, Richard W.; Fujimoto, Bryant S.; Kensel-Hammes, P.; Belnap, David M.; Bajjalieh, Sandra M.; Chiu, Daniel T.

    2009-01-01

    The size of a synaptic vesicle (SV) is generally thought to be determined by the amount of lipid and membrane protein it contains. Once formed, it is thought to remain constant in size. Using fluorescence correlation spectroscopy and cryogenic electron microscopy, we show that glutamatergic vesicles reversibly increase their size upon filling with glutamate. The increase (∼25% in diameter) corresponds to an increase in surface area of ∼50% and in volume of ∼100%. This large size increase implies a large structural change in the SV upon loading with neurotransmitters. Vesicles lacking SV protein 2A (SV2A) did not manifest a change in size after loading with glutamate, indicating that SV2A is required for this phenomenon. PMID:19883601

  11. Reduced Expression of the Vesicular Acetylcholine Transporter and Neurotransmitter Content Affects Synaptic Vesicle Distribution and Shape in Mouse Neuromuscular Junction

    PubMed Central

    Rodrigues, Hermann A.; Fonseca, Matheus de C.; Camargo, Wallace L.; Lima, Patrícia M. A.; Martinelli, Patrícia M.; Naves, Lígia A.; Prado, Vânia F.; Prado, Marco A. M.; Guatimosim, Cristina

    2013-01-01

    In vertebrates, nerve muscle communication is mediated by the release of the neurotransmitter acetylcholine packed inside synaptic vesicles by a specific vesicular acetylcholine transporter (VAChT). Here we used a mouse model (VAChT KDHOM) with 70% reduction in the expression of VAChT to investigate the morphological and functional consequences of a decreased acetylcholine uptake and release in neuromuscular synapses. Upon hypertonic stimulation, VAChT KDHOM mice presented a reduction in the amplitude and frequency of miniature endplate potentials, FM 1–43 staining intensity, total number of synaptic vesicles and altered distribution of vesicles within the synaptic terminal. In contrast, under electrical stimulation or no stimulation, VAChT KDHOM neuromuscular junctions did not differ from WT on total number of vesicles but showed altered distribution. Additionally, motor nerve terminals in VAChT KDHOM exhibited small and flattened synaptic vesicles similar to that observed in WT mice treated with vesamicol that blocks acetylcholine uptake. Based on these results, we propose that decreased VAChT levels affect synaptic vesicle biogenesis and distribution whereas a lower ACh content affects vesicles shape. PMID:24260111

  12. Reduced expression of the vesicular acetylcholine transporter and neurotransmitter content affects synaptic vesicle distribution and shape in mouse neuromuscular junction.

    PubMed

    Rodrigues, Hermann A; Fonseca, Matheus de C; Camargo, Wallace L; Lima, Patrícia M A; Martinelli, Patrícia M; Naves, Lígia A; Prado, Vânia F; Prado, Marco A M; Guatimosim, Cristina

    2013-01-01

    In vertebrates, nerve muscle communication is mediated by the release of the neurotransmitter acetylcholine packed inside synaptic vesicles by a specific vesicular acetylcholine transporter (VAChT). Here we used a mouse model (VAChT KD(HOM)) with 70% reduction in the expression of VAChT to investigate the morphological and functional consequences of a decreased acetylcholine uptake and release in neuromuscular synapses. Upon hypertonic stimulation, VAChT KD(HOM) mice presented a reduction in the amplitude and frequency of miniature endplate potentials, FM 1-43 staining intensity, total number of synaptic vesicles and altered distribution of vesicles within the synaptic terminal. In contrast, under electrical stimulation or no stimulation, VAChT KD(HOM) neuromuscular junctions did not differ from WT on total number of vesicles but showed altered distribution. Additionally, motor nerve terminals in VAChT KD(HOM) exhibited small and flattened synaptic vesicles similar to that observed in WT mice treated with vesamicol that blocks acetylcholine uptake. Based on these results, we propose that decreased VAChT levels affect synaptic vesicle biogenesis and distribution whereas a lower ACh content affects vesicles shape. PMID:24260111

  13. 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

  14. Diacylglycerol, phosphatidic acid, and their metabolic enzymes in synaptic vesicle recycling.

    PubMed

    Tu-Sekine, Becky; Goldschmidt, Hana; Raben, Daniel M

    2015-01-01

    The synaptic vesicle (SV) cycle includes exocytosis of vesicles loaded with a neurotransmitter such as glutamate, coordinated recovery of SVs by endocytosis, refilling of vesicles, and subsequent release of the refilled vesicles from the presynaptic bouton. SV exocytosis is tightly linked with endocytosis, and variations in the number of vesicles, and/or defects in the refilling of SVs, will affect the amount of neurotransmitter available for release (Sudhof, 2004). There is increasing interest in the roles synaptic vesicle lipids and lipid metabolizing enzymes play in this recycling. Initial emphasis was placed on the role of polyphosphoinositides in SV cycling as outlined in a number of reviews (Lim and Wenk, 2009; Martin, 2012; Puchkov and Haucke, 2013; Rohrbough and Broadie, 2005). Other lipids are now recognized to also play critical roles. For example, PLD1 (Humeau et al., 2001; Rohrbough and Broadie, 2005) and some DGKs (Miller et al., 1999; Nurrish et al., 1999) play roles in neurotransmission which is consistent with the critical roles for phosphatidic acid (PtdOH) and diacylglycerol (DAG) in the regulation of SV exo/endocytosis (Cremona et al., 1999; Exton, 1994; Huttner and Schmidt, 2000; Lim and Wenk, 2009; Puchkov and Haucke, 2013; Rohrbough and Broadie, 2005). PLD generates phosphatidic acid by catalyzing the hydrolysis of phosphatidylcholine (PtdCho) and in some systems this PtdOH is de-phosphorylated to generate DAG. In contrast, DGK catalyzes the phosphorylation of DAG thereby converting it into PtdOH. While both enzymes are poised to regulate the levels of DAG and PtdOH, therefore, they both lead to the generation of PtdOH and could have opposite effects on DAG levels. This is particularly important for SV cycling as PtdOH and DAG are both needed for evoked exocytosis (Lim and Wenk, 2009; Puchkov and Haucke, 2013; Rohrbough and Broadie, 2005). Two lipids and their involved metabolic enzymes, two sphingolipids have also been implicated in

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

    PubMed

    Wang, Xueyong; Pinter, Martin J; Rich, Mark M

    2016-01-20

    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 Ca(2+)-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 Ca(2+)-dependent

  16. 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

  17. Electrical synapse formation disrupts calcium-dependent exocytosis, but not vesicle mobilization.

    PubMed

    Neunuebel, Joshua P; Zoran, Mark J

    2005-06-01

    Electrical coupling exists prior to the onset of chemical connectivity at many developing and regenerating synapses. At cholinergic synapses in vitro, trophic factors facilitated the formation of electrical synapses and interfered with functional neurotransmitter release in response to photolytic elevations of intracellular calcium. In contrast, neurons lacking trophic factor induction and electrical coupling possessed flash-evoked transmitter release. Changes in cytosolic calcium and postsynaptic responsiveness to acetylcholine were not affected by electrical coupling. These data indicate that transient electrical synapse formation delayed chemical synaptic transmission by imposing a functional block between the accumulation of presynaptic calcium and synchronized, vesicular release. Despite the inability to release neurotransmitter, neurons that had possessed strong electrical coupling recruited secretory vesicles to sites of synaptic contact. These results suggest that the mechanism by which neurotransmission is disrupted during electrical synapse formation is downstream of both calcium influx and synaptic vesicle mobilization. Therefore, electrical synaptogenesis may inhibit synaptic vesicles from acquiring a readily releasable state. We hypothesize that gap junctions might negatively interact with exocytotic processes, thereby diminishing chemical neurotransmission. PMID:15765535

  18. 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

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

    PubMed

    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

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

    PubMed

    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.

  1. Individual Neuronal Subtypes Exhibit Diversity in CNS Myelination Mediated by Synaptic Vesicle Release.

    PubMed

    Koudelka, Sigrid; Voas, Matthew G; Almeida, Rafael G; Baraban, Marion; Soetaert, Jan; Meyer, Martin P; Talbot, William S; Lyons, David A

    2016-06-01

    Regulation of myelination by oligodendrocytes in the CNS has important consequences for higher-order nervous system function (e.g., [1-4]), and there is growing consensus that neuronal activity regulates CNS myelination (e.g., [5-9]) through local axon-oligodendrocyte synaptic-vesicle-release-mediated signaling [10-12]. Recent analyses have indicated that myelination along axons of distinct neuronal subtypes can differ [13, 14], but it is not known whether regulation of myelination by activity is common to all neuronal subtypes or only some. This limits insight into how specific neurons regulate their own conduction. Here, we use a novel fluorescent fusion protein reporter to study myelination along the axons of distinct neuronal subtypes over time in zebrafish. We find that the axons of reticulospinal and commissural primary ascending (CoPA) neurons are among the first myelinated in the zebrafish CNS. To investigate how activity regulates myelination by different neuronal subtypes, we express tetanus toxin (TeNT) in individual reticulospinal or CoPA neurons to prevent synaptic vesicle release. We find that the axons of individual tetanus toxin expressing reticulospinal neurons have fewer myelin sheaths than controls and that their myelin sheaths are 50% shorter than controls. In stark contrast, myelination along tetanus-toxin-expressing CoPA neuron axons is entirely normal. These results indicate that while some neuronal subtypes modulate myelination by synaptic vesicle release to a striking degree in vivo, others do not. These data have implications for our understanding of how different neurons regulate myelination and thus their own function within specific neuronal circuits.

  2. 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.

  3. 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.

  4. 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

  5. SAD-B Phosphorylation of CAST Controls Active Zone Vesicle Recycling for Synaptic Depression.

    PubMed

    Mochida, Sumiko; Hida, Yamato; Tanifuji, Shota; Hagiwara, Akari; Hamada, Shun; Abe, Manabu; Ma, Huan; Yasumura, Misato; Kitajima, Isao; Sakimura, Kenji; Ohtsuka, Toshihisa

    2016-09-13

    Short-term synaptic depression (STD) is a common form of activity-dependent plasticity observed widely in the nervous system. Few molecular pathways that control STD have been described, but the active zone (AZ) release apparatus provides a possible link between neuronal activity and plasticity. Here, we show that an AZ cytomatrix protein CAST and an AZ-associated protein kinase SAD-B coordinately regulate STD by controlling reloading of the AZ with release-ready synaptic vesicles. SAD-B phosphorylates the N-terminal serine (S45) of CAST, and S45 phosphorylation increases with higher firing rate. A phosphomimetic CAST (S45D) mimics CAST deletion, which enhances STD by delaying reloading of the readily releasable pool (RRP), resulting in a pool size decrease. A phosphonegative CAST (S45A) inhibits STD and accelerates RRP reloading. Our results suggest that the CAST/SAD-B reaction serves as a brake on synaptic transmission by temporal calibration of activity and synaptic depression via RRP size regulation. PMID:27626661

  6. SAD-B Phosphorylation of CAST Controls Active Zone Vesicle Recycling for Synaptic Depression.

    PubMed

    Mochida, Sumiko; Hida, Yamato; Tanifuji, Shota; Hagiwara, Akari; Hamada, Shun; Abe, Manabu; Ma, Huan; Yasumura, Misato; Kitajima, Isao; Sakimura, Kenji; Ohtsuka, Toshihisa

    2016-09-13

    Short-term synaptic depression (STD) is a common form of activity-dependent plasticity observed widely in the nervous system. Few molecular pathways that control STD have been described, but the active zone (AZ) release apparatus provides a possible link between neuronal activity and plasticity. Here, we show that an AZ cytomatrix protein CAST and an AZ-associated protein kinase SAD-B coordinately regulate STD by controlling reloading of the AZ with release-ready synaptic vesicles. SAD-B phosphorylates the N-terminal serine (S45) of CAST, and S45 phosphorylation increases with higher firing rate. A phosphomimetic CAST (S45D) mimics CAST deletion, which enhances STD by delaying reloading of the readily releasable pool (RRP), resulting in a pool size decrease. A phosphonegative CAST (S45A) inhibits STD and accelerates RRP reloading. Our results suggest that the CAST/SAD-B reaction serves as a brake on synaptic transmission by temporal calibration of activity and synaptic depression via RRP size regulation.

  7. 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. PMID:27025060

  8. Cannabinoid agonists rearrange synaptic vesicles at excitatory synapses and depress motoneuron activity in vivo.

    PubMed

    García-Morales, Victoria; Montero, Fernando; Moreno-López, Bernardo

    2015-05-01

    Impairment of motor skills is one of the most common acute adverse effects of cannabis. Related studies have focused mainly on psychomotor alterations, and little is known about the direct impact of cannabinoids (CBs) on motoneuron physiology. As key modulators of synaptic function, CBs regulate multiple neuronal functions and behaviors. Presynaptic CB1 mediates synaptic strength depression by inhibiting neurotransmitter release, via a poorly understood mechanism. The present study examined the effect of CB agonists on excitatory synaptic inputs incoming to hypoglossal motoneurons (HMNs) in vitro and in vivo. The endocannabinoid anandamide (AEA) and the synthetic CB agonist WIN 55,212-2 rapidly and reversibly induced short-term depression (STD) of glutamatergic synapses on motoneurons by a presynaptic mechanism. Presynaptic effects were fully reversed by the CB1-selective antagonist AM281. Electrophysiological and electron microscopy analysis showed that WIN 55,212-2 reduced the number of synaptic vesicles (SVs) docked to active zones in excitatory boutons. Given that AM281 fully abolished depolarization-induced depression of excitation, motoneurons can be feasible sources of CBs, which in turn act as retrograde messengers regulating synaptic function. Finally, microiontophoretic application of the CB agonist O-2545 reversibly depressed, presumably via CB1, glutamatergic inspiratory-related activity of HMNs in vivo. Therefore, evidence support that CBs, via presynaptic CB1, induce excitatory STD by reducing the readily releasable pool of SVs at excitatory synapses, then attenuating motoneuron activity. These outcomes contribute a possible mechanistic basis for cannabis-associated motor performance disturbances such as ataxia, dysarthria and dyscoordination.

  9. Cannabinoid agonists rearrange synaptic vesicles at excitatory synapses and depress motoneuron activity in vivo.

    PubMed

    García-Morales, Victoria; Montero, Fernando; Moreno-López, Bernardo

    2015-05-01

    Impairment of motor skills is one of the most common acute adverse effects of cannabis. Related studies have focused mainly on psychomotor alterations, and little is known about the direct impact of cannabinoids (CBs) on motoneuron physiology. As key modulators of synaptic function, CBs regulate multiple neuronal functions and behaviors. Presynaptic CB1 mediates synaptic strength depression by inhibiting neurotransmitter release, via a poorly understood mechanism. The present study examined the effect of CB agonists on excitatory synaptic inputs incoming to hypoglossal motoneurons (HMNs) in vitro and in vivo. The endocannabinoid anandamide (AEA) and the synthetic CB agonist WIN 55,212-2 rapidly and reversibly induced short-term depression (STD) of glutamatergic synapses on motoneurons by a presynaptic mechanism. Presynaptic effects were fully reversed by the CB1-selective antagonist AM281. Electrophysiological and electron microscopy analysis showed that WIN 55,212-2 reduced the number of synaptic vesicles (SVs) docked to active zones in excitatory boutons. Given that AM281 fully abolished depolarization-induced depression of excitation, motoneurons can be feasible sources of CBs, which in turn act as retrograde messengers regulating synaptic function. Finally, microiontophoretic application of the CB agonist O-2545 reversibly depressed, presumably via CB1, glutamatergic inspiratory-related activity of HMNs in vivo. Therefore, evidence support that CBs, via presynaptic CB1, induce excitatory STD by reducing the readily releasable pool of SVs at excitatory synapses, then attenuating motoneuron activity. These outcomes contribute a possible mechanistic basis for cannabis-associated motor performance disturbances such as ataxia, dysarthria and dyscoordination. PMID:25595101

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

    PubMed

    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.

  11. Vesicular Monoamine and Glutamate Transporters Select Distinct Synaptic Vesicle Recycling Pathways

    PubMed Central

    Onoa, Bibiana; Li, Haiyan; Gagnon-Bartsch, Johann A.; Elias, Laura A. B.; Edwards, Robert H.

    2011-01-01

    Previous work has characterized the properties of neurotransmitter release at excitatory and inhibitory synapses, but we know remarkably little about the properties of monoamine release because these neuromodulators do not generally produce a fast ionotropic response. Since dopamine and serotonin neurons can also release glutamate in vitro and in vivo, we have used the vesicular monoamine transporter VMAT2 and the vesicular glutamate transporter VGLUT1 to compare the localization and recycling of synaptic vesicles that store, respectively, monoamines and glutamate. First, VMAT2 segregates partially from VGLUT1 in the boutons of midbrain dopamine neurons, indicating the potential for distinct release sites. Second, endocytosis after stimulation is slower for VMAT2 than VGLUT1. During the stimulus, however, the endocytosis of VMAT2 (but not VGLUT1) accelerates dramatically in midbrain dopamine but not hippocampal neurons, indicating a novel, cell-specific mechanism to sustain high rates of release. On the other hand, we find that in both midbrain dopamine and hippocampal neurons, a substantially smaller proportion of VMAT2 than VGLUT1 is available for evoked release, and VMAT2 shows considerably more dispersion along the axon after exocytosis than VGLUT1. Even when expressed in the same neuron, the two vesicular transporters thus target to distinct populations of synaptic vesicles, presumably due to their selection of distinct recycling pathways. PMID:20534840

  12. 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

  13. SCAMP5 plays a critical role in synaptic vesicle endocytosis during high neuronal activity.

    PubMed

    Zhao, Haiyan; Kim, Yoonju; Park, Joohyun; Park, Daehun; Lee, Sang-Eun; Chang, Iree; Chang, Sunghoe

    2014-07-23

    Secretory carrier membrane protein 5 (SCAMP5), a recently identified candidate gene for autism, is brain specific and highly abundant in synaptic vesicles (SVs), but its function is currently unknown. Here, we found that knockdown (KD) of endogenous SCAMP5 by SCAMP5-specific shRNAs in cultured rat hippocampal neurons resulted in a reduction in total vesicle pool size as well as in recycling pool size, but the recycling/resting pool ratio was significantly increased. SCAMP5 KD slowed endocytosis after stimulation, but impaired it severely during strong stimulation. We also found that KD dramatically lowered the threshold of activity at which SV endocytosis became unable to compensate for the ongoing exocytosis occurring during a stimulus. Reintroducing shRNA-resistant SCAMP5 reversed these endocytic defects. Therefore, our results suggest that SCAMP5 functions during high neuronal activity when a heavy load is imposed on endocytosis. Our data also raise the possibility that the reduction in expression of SCAMP5 in autistic patients may be related to the synaptic dysfunction observed in autism.

  14. 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

  15. Measurement of quantal secretion induced by ouabain and its correlation with depletion of synaptic vesicles.

    PubMed

    Haimann, C; Torri-Tarelli, F; Fesce, R; Ceccarelli, B

    1985-11-01

    Ouabain (0.1 and 0.05 mM) was applied to frog cutaneous pectoris nerve-muscle preparations bathed in modified Ringer's solution containing either 1.8 mM Ca2+ (and 4 mM Mg2+) or no added Ca2+ (4 mM Mg2+ and 1 mM EGTA). During the intense quantal release of acetylcholine (ACh) induced by ouabain, the parameters of the miniature endplate potentials (mepps) were deduced from the variance, skew, and power spectra of the endplate recordings by applying a recently described modification of classical fluctuation analysis. Often the high frequency of mepps is not stationary; therefore, the signal was high-pass filtered (time constant of the resistance-capacitance filter of 2 ms) to remove the errors introduced by nonstationarity. When ouabain was applied in the presence of Ca2+, mepp frequency started to rise exponentially after a lag of 1.5-2 h, reached an average peak frequency of 1,300/s in approximately 30 min, and then suddenly subsided to low level (10/s). In Ca2+-free solution, after a shorter lag (1-1.5 h), mepp frequency rose to peak rate of 700/s in approximately 20 min and then gradually subsided. In spite of the different time course of secretion in the two experimental conditions, the cumulative quantal release was not significantly different (7.4 +/- 1.3 X 10(5) in Ca2+-containing and 8.8 +/- 2.7 X 10(5) in Ca2+-free solutions). 60 min after the peak secretion, the muscles were fixed for observation in the electron microscope. Morphometric analysis on micrographs of neuromuscular junctions revealed in both cases a profound depletion of synaptic vesicles and deep infoldings of presynaptic membrane. This rapid depletion and the lack of uptake of horseradish peroxidase suggest that ouabain impairs the recycling process that tends to conserve the vesicle population during intense secretion of neurotransmitter. The good correlation observed between the reduction in the store of synaptic vesicles and the total number of quanta of ACh secreted in the absence of a

  16. 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

  17. 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.

  18. 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

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

    PubMed

    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.

  20. 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

  1. 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

  2. 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

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

    PubMed Central

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

    2014-01-01

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

  4. 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.

  5. Formation and structural characteristics of thermosensitive multiblock copolymer vesicles.

    PubMed

    Ma, Shiying; Xiao, Mengying; Wang, Rong

    2013-12-23

    The spontaneous vesicle formation of ABABA-type amphiphilic multiblock copolymers bearing thermosensitive hydrophilic A-block in a selective solvent is studied using dissipative particle dynamics (DPD) approach. The formation process of vesicle through nucleation and growth pathway is observed by varying the temperature. The simulation results show that spherical micelle takes shape at high temperature. As temperature decreases, vesicles with small aqueous cavity appear and the cavity expands as well as the membrane thickness decreases with the temperature further decreasing. This finding is in agreement with the experimental observation. Furthermore, by continuously varying the temperature and the length of the hydrophobic block, a phase diagram is constructed, which can indicate the thermodynamically stable region for vesicles. The morphological phase diagram shows that vesicles can form in a larger parameter scope. The relationship between the hydrophilic and hydrophobic block length versus the aqueous cavity size and vesicle size are revealed. Simulation results demonstrate that the copolymers with shorter hydrophobic blocks length or the higher hydrophilicity are more likely to form vesicles with larger aqueous cavity size and vesicle size as well as thinner wall thickness. However, the increase in A-block length results to form vesicles with smaller aqueous cavity size and larger vesicle size. PMID:24304193

  6. A catalytic independent function of the deubiquitinating enzyme USP14 regulates hippocampal synaptic short-term plasticity and vesicle number

    PubMed Central

    Walters, Brandon J; Hallengren, Jada J; Theile, Christopher S; Ploegh, Hidde L; Wilson, Scott M; Dobrunz, Lynn E

    2014-01-01

    AbstractThe ubiquitin proteasome system is required for the rapid and precise control of protein abundance that is essential for synaptic function. USP14 is a proteasome-bound deubiquitinating enzyme that recycles ubiquitin and regulates synaptic short-term synaptic plasticity. We previously reported that loss of USP14 in axJ mice causes a deficit in paired pulse facilitation (PPF) at hippocampal synapses. Here we report that USP14 regulates synaptic function through a novel, deubiquitination-independent mechanism. Although PPF is usually inversely related to release probability, USP14 deficiency impairs PPF without altering basal release probability. Instead, the loss of USP14 causes a large reduction in the number of synaptic vesicles. Over-expression of a catalytically inactive form of USP14 rescues the PPF deficit and restores synaptic vesicle number, indicating that USP14 regulates presynaptic structure and function independently of its role in deubiquitination. Finally, the PPF deficit caused by loss of USP14 can be rescued by pharmacological inhibition of proteasome activity, suggesting that inappropriate protein degradation underlies the PPF impairment. Overall, we demonstrate a novel, deubiquitination-independent function for USP14 in influencing synaptic architecture and plasticity. PMID:24218545

  7. 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

  8. Endocytic structures and synaptic vesicle recycling at a central synapse in awake rats.

    PubMed

    Körber, Christoph; Horstmann, Heinz; Sätzler, Kurt; Kuner, Thomas

    2012-12-01

    The synaptic vesicle (SV) cycle has been studied extensively in cultured cells and slice preparations, but not much is known about the roles and relative contributions of endocytic pathways and mechanisms of SV recycling in vivo, under physiological patterns of activity. We employed horseradish peroxidase (HRP) as an in vivo marker of endocytosis at the calyx of Held synapse in the awake rat. Ex vivo serial section scanning electron microscopy and 3D reconstructions revealed two categories of labelled structures: HRP-filled SVs and large cisternal endosomes. Inhibition of adaptor protein complexes 1 and 3 (AP-1, AP-3) by in vivo application of Brefeldin A (BFA) disrupted endosomal SV budding while SV recycling via clathrin-mediated endocytosis (CME) remained unaffected. In conclusion, our study establishes cisternal endosomes as an intermediate of the SV cycle and reveals CME and endosomal budding as the predominant mechanisms of SV recycling in a tonically active central synapse in vivo.

  9. Fractional vesamicol receptor occupancy and acetylcholine active transport inhibition in synaptic vesicles.

    PubMed

    Kaufman, R; Rogers, G A; Fehlmann, C; Parsons, S M

    1989-09-01

    Vesamicol [(-)-(trans)-2-(4-phenylpiperidino)cyclohexanol] receptor binding and inhibition of acetylcholine (AcCh) active transport by cholinergic synaptic vesicles that were isolated from Torpedo electric organ were studied for 23 vesamicol enantiomers, analogues, and other drugs. Use of trace [3H]vesamicol and [14C]AcCh allowed simultaneous determination of the concentrations of enantiomer, analogue, or drug required to half-saturate the vesamicol receptor (Ki) and to half-inhibit transport (IC50), respectively. Throughout a wide range of potencies for different compounds, the Ki/IC50 ratios varied from 1.5 to 24. Compounds representative of the diverse structures studied, namely deoxyvesamicol, chloroquine, and levorphanol, were competitive inhibitors of vesamicol binding. It is concluded that many drugs can bind to the vesamicol receptor and binding to only a small fraction of the receptors can result in AcCh active transport inhibition. Possible mechanisms for this effect are discussed. PMID:2550778

  10. Synaptic Vesicle Endocytosis and Endosomal Recycling in Central Nerve Terminals: Discrete Trafficking Routes?

    PubMed

    Cousin, Michael A

    2015-08-01

    Synaptic vesicle (SV) retrieval from the presynaptic plasma membrane occurs via a variety of different and complementary modes. The dominant retrieval mode during high-intensity stimulation is activity-dependent bulk endocytosis (ADBE). ADBE involves the generation of endosomes direct from the plasma membrane which then donate membrane and cargo to form SVs that replenish the reserve SV pool. Recent evidence has suggested that ADBE may involve an additional endosomal processing step to produce a mature, functional SV. This suggests that ADBE may utilize key molecules or indeed whole pathways from classical endocytic recycling routes that are ubiquitous across all cell types. This review will assess the current evidence for a contribution of endocytic recycling to the SV life cycle, with a particular focus on ADBE. In doing so it highlights points where both routes may either converge or exploit existing mechanisms to ensure efficient generation of SVs during high-intensity stimulation.

  11. 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. PMID:24882013

  12. Platelet Activating Factor Enhances Synaptic Vesicle Exocytosis Via PKC, Elevated Intracellular Calcium, and Modulation of Synapsin 1 Dynamics and Phosphorylation

    PubMed Central

    Hammond, Jennetta W.; Lu, Shao-Ming; Gelbard, Harris A.

    2016-01-01

    Platelet activating factor (PAF) is an inflammatory phospholipid signaling molecule implicated in synaptic plasticity, learning and memory and neurotoxicity during neuroinflammation. However, little is known about the intracellular mechanisms mediating PAF’s physiological or pathological effects on synaptic facilitation. We show here that PAF receptors are localized at the synapse. Using fluorescent reporters of presynaptic activity we show that a non-hydrolysable analog of PAF (cPAF) enhances synaptic vesicle release from individual presynaptic boutons by increasing the size or release of the readily releasable pool and the exocytosis rate of the total recycling pool. cPAF also activates previously silent boutons resulting in vesicle release from a larger number of terminals. The underlying mechanism involves elevated calcium within presynaptic boutons and protein kinase C activation. Furthermore, cPAF increases synapsin I phosphorylation at sites 1 and 3, and increases dispersion of synapsin I from the presynaptic compartment during stimulation, freeing synaptic vesicles for subsequent release. These findings provide a conceptual framework for how PAF, regardless of its cellular origin, can modulate synapses during normal and pathologic synaptic activity. PMID:26778968

  13. Botulinum Neurotoxins Can Enter Cultured Neurons Independent of Synaptic Vesicle Recycling

    PubMed Central

    Pellett, Sabine; Tepp, William H.; Scherf, Jacob M.; Johnson, Eric A.

    2015-01-01

    Botulinum neurotoxins (BoNTs) are the causative agent of the severe and long-lasting disease botulism. At least seven different serotypes of BoNTs (denoted A-G) have been described. All BoNTs enter human or animal neuronal cells via receptor mediated endocytosis and cleave cytosolic SNARE proteins, resulting in a block of synaptic vesicle exocytosis, leading to the flaccid paralysis characteristic of botulism. Previous data have indicated that once a neuronal cell has been intoxicated by a BoNT, further entry of the same or other BoNTs is prevented due to disruption of synaptic vesicle recycling. However, it has also been shown that cultured neurons exposed to BoNT/A are still capable of taking up BoNT/E. In this report we show that in general BoNTs can enter cultured human or mouse neuronal cells that have previously been intoxicated with another BoNT serotype. Quantitative analysis of cell entry by assessing SNARE cleavage revealed none or only a minor difference in the efficiency of uptake of BoNTs into previously intoxicated neurons. Examination of the endocytic entry pathway by specific endocytosis inhibitors indicated that BoNTs are taken up by clathrin coated pits in both non pre-exposed and pre-exposed neurons. LDH release assays indicated that hiPSC derived neurons exposed consecutively to two different BoNT serotypes remained viable and healthy except in the case of BoNT/E or combinations of BoNT/E with BoNT/B, /D, or /F. Overall, our data indicate that previous intoxication of neuronal cells with BoNT does not inhibit further uptake of BoNTs. PMID:26207366

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

    PubMed

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

    2015-08-11

    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, Ca(2+)-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.

  15. 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

  16. The synaptic vesicle protein synaptophysin: purification and characterization of its channel activity.

    PubMed Central

    Gincel, Dan; Shoshan-Barmatz, Varda

    2002-01-01

    The synaptic vesicle protein synaptophysin was solubilized from rat brain synaptosomes with a relatively low concentration of Triton X-100 (0.2%) and was highly purified (above 95%) using a rapid single chromatography step on hydroxyapatite/celite resin. Purified synaptophysin was reconstituted into a planar lipid bilayer and the channel activity of synaptophysin was characterized. In asymmetric KCl solutions (cis 300 mM/trans 100 mM), synaptophysin formed a fast-fluctuating channel with a conductance of 414 +/- 13 pS at +60 mV. The open probability of synaptophysin channels was decreased upon depolarization, and channels were found to be cation-selective. Synaptophysin channels showed higher selectivity for K(+) over Cl(-) (P(K(+))/P(Cl(-)) > 8) and preferred K(+) over Li(+), Na(+), Rb(+), Cs(+), or choline(+). The synaptophysin channel is impermeable to Ca(2+), which has no effect on its channel activity. This study is the second demonstration of purified synaptophysin channel activity, but the first biophysical characterization of its channel properties. The availability of large amounts of purified synaptophysin and of its characteristic channel properties might help to establish the role of synaptophysin in synaptic transmission. PMID:12496091

  17. Temporal coincidence between synaptic vesicle fusion and quantal secretion of acetylcholine

    PubMed Central

    1985-01-01

    We applied the quick-freezing technique to investigate the precise temporal coincidence between the onset of quantal secretion and the appearance of fusions of synaptic vesicles with the prejunctional membrane. Frog cutaneous pectoris nerve-muscle preparations were soaked in modified Ringer's solution with 1 mM 4-aminopyridine, 10 mM Ca2+, and 10(-4) M d-Tubocurarine and quick-frozen 1-10 ms after a single supramaximal shock. The frozen muscles were then either freeze- fractured or cryosubstituted in acetone with 13% OsO4 and processed for thin section electron microscopy. Temporal resolution of less than 1 ms can be achieved using a quick-freeze device that increases the rate of freezing of the muscle after it strikes the chilled copper block (15 degrees K) and that minimizes the precooling of the muscle during its descent toward the block. We minimized variations in transmission time by examining thin sections taken only from the medial edge of the muscle, which was at a fixed distance from the point of stimulation of the nerve. The ultrastructure of the cryosubstituted preparations was well preserved to a depth of 5 - 10 micron, and within this narrow band vesicles were found fused with the axolemma after a minimum delay of 2.5 ms after stimulation of the nerve. Since the total transmission time to this edge of the muscle was approximately 3 ms, these results indicate that the vesicles fuse with the axolemma precisely at the same time the quanta are released. Freeze-fracture does not seem to be an adequate experimental technique for this work because in the well- preserved band of the muscle the fracture plane crosses, but does not cleave, the inner hydrophobic domain of the plasmalemma. Fracture faces may form in deeper regions of the muscle where tissue preservation is unsatisfactory and freezing is delayed. PMID:2995407

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

    PubMed

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

    2010-11-18

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

  19. 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

  20. Identification of a synaptic vesicle-specific membrane protein with a wide distribution in neuronal and neurosecretory tissue

    PubMed Central

    1981-01-01

    Two different monoclonal antibodies, characterized initially as binding synaptic terminal regions of rat brain, bind a 65,000-dalton protein, which is exposed on the outer surface of brain synaptic vesicles. Immunocytochemical experiments at the electron microscope level demonstrate that these antibodies bind the vesicles in many different types of nerve terminals. The antibodies have been used successfully to purify synaptic vesicles from crude brain homogenates by immunoprecipitation onto the surface of polyacrylamide beads. The profiles of the structures precipitated by these beads are almost exclusively vesicular, confirming the vesicle-specificity of the antibodies. In SDS gels, the antibodies bind a single protein of 65,000 daltons. The two antibodies are not identical, but compete for binding sites on this protein. Immune competition experiments also demonstrate that the antigenic components on the 65,000-dalton protein are widely distributed in neuronal and neural secretory tissues. Detectable antigen is not found in uninnervated tissue--blood cells and extrajunctional muscle. Low levels are found in nonneural secretory tissues; it is not certain whether this reflects the presence of low amounts of the antigen on all the exocytotic vesicles in these tissues or whether the antigen is found only in neuronal fibers within these tissues. The molecular weight and at least two antigenic determinants of the 65,000-dalton protein are highly conserved throughout vertebrate phylogeny. The two antibodies recognize a 65,000-dalton protein present in shark, amphibia, birds, and mammals. The highly conserved nature of the determinants on this protein and their specific localization on secretory vesicles of many different types suggest that this protein may be essential for the normal function of neuronal secretory vesicles. PMID:7298720

  1. 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.

  2. 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.

  3. Selective MPP+ uptake into synaptic dopamine vesicles: possible involvement in MPTP neurotoxicity.

    PubMed Central

    Del Zompo, M.; Piccardi, M. P.; Ruiu, S.; Quartu, M.; Gessa, G. L.; Vaccari, A.

    1993-01-01

    1. In the present study we provide evidence for a saturable, Mg2+/ATP- and temperature-dependent, tetrabenazine-, dopamine-, and amphetamine-sensitive uptake of 1-methyl-4-phenylpyridinium ion (MPP+) in synaptic vesicles from mouse striatum. 2. Similarity in the properties of the vesicular uptake suggests that in the striatum dopamine and MPP+ share the vesicular carrier. 3. The presence of MPP+ vesicular uptake in dopamine-rich regions such as striatum, olfactory, tubercles and hypothalamus, as well as its absence in cerebellum, cortex and pons-medulla, suggest that monoamine vesicular carriers differ between highly and poorly dopamine-innervated regions. 4. The restriction of active MPP+ uptake to the dopaminergic regions, which reflects the previously shown distribution of [3H]-MPP+ binding sites in mouse brain membranes, indicates MPP+ as a marker of the vesicular carrier for dopamine in dopaminergic neurones. 5. A role in MPP+ neurotoxicity is suggested for this region-specific, vesicular storage of the toxin. Images Figure 1 PMID:8102929

  4. Monitoring Synaptic Vesicle Protein Sorting with Enhanced Horseradish Peroxidase in the Electron Microscope.

    PubMed

    Schikorski, Thomas

    2016-01-01

    Protein sorting is the fundamental cellular process that creates and maintains cell organelles and subcellular structures. The synaptic vesicle (SV) is a unique cell organelle that contains a plethora of specific SV proteins and its protein composition is crucial for its function. Thus understanding the mechanisms that sort proteins to SVs and other cell organelles is central to neuroscience and cell biology.While in the past protein sorting was studied in the fluorescence and confocal microscope, we here present a protocol that reveals SV protein trafficking and sorting in the electron microscope (EM). The protocol exploits tagging SV proteins with a new genetically encoded label for EM: enhanced horseradish peroxidase (eHRP). eHRP gained its high sensitivity through direct evolution of its catalytic activity and is detectable in the EM and LM after expression in neurons and other mammalian cells. The protocol describes the use of eHRP, labeling of SVs in cultured hippocampal neurons, and analysis via serial section reconstruction. PMID:27515091

  5. Synaptic vesicle generation from activity-dependent bulk endosomes requires calcium and calcineurin.

    PubMed

    Cheung, Giselle; Cousin, Michael A

    2013-02-20

    Activity-dependent bulk endocytosis (ADBE) is the dominant mode of synaptic vesicle (SV) endocytosis during high-frequency stimulation in central nerve terminals. ADBE generates endosomes direct from the plasma membrane, meaning that high concentrations of calcium will be present in their interior due to fluid phase uptake from the extracellular space. Morphological and fluorescent assays were used to track the generation of SVs from bulk endosomes in primary neuronal culture. This process was functionally uncoupled from both SV exocytosis and plasma membrane retrieval events by intervening only after SV fusion and endocytosis were completed. Either intracellular (BAPTA-AM) or intra-endosomal (Rhod-dextran) calcium chelation inhibited SV generation from bulk endosomes, indicating that calcium efflux from this compartment is critical for this process. The V-type ATPase antagonist bafilomycin A1 also arrested SV generation from bulk endosomes, indicating endosomal acidification may be required for calcium efflux. Finally, pharmacological inhibition of the calcium-dependent protein phosphatase calcineurin blocked endosomal SV generation, identifying it as a key downstream effector in this process. These results reveal a novel and key role for the fluid phase uptake of extracellular calcium and its subsequent efflux in the SV lifecycle.

  6. 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.

  7. 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

  8. Monitoring of vacuolar-type H+ ATPase-mediated proton influx into synaptic vesicles.

    PubMed

    Egashira, Yoshihiro; Takase, Miki; Takamori, Shigeo

    2015-02-25

    During synaptic vesicle (SV) recycling, the vacuolar-type H(+) ATPase creates a proton electrochemical gradient (ΔμH(+)) that drives neurotransmitter loading into SVs. Given the low estimates of free luminal protons, it has been envisioned that the influx of a limited number of protons suffices to establish ΔμH(+). Consistent with this, the time constant of SV re-acidification was reported to be <5 s, much faster than glutamate loading (τ of ∼ 15 s) and thus unlikely to be rate limiting for neurotransmitter loading. However, such estimates have relied on pHluorin-based probes that lack sensitivity in the lower luminal pH range. Here, we reexamined re-acidification kinetics using the mOrange2-based probe that should report the SV pH more accurately. In recordings from cultured mouse hippocampal neurons, we found that re-acidification took substantially longer (τ of ∼ 15 s) than estimated previously. In addition, we found that the SV lumen exhibited a large buffering capacity (∼ 57 mm/pH), corresponding to an accumulation of ∼ 1200 protons during re-acidification. Together, our results uncover hitherto unrecognized robust proton influx and storage in SVs that can restrict the rate of neurotransmitter refilling.

  9. 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.

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

    PubMed

    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.

  11. Vesicle Size Regulates Nanotube Formation in the Cell

    PubMed Central

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

    2016-01-01

    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. PMID:27052881

  12. 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.

  13. Ultrastructural readout of functional synaptic vesicle pools in hippocampal slices based on FM dye labeling and photoconversion.

    PubMed

    Marra, Vincenzo; Burden, Jemima J; Crawford, Freya; Staras, Kevin

    2014-01-01

    Fast activity-driven turnover of neurotransmitter-filled vesicles at presynaptic terminals is a crucial step in information transfer in the CNS. Characterization of the relationship between the nanoscale organization of synaptic vesicles and their functional properties during transmission is currently of interest. Here we outline a procedure for ultrastructural investigation of functional vesicles in synapses from native mammalian brain tissue. FM dye is injected into the target region of a brain slice and upstream axons are electrically activated to stimulate vesicle turnover and dye uptake. In the presence of diaminobenzidine (DAB), photoactivation of dye-filled vesicles yields an osmiophilic precipitate that is visible in electron micrographs. When combined with serial-section electron microscopy, fundamental ultrastructure-function relationships of presynaptic terminals in native circuits are revealed. We outline the utility of this protocol for the 3D reconstruction of a recycling vesicle pool in CA3-CA1 synapses from an acute hippocampal slice and for the characterization of its anatomically defined docked pool. This protocol requires 6-7 d.

  14. Synaptic vesicles in electromotoneurones. I. Axonal transport, site of transmitter uptake and processing of a core proteoglycan during maturation.

    PubMed Central

    Kiene, M L; Stadler, H

    1987-01-01

    We were able by using an in vivo pulse-label technique to trace part of the life cycle of a secretory organelle, the acetylcholine-storing synaptic vesicle from electromotoneurones of Torpedo marmorata. This technique uses [35S]sulphate incorporation into the cell bodies of the electromotoneurones which results in radioactive labelling of a synaptic vesicle heparansulphate proteoglycan--a major core component. Vesicles are anterogradely transported in the axons at a fast rate as 'empty' organelles (VP0 population). In the nerve terminal, maturation of the granule to a population (VP1) fully charged with acetylcholine and ATP occurs. Finally after a longer time interval a change to a third population (VP2) is observed. This population is reduced in diameter as compared to VP0 and VP1 suggesting, in agreement with earlier reports, that it has undergone exo-endocytosis. The changes from VP0 to VP1 and VP2 are accompanied by a degradation of the core proteoglycan as measured by gel filtration of the 35S-labelled compound. The results show that vesicles are axonally transported as preformed organelles, exist in the neurone at least in three different populations and that the nerve terminal is the major site of transmitter uptake. Images Fig. 1. Fig. 4. Fig. 6. PMID:2444433

  15. Sidedness and chemical and kinetic properties of the vesamicol receptor of cholinergic synaptic vesicles

    SciTech Connect

    Kornreich, W.D.; Parsons, S.M.

    1988-07-12

    Cholinergic synaptic vesicles isolated from Torpedo electric organ contain a receptor for the compound l-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183), which then occupied blocks storage of acetylcholine (AcCh). The inside or outside orientation of the receptor and its chemical and ligand binding kinetics characteristics were studied. Binding of (/sup 3/H)vesamicol to the receptor is inhibited efficiently by the protein modification reagents 4-(chloromercuri)benzenesulfonate and N,N'-dicyclohexylcarbodiimide and by protease treatment of cholate-solubilized receptor. The receptor in native vesicles is resistant to irreversible inactivation by proteases, elevated temperature, or pH extremes. (/sup 3/H)Vesamicol binding depends on deprotonation of a group of pK/sub a/sub 1// = 6.26 +/- 0.03 and protonation of a group of pK/sub a/sub 2// = 10.60 +/- 0.04, which is probably the tertiary amine of the drug molecule itself. The membrane-impermeant zwitterionic vesamicol analgoue dl-trans-4-oxo-4-((5,6,7,8-tetrahydro-6-hydroxy-7-(4-phenyl-1-piperidinyl)-1-naphthalenyl)amino)butanoic acid (TPNB) is an effective inhibitor of AcCh active transport. At 0/sup 0/C, 10 ..mu..M unlabeled vesamicol displaced 36 +/- 2% of a low concentration of bound (/sup 3/H)vesamicol at 0.16 +/- 0.02 min/sup -1/ and 64 +/- 2% at 0.013 +/- 0.001 min/sup -1/. One micromolar unlabeled vesamicol behaved similarly. Several types of receptor heterogeneity are consistent with the data. It is concluded that the vesamicol receptor is a stable protein often exhibiting heterogeneity, which faces the cytoplasmic compartment of the cholinergic nerve terminal. It probably contains a binding site carboxylate in a hydrophobic environment, which ion pairs with the protonated tertiary ammonium group of the drug. It also contains a cytoplasmically oriented sulfhydryl group, which is linked to but not part of the binding site.

  16. Wnts in action: from synapse formation to synaptic maintenance

    PubMed Central

    Dickins, Ellen M.; Salinas, Patricia C.

    2013-01-01

    A proper balance between synapse assembly and disassembly is crucial for the formation of functional neuronal circuits and synaptic plasticity in the adult brain. During development, synaptogenesis generates a vast excess of synapses, which are subsequently eliminated. Importantly, aberrant synaptic disassembly during development underpins many neurological disorders. Wnt secreted proteins are robust synaptogenic factors that regulate synapse assembly and function in the developing and mature brain. Recent studies show that Wnt blockade with the antagonist Dickkopf-1 (Dkk1) induces the rapid disassembly of synapses in mature neurons. Importantly, Dkk1 mediates synaptic loss induced by Amyloid-ß, a key pathogenic molecule in Alzheimer’s disease (AD). These findings provide new insights into the potential contribution of dysfunctional Wnt signaling to synaptic loss observed in neurodegenerative diseases. In this review, we discuss the role of Wnt signaling in vertebrate synaptic assembly, function and maintenance, and consider how dysfunction of Wnt signaling could contribute to synaptic disassembly in neurodegenerative diseases such as AD. PMID:24223536

  17. A Fine Balance of Synaptophysin Levels Underlies Efficient Retrieval of Synaptobrevin II to Synaptic Vesicles

    PubMed Central

    Gordon, Sarah L.; Harper, Callista B.; Smillie, Karen J.; Cousin, Michael A.

    2016-01-01

    Synaptobrevin II (sybII) is a vesicular soluble NSF attachment protein receptor (SNARE) protein that is essential for neurotransmitter release, and thus its correct trafficking to synaptic vesicles (SVs) is critical to render them fusion competent. The SV protein synaptophysin binds to sybII and facilitates its retrieval to SVs during endocytosis. Synaptophysin and sybII are the two most abundant proteins on SVs, being present in a 1:2 ratio. Synaptophysin and sybII are proposed to form a large multimeric complex, and the copy number of the proteins in this complex is also in a 1:2 ratio. We investigated the importance of this ratio between these proteins for the localisation and trafficking of sybII in central neurons. SybII was overexpressed in mouse hippocampal neurons at either 1.6 or 2.15–2.35-fold over endogenous protein levels, in the absence or presence of varying levels of synaptophysin. In the absence of exogenous synaptophysin, exogenous sybII was dispersed along the axon, trapped on the plasma membrane and retrieved slowly during endocytosis. Co-expression of exogenous synaptophysin rescued all of these defects. Importantly, the expression of synaptophysin at nerve terminals in a 1:2 ratio with sybII was sufficient to fully rescue normal sybII trafficking. These results demonstrate that the balance between synaptophysin and sybII levels is critical for the correct targeting of sybII to SVs and suggests that small alterations in synaptophysin levels might affect the localisation of sybII and subsequent presynaptic performance. PMID:26871701

  18. 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

  19. A Fine Balance of Synaptophysin Levels Underlies Efficient Retrieval of Synaptobrevin II to Synaptic Vesicles.

    PubMed

    Gordon, Sarah L; Harper, Callista B; Smillie, Karen J; Cousin, Michael A

    2016-01-01

    Synaptobrevin II (sybII) is a vesicular soluble NSF attachment protein receptor (SNARE) protein that is essential for neurotransmitter release, and thus its correct trafficking to synaptic vesicles (SVs) is critical to render them fusion competent. The SV protein synaptophysin binds to sybII and facilitates its retrieval to SVs during endocytosis. Synaptophysin and sybII are the two most abundant proteins on SVs, being present in a 1:2 ratio. Synaptophysin and sybII are proposed to form a large multimeric complex, and the copy number of the proteins in this complex is also in a 1:2 ratio. We investigated the importance of this ratio between these proteins for the localisation and trafficking of sybII in central neurons. SybII was overexpressed in mouse hippocampal neurons at either 1.6 or 2.15-2.35-fold over endogenous protein levels, in the absence or presence of varying levels of synaptophysin. In the absence of exogenous synaptophysin, exogenous sybII was dispersed along the axon, trapped on the plasma membrane and retrieved slowly during endocytosis. Co-expression of exogenous synaptophysin rescued all of these defects. Importantly, the expression of synaptophysin at nerve terminals in a 1:2 ratio with sybII was sufficient to fully rescue normal sybII trafficking. These results demonstrate that the balance between synaptophysin and sybII levels is critical for the correct targeting of sybII to SVs and suggests that small alterations in synaptophysin levels might affect the localisation of sybII and subsequent presynaptic performance.

  20. 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.

  1. 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.

  2. 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

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

    PubMed

    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 (Sv2a(L174Q) rat) carrying a missense mutation of the Sv2a gene and showed that the Sv2a(L174Q) 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 Sv2a(L174Q) rats. Sv2a(L174Q) 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 Sv2a(L174Q) rats. In vivo microdialysis study showed that the Sv2a(L174Q) 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

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

    PubMed

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

    2015-03-01

    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.

  5. 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

  6. pH-dependent gas vesicle formation in Microcystis.

    PubMed

    Gao, Hong; Zhu, Tao; Xu, Min; Wang, Shuai; Xu, Xudong; Kong, Renqiu

    2016-09-01

    In lakes with seasonal cyanobacterial blooms, the pH fluctuates from slightly above 7 to around 10. In this study, we found that the abundance of gas vesicles in Microcystis species, in parallel to the buoyancy of cells, increased in response to elevation of the extracellular pH. Within 48 h after pH upshift, gas vesicle protein genes (gvp) were upregulated at both mRNA and protein levels due to reduced decay of gvp transcripts. The effect of pH on GvpC level was basically unaffected by inorganic carbon availability. This is the first report that long-term pH range plays a role in controlling gas vesicle formation in certain Microcystis species. PMID:27543911

  7. pH-dependent gas vesicle formation in Microcystis.

    PubMed

    Gao, Hong; Zhu, Tao; Xu, Min; Wang, Shuai; Xu, Xudong; Kong, Renqiu

    2016-09-01

    In lakes with seasonal cyanobacterial blooms, the pH fluctuates from slightly above 7 to around 10. In this study, we found that the abundance of gas vesicles in Microcystis species, in parallel to the buoyancy of cells, increased in response to elevation of the extracellular pH. Within 48 h after pH upshift, gas vesicle protein genes (gvp) were upregulated at both mRNA and protein levels due to reduced decay of gvp transcripts. The effect of pH on GvpC level was basically unaffected by inorganic carbon availability. This is the first report that long-term pH range plays a role in controlling gas vesicle formation in certain Microcystis species.

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

    PubMed

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

    2014-02-15

    Synaptic 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.

  9. 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

  10. 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

    2016-01-01

    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

  11. 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

  12. 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.

  13. 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. PMID:25617730

  14. Phase Transition in Postsynaptic Densities Underlies Formation of Synaptic Complexes and Synaptic Plasticity.

    PubMed

    Zeng, Menglong; Shang, Yuan; Araki, Yoichi; Guo, Tingfeng; Huganir, Richard L; Zhang, Mingjie

    2016-08-25

    Postsynaptic densities (PSDs) are membrane semi-enclosed, submicron protein-enriched cellular compartments beneath postsynaptic membranes, which constantly exchange their components with bulk aqueous cytoplasm in synaptic spines. Formation and activity-dependent modulation of PSDs is considered as one of the most basic molecular events governing synaptic plasticity in the nervous system. In this study, we discover that SynGAP, one of the most abundant PSD proteins and a Ras/Rap GTPase activator, forms a homo-trimer and binds to multiple copies of PSD-95. Binding of SynGAP to PSD-95 induces phase separation of the complex, forming highly concentrated liquid-like droplets reminiscent of the PSD. The multivalent nature of the SynGAP/PSD-95 complex is critical for the phase separation to occur and for proper activity-dependent SynGAP dispersions from the PSD. In addition to revealing a dynamic anchoring mechanism of SynGAP at the PSD, our results also suggest a model for phase-transition-mediated formation of PSD. PMID:27565345

  15. Characterization of a Synaptic Vesicle Binding Motif on the Distal CaV2.2 Channel C-terminal.

    PubMed

    Gardezi, Sabiha R; Nath, Arup R; Li, Qi; Stanley, Elise F

    2016-01-01

    Neurotransmitter is released from synaptic vesicles (SVs) that are gated to fuse with the presynaptic membrane by calcium ions that enter through voltage-gated calcium channels (CaVs). There is compelling evidence that SVs associate closely with the CaVs but the molecular linking mechanisms remain poorly understood. Using a cell-free, synaptic vesicle-pull-down assay method (SV-PD) we have recently demonstrated that SVs can bind both to the intact CaV2.2 channel and also to a fusion protein comprising the distal third, C3 segment, of its long C-terminal. This site was localized to a 49 amino acid region just proximal to the C-terminal tip. To further restrict the SV binding site we generated five, 10 amino acid mimetic blocking peptides spanning this region. Of these, HQARRVPNGY effectively inhibited SV-PD and also inhibited SV recycling when cryoloaded into chick brain nerve terminals (synaptosomes). Further, SV-PD was markedly reduced using a C3 fusion protein that lacked the HQARRVPNGY sequence, C3HQless. We zeroed in on the SV binding motif within HQARRVPNGY by means of a palette of mutant blocking peptides. To our surprise, peptides that lacked the highly conserved VPNGY sequence still blocked SV-PD. However, substitution of the HQ and RR amino acids markedly reduced block. Of these, the RR pair was essential but not sufficient as the full block was not observed without H suggesting a CaV2.2 SV binding motif of HxxRR. Interestingly, CaV2.1, the other primary presynaptic calcium channel, exhibits a similar motif, RHxRR, that likely serves the same function. Bioinformatic analysis showed that variations of this binding motif, +(+) xRR (where + is a positively charged aa H or R), are conserved from lung-fish to man. Further studies will be necessary to identify the C terminal motif binding partner on the SV itself and to determine the role of this molecular interaction in synaptic transmission. We hypothesize that the distal C-terminal participates in the capture

  16. Characterization of a Synaptic Vesicle Binding Motif on the Distal CaV2.2 Channel C-terminal

    PubMed Central

    Gardezi, Sabiha R.; Nath, Arup R.; Li, Qi; Stanley, Elise F.

    2016-01-01

    Neurotransmitter is released from synaptic vesicles (SVs) that are gated to fuse with the presynaptic membrane by calcium ions that enter through voltage-gated calcium channels (CaVs). There is compelling evidence that SVs associate closely with the CaVs but the molecular linking mechanisms remain poorly understood. Using a cell-free, synaptic vesicle-pull-down assay method (SV-PD) we have recently demonstrated that SVs can bind both to the intact CaV2.2 channel and also to a fusion protein comprising the distal third, C3 segment, of its long C-terminal. This site was localized to a 49 amino acid region just proximal to the C-terminal tip. To further restrict the SV binding site we generated five, 10 amino acid mimetic blocking peptides spanning this region. Of these, HQARRVPNGY effectively inhibited SV-PD and also inhibited SV recycling when cryoloaded into chick brain nerve terminals (synaptosomes). Further, SV-PD was markedly reduced using a C3 fusion protein that lacked the HQARRVPNGY sequence, C3HQless. We zeroed in on the SV binding motif within HQARRVPNGY by means of a palette of mutant blocking peptides. To our surprise, peptides that lacked the highly conserved VPNGY sequence still blocked SV-PD. However, substitution of the HQ and RR amino acids markedly reduced block. Of these, the RR pair was essential but not sufficient as the full block was not observed without H suggesting a CaV2.2 SV binding motif of HxxRR. Interestingly, CaV2.1, the other primary presynaptic calcium channel, exhibits a similar motif, RHxRR, that likely serves the same function. Bioinformatic analysis showed that variations of this binding motif, +(+) xRR (where + is a positively charged aa H or R), are conserved from lung-fish to man. Further studies will be necessary to identify the C terminal motif binding partner on the SV itself and to determine the role of this molecular interaction in synaptic transmission. We hypothesize that the distal C-terminal participates in the capture

  17. AXONAL AGRANULAR RETICULUM AND SYNAPTIC VESICLES IN CULTURED EMBRYONIC CHICK SYMPATHETIC NEURONS

    PubMed Central

    Teichberg, Saul; Holtzman, Eric

    1973-01-01

    Cultured chick embryonic sympathetic neurons contain an extensive axonal network of sacs and tubules of agranular reticulum. The reticulum is also seen branching into networks in axon terminals and varicosities. The axonal reticulum and perikaryal endoplasmic reticulum resemble one another in their content of cytochemically demonstrable enzyme activities (G6Pase and IDPase) and in their characteristic membrane thicknesses (narrower than plasma membrane or some Golgi membranes). From the reticulum, both along the axon and at terminals, there appear to form dense-cored vesicles ranging in size from 400 to 1,000 Å in diameter. These vesicles behave pharmacologically and cytochemically like the classes of large and small catecholamine storage vesicles found in several adrenergic systems; for example, they can accumulate exogenous 5-hydroxydopamine. In addition, dense-cored vesicles at the larger (1,000 Å) end of the size spectrum appear to arise within perikaryal membrane systems associated with the Golgi apparatus; this is true also of very large (800–3,500 Å) dense-cored vesicles found in some perikarya. PMID:4347980

  18. Atg9 Vesicles Recruit Vesicle-tethering Proteins Trs85 and Ypt1 to the Autophagosome Formation Site*

    PubMed Central

    Kakuta, Soichiro; Yamamoto, Hayashi; Negishi, Lumi; Kondo-Kakuta, Chika; Hayashi, Nobuhiro; Ohsumi, Yoshinori

    2012-01-01

    Atg9 is a transmembrane protein that is essential for autophagy. In the budding yeast Saccharomyces cerevisiae, it has recently been revealed that Atg9 exists on cytoplasmic small vesicles termed Atg9 vesicles. To identify the components of Atg9 vesicles, we purified the Atg9 vesicles and subjected them to mass spectrometry. We found that their protein composition was distinct from other organellar membranes and that Atg9 and Atg27 in particular are major components of Atg9 vesicles. In addition to these two components, Trs85, a specific subunit of the transport protein particle III (TRAPPIII) complex, and the Rab GTPase Ypt1 were also identified. Trs85 directly interacts with Atg9, and the Trs85-containing TRAPPIII complex facilitates the association of Ypt1 onto Atg9 vesicles. We also showed that Trs85 and Ypt1 are localized to the preautophagosomal structure in an Atg9-dependent manner. Our data suggest that Atg9 vesicles recruit the TRAPPIII complex and Ypt1 to the preautophagosomal structure. The vesicle-tethering machinery consequently acts in the process of autophagosome formation. PMID:23129774

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

    PubMed

    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

    2016-02-16

    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.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    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. 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.

  3. 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. PMID:25913068

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

    PubMed

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

    2016-09-20

    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

  5. The Redistribution of Drosophila Vesicular Monoamine Transporter Mutants from Synaptic Vesicles to Large Dense-Core Vesicles Impairs Amine-Dependent Behaviors

    PubMed Central

    Grygoruk, Anna; Chen, Audrey; Martin, Ciara A.; Lawal, Hakeem O.; Fei, Hao; Gutierrez, Gabriel; Biedermann, Traci; Najibi, Rod; Hadi, Richard; Chouhan, Amit K.; Murphy, Niall P.; Schweizer, Felix E.; Macleod, Gregory T.; Maidment, Nigel T.

    2014-01-01

    Monoamine neurotransmitters are stored in both synaptic vesicles (SVs), which are required for release at the synapse, and large dense-core vesicles (LDCVs), which mediate extrasynaptic release. The contributions of each type of vesicular release to specific behaviors are not known. To address this issue, we generated mutations in the C-terminal trafficking domain of the Drosophila vesicular monoamine transporter (DVMAT), which is required for the vesicular storage of monoamines in both SVs and LDCVs. Deletion of the terminal 23 aa (DVMAT-Δ3) reduced the rate of endocytosis and localization of DVMAT to SVs, but supported localization to LDCVs. An alanine substitution mutation in a tyrosine-based motif (DVMAT-Y600A) also reduced sorting to SVs and showed an endocytic deficit specific to aminergic nerve terminals. Redistribution of DVMAT-Y600A from SV to LDCV fractions was also enhanced in aminergic neurons. To determine how these changes might affect behavior, we expressed DVMAT-Δ3 and DVMAT-Y600A in a dVMAT null genetic background that lacks endogenous dVMAT activity. When expressed ubiquitously, DVMAT-Δ3 showed a specific deficit in female fertility, whereas DVMAT-Y600A rescued behavior similarly to DVMAT-wt. In contrast, when expressed more specifically in octopaminergic neurons, both DVMAT-Δ3 and DVMAT-Y600A failed to rescue female fertility, and DVMAT-Y600A showed deficits in larval locomotion. DVMAT-Y600A also showed more severe dominant effects than either DVMAT-wt or DVMAT-Δ3. We propose that these behavioral deficits result from the redistribution of DVMAT from SVs to LDCVs. By extension, our data suggest that the balance of amine release from SVs versus that from LDCVs is critical for the function of some aminergic circuits. PMID:24828646

  6. The redistribution of Drosophila vesicular monoamine transporter mutants from synaptic vesicles to large dense-core vesicles impairs amine-dependent behaviors.

    PubMed

    Grygoruk, Anna; Chen, Audrey; Martin, Ciara A; Lawal, Hakeem O; Fei, Hao; Gutierrez, Gabriel; Biedermann, Traci; Najibi, Rod; Hadi, Richard; Chouhan, Amit K; Murphy, Niall P; Schweizer, Felix E; Macleod, Gregory T; Maidment, Nigel T; Krantz, David E

    2014-05-14

    Monoamine neurotransmitters are stored in both synaptic vesicles (SVs), which are required for release at the synapse, and large dense-core vesicles (LDCVs), which mediate extrasynaptic release. The contributions of each type of vesicular release to specific behaviors are not known. To address this issue, we generated mutations in the C-terminal trafficking domain of the Drosophila vesicular monoamine transporter (DVMAT), which is required for the vesicular storage of monoamines in both SVs and LDCVs. Deletion of the terminal 23 aa (DVMAT-Δ3) reduced the rate of endocytosis and localization of DVMAT to SVs, but supported localization to LDCVs. An alanine substitution mutation in a tyrosine-based motif (DVMAT-Y600A) also reduced sorting to SVs and showed an endocytic deficit specific to aminergic nerve terminals. Redistribution of DVMAT-Y600A from SV to LDCV fractions was also enhanced in aminergic neurons. To determine how these changes might affect behavior, we expressed DVMAT-Δ3 and DVMAT-Y600A in a dVMAT null genetic background that lacks endogenous dVMAT activity. When expressed ubiquitously, DVMAT-Δ3 showed a specific deficit in female fertility, whereas DVMAT-Y600A rescued behavior similarly to DVMAT-wt. In contrast, when expressed more specifically in octopaminergic neurons, both DVMAT-Δ3 and DVMAT-Y600A failed to rescue female fertility, and DVMAT-Y600A showed deficits in larval locomotion. DVMAT-Y600A also showed more severe dominant effects than either DVMAT-wt or DVMAT-Δ3. We propose that these behavioral deficits result from the redistribution of DVMAT from SVs to LDCVs. By extension, our data suggest that the balance of amine release from SVs versus that from LDCVs is critical for the function of some aminergic circuits. PMID:24828646

  7. Mucin-vesicle interactions in model bile: evidence for vesicle aggregation and fusion before cholesterol crystal formation.

    PubMed

    Afdhal, N H; Niu, N; Nunes, D P; Bansil, R; Cao, X X; Gantz, D; Small, D M; Offner, G D

    1995-09-01

    Nucleation of cholesterol monohydrate crystals from bile is a critical step in the formation of cholesterol gallstones. Measurement of nucleation in model bile system and the characteristics of the initial nucleus have proven elusive. In this study we have used three separate physical chemical techniques to examine vesicle aggregation and fusion, including dynamic light scattering (DLS), transmission electron microscopy (TEM), and fluorescent biochemical assays. These assays enabled us to quantify the effect of biliary proteins, such as gallbladder mucin, on vesicle fusion and aggregation. In the absence of mucin, fusion is a relatively slow process occurring over 24 hours, whereas physiological concentrations of mucin are able to accelerate almost complete fusion of vesicles within 6 hours. Vesicle fusion and aggregation as characterized by TEM result in the formation of aggregates of multilamellar vesicles and giant fusion bodies associated with a background of mucin. These mucin-vesicle aggregate bodies may represent true nuclei and precede cholesterol monohydrate crystal nucleation. In future studies, these vesicle fusion assays can be used to quantitatively examine the effect of putative pro- and anti-nucleating proteins on the earliest steps of cholesterol crystal nucleation.

  8. 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. PMID:25869133

  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. 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

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

    PubMed

    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

    2016-03-01

    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

  12. 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. PMID:27443396

  13. Thermodynamically stable vesicle formation and vesicle-to-micelle transition of single-tailed anionic surfactant in water.

    PubMed

    Sakai, Takaya; Ikoshi, Risa; Toshida, Natsuko; Kagaya, Mariko

    2013-05-01

    The aggregation behavior of sodium 3,6,9,12,15-pentaoxa-heptacosanoate (AEC4-Na) in aqueous solution with increase of the concentration at 25 °C was investigated using differential scanning calorimetry, equilibrium surface tension, solubilization of an oil-soluble dye, steady-state fluorescence, dynamic light scattering, and freeze-fractured transmission electron microscopy. Vesicle formation of AEC4-Na preceded micelle formation below the critical micelle concentration (cmc). The vesicle-to-micelle transition was observed through a narrow concentration region above the cmc. The mean diameters of the vesicles and micelles were not affected by the concentration. All solutions over a wide range of concentrations were homogeneously transparent with a low Krafft point below 0 °C. These results indicate that the AEC4-Na vesicles have a thermodynamically stable structure. Vesicle formation may be caused by a pseudobinary mixed surfactant system composed of monomeric AEC4-Na and an acid soap that consists of a dimer complex formed between AEC4-Na and unneutralized AEC4-Na. The thermodynamic stability would then result from the inhibition of close intermolecular aggregation and flexibility of the molecular shape in the vesicles due to the oxyethylene units in AEC4-Na.

  14. 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.

  15. 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

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

    PubMed Central

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

    2002-01-01

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

  17. 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.

  18. Vesicle uncoating regulated by SH3-SH3 domain-mediated complex formation between endophilin and intersectin at synapses

    PubMed Central

    Pechstein, Arndt; Gerth, Fabian; Milosevic, Ira; Jäpel, Maria; Eichhorn-Grünig, Marielle; Vorontsova, Olga; Bacetic, Jelena; Maritzen, Tanja; Shupliakov, Oleg; Freund, Christian; Haucke, Volker

    2015-01-01

    Neurotransmission involves the exo-endocytic cycling of synaptic vesicle (SV) membranes. Endocytic membrane retrieval and clathrin-mediated SV reformation require curvature-sensing and membrane-bending BAR domain proteins such as endophilin A. While their ability to sense and stabilize curved membranes facilitates membrane recruitment of BAR domain proteins, the precise mechanisms by which they are targeted to specific sites of SV recycling has remained unclear. Here, we demonstrate that the multi-domain scaffold intersectin 1 directly associates with endophilin A to facilitate vesicle uncoating at synapses. Knockout mice deficient in intersectin 1 accumulate clathrin-coated vesicles at synapses, a phenotype akin to loss of endophilin function. Intersectin 1/endophilin A1 complex formation is mediated by direct binding of the SH3B domain of intersectin to a non-canonical site on the SH3 domain of endophilin A1. Consistent with this, intersectin-binding defective mutant endophilin A1 fails to rescue clathrin accumulation at neuronal synapses derived from endophilin A1-3 triple knockout (TKO) mice. Our data support a model in which intersectin aids endophilin A recruitment to sites of clathrin-mediated SV recycling, thereby facilitating vesicle uncoating. PMID:25520322

  19. Is Parkinson's disease a vesicular dopamine storage disorder? Evidence from a study in isolated synaptic vesicles of human and nonhuman primate striatum.

    PubMed

    Pifl, Christian; Rajput, Alex; Reither, Harald; Blesa, Javier; Cavada, Carmen; Obeso, José A; Rajput, Ali H; Hornykiewicz, Oleh

    2014-06-11

    The cause of degeneration of nigrostriatal dopamine (DA) neurons in idiopathic Parkinson's disease (PD) is still unknown. Intraneuronally, DA is largely confined to synaptic vesicles where it is protected from metabolic breakdown. In the cytoplasm, however, free DA can give rise to formation of cytotoxic free radicals. Normally, the concentration of cytoplasmic DA is kept at a minimum by continuous pumping activity of the vesicular monoamine transporter (VMAT)2. Defects in handling of cytosolic DA by VMAT2 increase levels of DA-generated oxy radicals ultimately resulting in degeneration of DAergic neurons. Here, we isolated for the first time, DA storage vesicles from the striatum of six autopsied brains of PD patients and four controls and measured several indices of vesicular DA storage mechanisms. We found that (1) vesicular uptake of DA and binding of the VMAT2-selective label [(3)H]dihydrotetrabenazine were profoundly reduced in PD by 87-90% and 71-80%, respectively; (2) after correcting for DA nerve terminal loss, DA uptake per VMAT2 transport site was significantly reduced in PD caudate and putamen by 53 and 55%, respectively; (3) the VMAT2 transport defect appeared specific for PD as it was not present in Macaca fascicularis (7 MPTP and 8 controls) with similar degree of MPTP-induced nigrostriatal neurodegeneration; and (4) DA efflux studies and measurements of acidification in the vesicular preparations suggest that the DA storage impairment was localized at the VMAT2 protein itself. We propose that this VMAT2 defect may be an early abnormality promoting mechanisms leading to nigrostriatal DA neuron death in PD.

  20. 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.

  1. 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

  2. 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.

  3. Overlapping functions of stonin 2 and SV2 in sorting of the calcium sensor synaptotagmin 1 to synaptic vesicles

    PubMed Central

    Kaempf, Natalie; Kochlamazashvili, Gaga; Puchkov, Dmytro; Maritzen, Tanja; Bajjalieh, Sandra M.; Kononenko, Natalia L.; Haucke, Volker

    2015-01-01

    Neurotransmission involves the calcium-regulated exocytic fusion of synaptic vesicles (SVs) and the subsequent retrieval of SV membranes followed by reformation of properly sized and shaped SVs. An unresolved question is whether each SV protein is sorted by its own dedicated adaptor or whether sorting is facilitated by association between different SV proteins. We demonstrate that endocytic sorting of the calcium sensor synaptotagmin 1 (Syt1) is mediated by the overlapping activities of the Syt1-associated SV glycoprotein SV2A/B and the endocytic Syt1-adaptor stonin 2 (Stn2). Deletion or knockdown of either SV2A/B or Stn2 results in partial Syt1 loss and missorting of Syt1 to the neuronal surface, whereas deletion of both SV2A/B and Stn2 dramatically exacerbates this phenotype. Selective missorting and degradation of Syt1 in the absence of SV2A/B and Stn2 impairs the efficacy of neurotransmission at hippocampal synapses. These results indicate that endocytic sorting of Syt1 to SVs is mediated by the overlapping activities of SV2A/B and Stn2 and favor a model according to which SV protein sorting is guarded by both cargo-specific mechanisms as well as association between SV proteins. PMID:26015569

  4. 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

  5. Ca2+ channel to synaptic vesicle distance accounts for the readily releasable pool kinetics at a functionally mature auditory synapse.

    PubMed

    Chen, Zuxin; Das, Brati; Nakamura, Yukihiro; DiGregorio, David A; Young, Samuel M

    2015-02-01

    Precise regulation of synaptic vesicle (SV) release at the calyx of Held is critical for auditory processing. At the prehearing calyx of Held, synchronous and asynchronous release is mediated by fast and slow releasing SVs within the readily releasable pool (RRP). However, the posthearing calyx has dramatically different release properties. Whether developmental alterations in RRP properties contribute to the accelerated release time course found in posthearing calyces is not known. To study these questions, we performed paired patch-clamp recordings, deconvolution analysis, and numerical simulations of buffered Ca(2+) diffusion and SV release in postnatal day (P) 16-19 mouse calyces, as their release properties resemble mature calyces of Held. We found the P16-P19 calyx RRP consists of two pools: a fast pool (τ ≤ 0.9 ms) and slow pool (τ ∼4 ms), in which release kinetics and relative composition of the two pools were unaffected by 5 mm EGTA. Simulations of SV release from the RRP revealed that two populations of SVs were necessary to reproduce the experimental release rates: (1) SVs located close (∼5-25 nm) and (2) more distal (25-100 nm) to VGCC clusters. This positional coupling was confirmed by experiments showing 20 mm EGTA preferentially blocked distally coupled SVs. Lowering external [Ca(2+)] to in vivo levels reduced only the fraction SVs released from the fast pool. Therefore, we conclude that a dominant parameter regulating the mature calyx RRP release kinetics is the distance between SVs and VGCC clusters.

  6. 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.

  7. Mutations in the Drosophila pushover gene confer increased neuronal excitability and spontaneous synaptic vesicle fusion

    SciTech Connect

    Richards, S.; Hillman, T.; Stern, M.

    1996-04-01

    We describe the identification of a gene called pushover (push), which affects both behavior and synaptic transmission at the neuromuscular junction. Adults carrying either of two mutations in push exhibit sluggishness, uncoordination, a defective escape response, and male sterility. Larvae defective in push exhibit increased release of transmitter at the neuromuscular junction. In particular, the frequency of spontaneous transmitter release and the amount of transmitter release evoked by nerve stimulation are each increased two- to threefold in push mutants at the lowest external [(Ca{sup 2+})] tested (0.15 mM). Furthermore, these mutants are more sensitive than wild type to application of the potassium channel-blocking drug quinidine: following quinidine application, push mutants, but not wild-type, display repetitive firing of the motor axon, leading to repetitive muscle postsynaptic potentials. The push gene thus might affect both neuronal excitability and the transmitter release process. Complementation tests and recombinational mapping suggest that the push mutations are allelic to a previously identified P-element-induced mutation, which also causes behavorial abnormalities and male sterility. 43 refs., 5 figs., 1 tab.

  8. 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.

  9. Prebiotic Vesicle Formation and the Necessity of Salts.

    PubMed

    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. PMID:26590931

  10. 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.

  11. 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.

  12. [Investigation vesicle cycle in nerve formations in somatic muscle of the earthworm Lumbricus terrestris].

    PubMed

    Volkov, M E; Petrov, A M; Volkov, E M; Zefirov, A L

    2011-01-01

    Luminous spots with a diameter of 1-2 microm, which are clusters of "synaptic buds", were revealed in the muscular wall of the earthworm using endocytotic fluorescent dyes FM1-43, FM2-10 and FM4-64. Application of the membrane probe Dil that is capable of being subjected to anterograde axonal transport to abdominal ganglia of the nervous chain, and subsequent (in a day) staining of nerve formations by endocytotic dye FM4-64 showed complete imposition of the emission data of the dyes that fluoresce in different parts of the spectrum. Using fluorescent marker DiBAC4(3) showed an increased emission of neural elements with increasing concentration of K+ in the extracellular environment. Application of FM2-10 showed that the higher concentration of K+ in solution, and hence the depolarization of the nerve cells, the faster the upload of the dye, and vice versa, the process slowed down in the absence of K+ in the medium. The seizure and removal of FM2-10 were blocked in calcium-free solutions in the presence of Ca2+ buffers, BABTA or BABTA-AM, but only after a preliminary 40 min incubation. The processes of exo- and endocytosis occurred in the clusters of synaptic "buds" and were preserved in conditions of "rest". This vesicle cycle depends on membrane potential and concentration of K+ and Ca2+, and, it is very likely that the calcium sensor operates on the principle "all or nothing". PMID:22232936

  13. 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, Ken; Katsaras, John

    2011-01-01

    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 applications that

  14. Rough glass surface-mediated formation of vesicles from lauryl sulfobetaine micellar solutions.

    PubMed

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

    2014-10-01

    We report novel vesicles composed of the zwitterionic surfactant lauryl sulfobetaine (LSB), which is a simple single-tailed surfactant (STS). The novel vesicles spontaneously formed from LSB micellar solutions with the mediation of a rough glass surface (RGS) in the absence of any cosurfactants or additives. Importantly, the obtained STS vesicles displayed good stability upon long-term storage, exposure to high temperature, and freeze-thawing after the RGS was removed. The pH of the LSB solution (4.0-9.0) and the presence of NaCl (1.0 × 10(-5) and 1.0 × 10(-4) mol/L) in the LSB solution had no obvious influence on the formation and stability of the vesicles. The adsorption configuration of LSB on the RGS was investigated via water contact angle measurements and atomic force microscope observations. The results showed that LSB adsorption bilayers could form on the RGS, and the bilayer adsorption of LSB on the RGS and the roughness of the solid surface played a key role in the vesicle formation. A possible mechanism for the RGS-mediated formation of LSB vesicles is proposed: LSB micelles and molecules adsorb on the RGS to form curved bilayers, and the curved bilayers are then detached from the RGS and close to form vesicles. To the best of our knowledge, this is the first report of LSB alone forming vesicles. This finding extends our understanding of the nature of vesicle systems.

  15. Rough glass surface-mediated formation of vesicles from lauryl sulfobetaine micellar solutions.

    PubMed

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

    2014-10-01

    We report novel vesicles composed of the zwitterionic surfactant lauryl sulfobetaine (LSB), which is a simple single-tailed surfactant (STS). The novel vesicles spontaneously formed from LSB micellar solutions with the mediation of a rough glass surface (RGS) in the absence of any cosurfactants or additives. Importantly, the obtained STS vesicles displayed good stability upon long-term storage, exposure to high temperature, and freeze-thawing after the RGS was removed. The pH of the LSB solution (4.0-9.0) and the presence of NaCl (1.0 × 10(-5) and 1.0 × 10(-4) mol/L) in the LSB solution had no obvious influence on the formation and stability of the vesicles. The adsorption configuration of LSB on the RGS was investigated via water contact angle measurements and atomic force microscope observations. The results showed that LSB adsorption bilayers could form on the RGS, and the bilayer adsorption of LSB on the RGS and the roughness of the solid surface played a key role in the vesicle formation. A possible mechanism for the RGS-mediated formation of LSB vesicles is proposed: LSB micelles and molecules adsorb on the RGS to form curved bilayers, and the curved bilayers are then detached from the RGS and close to form vesicles. To the best of our knowledge, this is the first report of LSB alone forming vesicles. This finding extends our understanding of the nature of vesicle systems. PMID:25220115

  16. Vesicle formation as a result of interaction between polymorphonuclear neutrophils and Staphylococcus aureus biofilm.

    PubMed

    Chebotar, Igor' V; Konchakova, Evgenia D; Maianskii, Andrey N

    2013-08-01

    Staphylococcus aureus, a major opportunistic pathogen, is a leading cause of biofilm-related infections in clinical practice. Staphylococcal biofilms are highly resistant to antibacterial medicines and immune effector cells. The main result of our work is the discovery of nano-vesicles in the supernatant of the human neutrophil-S. aureus biofilm system. We also found that phospholipase C treatment causes complete destruction of these vesicles. While the addition of proteinase K led to a partial structural disorganization of the vesicles, DNase treatment did not influence the vesicle structure. These observations allowed us to conclude that phospholipids and proteins play a structure-forming role in the formation of these nano-vesicles. The vesicles demonstrated anti-biofilm activities when tested against Staphylococcus epidermidis (strains 178M and 328/5) biofilms, but were ineffective for S. aureus (strains 5983/2, 5663 and 18A) biofilms.

  17. KIF1A, an Axonal Transporter of Synaptic Vesicles, Is Mutated in Hereditary Sensory and Autonomic Neuropathy Type 2

    PubMed Central

    Rivière, Jean-Baptiste; Ramalingam, Siriram; Lavastre, Valérie; Shekarabi, Masoud; Holbert, Sébastien; Lafontaine, Julie; Srour, Myriam; Merner, Nancy; Rochefort, Daniel; Hince, Pascale; Gaudet, Rébecca; Mes-Masson, Anne-Marie; Baets, Jonathan; Houlden, Henry; Brais, Bernard; Nicholson, Garth A.; Van Esch, Hilde; Nafissi, Shahriar; De Jonghe, Peter; Reilly, Mary M.; Timmerman, Vincent; Dion, Patrick A.; Rouleau, Guy A.

    2011-01-01

    Hereditary sensory and autonomic neuropathy type II (HSANII) is a rare autosomal-recessive disorder characterized by peripheral nerve degeneration resulting in a severe distal sensory loss. Although mutations in FAM134B and the HSN2 exon of WNK1 were associated with HSANII, the etiology of a substantial number of cases remains unexplained. In addition, the functions of WNK1/HSN2 and FAM134B and their role in the peripheral nervous system remain poorly understood. Using a yeast two-hybrid screen, we found that KIF1A, an axonal transporter of synaptic vesicles, interacts with the domain encoded by the HSN2 exon. In parallel to this screen, we performed genome-wide homozygosity mapping in a consanguineous Afghan family affected by HSANII and identified a unique region of homozygosity located on chromosome 2q37.3 and spanning the KIF1A gene locus. Sequencing of KIF1A in this family revealed a truncating mutation segregating with the disease phenotype. Subsequent sequencing of KIF1A in a series of 112 unrelated patients with features belonging to the clinical spectrum of ulcero-mutilating sensory neuropathies revealed truncating mutations in three additional families, thus indicating that mutations in KIF1A are a rare cause of HSANII. Similarly to WNK1 mutations, pathogenic mutations in KIF1A were almost exclusively restricted to an alternatively spliced exon. This study provides additional insights into the molecular pathogenesis of HSANII and highlights the potential biological relevance of alternative splicing in the peripheral sensory nervous system. PMID:21820098

  18. 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

  19. 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

    2015-01-01

    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.

  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

    2015-01-01

    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

  1. Lack of synaptic vesicle protein SV2B protects against amyloid-β₂₅₋₃₅-induced oxidative stress, cholinergic deficit and cognitive impairment in mice.

    PubMed

    Detrait, Eric; Maurice, Tangui; Hanon, Etienne; Leclercq, Karine; Lamberty, Yves

    2014-09-01

    SV2B is a synaptic protein widely distributed throughout the brain, which is part of the complex vesicle protein machinery involved in the regulation of synaptic vesicle endocytosis and exocytosis, and therefore in neurotransmitters release. The aims of the present work were twofold: (1) phenotype SV2B knockout mice (SV2B KO) in a battery of cognitive tests; and (2) examine their vulnerability to amyloid-β25-35 (Aβ25-35) peptide-induced toxicity. SV2B KO mice showed normal learning and memory abilities in absence of Aβ25-35 injection. SV2B KO mice were protected against the learning deficits induced after icv injection of an oligomeric preparation of amyloid-β25-35 peptide, as compared to wild-type littermates (SV2B WT). These mice failed to show Aβ25-35-induced impairments in a number of cognitive domains: working memory measured by a spontaneous alternation procedure, recognition memory measured by a novel object recognition task, spatial reference memory assessed in a Morris water-maze, and long-term contextual memory assessed in a inhibitory avoidance task. In addition, SV2B KO mice were protected against Aβ25-35-induced oxidative stress and decrease in ChAT activity in the hippocampus. These data suggest that SV2B could be a key modulator of amyloid toxicity at the synaptic site.

  2. 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

  3. Gel-Assisted Formation of Giant Unilamellar Vesicles

    PubMed Central

    Weinberger, Andreas; Tsai, Feng-Ching; Koenderink, Gijsje H.; Schmidt, Thais F.; Itri, Rosângela; Meier, Wolfgang; Schmatko, Tatiana; Schröder, André; Marques, Carlos

    2013-01-01

    Giant unilamellar vesicles or GUVs are systems of choice as biomimetic models of cellular membranes. Although a variety of procedures exist for making single walled vesicles of tens of microns in size, the range of lipid compositions that can be used to grow GUVs by the conventional methods is quite limited, and many of the available methods involve energy input that can damage the lipids or other molecules present in the growing solution for embedment in the membrane or in the vesicle interior. Here, we show that a wide variety of lipids or lipid mixtures can grow into GUVs by swelling lipid precursor films on top of a dried polyvinyl alcohol gel surface in a swelling buffer that can contain diverse biorelevant molecules. Moreover, we show that the encapsulation potential of this method can be enhanced by combining polyvinyl alcohol-mediated growth with inverse-phase methods, which allow (bio)molecule complexation with the lipids. PMID:23823234

  4. 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

  5. The hydration and ordering of lamellar block copolymer films prior to the formation of polymer vesicles

    NASA Astrophysics Data System (ADS)

    Kamata, Yohei; Parnell, Andrew; Dennison, Andrew; Barker, Robert; Gutfreund, Philipp; Skoda, Maximilian; Mai, Shaomin; Jones, Richard

    2012-02-01

    Polymersomes -- vesicles based on self-assembled bilayers in turn composed of amphiphilic copolymers -- are good candidates for molecular delivery systems; hydrophilic molecules can be enclosed within the aqueous core, to be released by a trigger, which disrupts the vesicle's wall. The key to the use of these polymer vesicles as effective molecular delivery system is in the ability to efficiently encapsulate a molecular payload within the vesicle. To understand the formation mechanism of polymer vesicles via the thin film rehydration method, we have evaluated the hydration and ordering of PEO-PBO diblock copolymer thin films in a controlled water vapor atmosphere. We have performed Neutron Reflectivity, Ellipsometry and Atomic Force Microscopy measurements during the hydration process. These results show that the film swells slowly in the initial stage. It then swells rapidly at a certain critical point and makes ordered structure at the same time. The lamellae are gradually oriented parallel to the substrate with increasing water absorption.

  6. 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.

  7. Vesicle formation in hydrocarbons assisted with microbial hydrolases and biosurfactants.

    PubMed

    Gnanamani, A; Kavitha, V; Sekaran, G; Rajakumar, G Suseela

    2008-12-01

    The present study demonstrates the role of microbial hydrolases in the transformation of hydrocarbons (soybean, sunflower, groundnut and gingelly oil, etc.) to vesicles. The combined effect of lipolytic enzyme generation and biosurfactants production during microbial growth at optimized media and environmental conditions mediates this transformation. Among the microbial species, Candida albicans exhibit complete transformation compared to Pseudomonads and Bacillus sps. Within hydrocarbons, only soybean and sunflower oils transformed to solid mass and no change with the remaining oils. Characterization of the vesicles revealed an increase in total weight by 160-180% compared to the original weight of hydrocarbon taken for the study and more than 73% increases in viscosity. Acid value and saponification value also showed an increase, respectively, by 78 and 84%. The bound water content estimated was 26%. Light microscopic analysis exhibit, presence of unilamellar and bi-lamellar structures. PMID:18829271

  8. Neural activity selects myosin IIB and VI with a specific time window in distinct dynamin isoform-mediated synaptic vesicle reuse pathways.

    PubMed

    Hayashida, Michikata; Tanifuji, Shota; Ma, Huan; Murakami, Noriko; Mochida, Sumiko

    2015-06-10

    Presynaptic nerve terminals must maintain stable neurotransmissions via synaptic vesicle (SV) resupply despite encountering wide fluctuations in the number and frequency of incoming action potentials (APs). However, the molecular mechanism linking variation in neural activity to SV resupply is unknown. Myosins II and VI are actin-based cytoskeletal motors that drive dendritic actin dynamics and membrane transport, respectively, at brain synapses. Here we combined genetic knockdown or molecular dysfunction and direct physiological measurement of fast synaptic transmission from paired rat superior cervical ganglion neurons in culture to show that myosins IIB and VI work individually in SV reuse pathways, having distinct dependency and time constants with physiological AP frequency. Myosin VI resupplied the readily releasable pool (RRP) with slow kinetics independently of firing rates but acted quickly within 50 ms after AP. Under high-frequency AP firing, myosin IIB resupplied the RRP with fast kinetics in a slower time window of 200 ms. Knockdown of both myosin and dynamin isoforms by mixed siRNA microinjection revealed that myosin IIB-mediated SV resupply follows amphiphysin/dynamin-1-mediated endocytosis, while myosin VI-mediated SV resupply follows dynamin-3-mediated endocytosis. Collectively, our findings show how distinct myosin isoforms work as vesicle motors in appropriate SV reuse pathways associated with specific firing patterns. PMID:26063922

  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. Atg9 vesicles are an important membrane source during early steps of autophagosome formation

    PubMed Central

    Yamamoto, Hayashi; Kakuta, Soichiro; Watanabe, Tomonobu M.; Kitamura, Akira; Sekito, Takayuki; Kondo-Kakuta, Chika; Ichikawa, Rie; Kinjo, Masataka

    2012-01-01

    During the process of autophagy, cytoplasmic materials are sequestered by double-membrane structures, the autophagosomes, and then transported to a lytic compartment to be degraded. One of the most fundamental questions about autophagy involves the origin of the autophagosomal membranes. In this study, we focus on the intracellular dynamics of Atg9, a multispanning membrane protein essential for autophagosome formation in yeast. We found that the vast majority of Atg9 existed on cytoplasmic mobile vesicles (designated Atg9 vesicles) that were derived from the Golgi apparatus in a process involving Atg23 and Atg27. We also found that only a few Atg9 vesicles were required for a single round of autophagosome formation. During starvation, several Atg9 vesicles assembled individually into the preautophagosomal structure, and eventually, they are incorporated into the autophagosomal outer membrane. Our findings provide conclusive linkage between the cytoplasmic Atg9 vesicles and autophagosomal membranes and offer new insight into the requirement for Atg9 vesicles at the early step of autophagosome formation. PMID:22826123

  11. 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.

  12. 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

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

    PubMed

    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 Ca(2+) transients at nerve terminals. Impaired SV release was efficiently rescued by elevation of extracellular Ca(2+), hinting at a link between DISC1 and voltage-gated Ca(2+) 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

  14. Thermodynamic Study on Vesicle Formation and Adsorption of Decyltrimethylammonium Decyl Sulfate.

    PubMed

    Villeneuve, Masumi; Kaneshina, Shoji; Aratono, Makoto

    2001-07-01

    The surface tension of an aqueous solution of decyltrimethylammonium decyl sulfate (DeTADeS) was measured as a function of temperature T at various molalities &mcirc; under atmospheric pressure. DeTADeS has been found to form equilibrium multilamellar vesicles (MLV) spontaneously. The surface density, the entropies of adsorption, and the entropy of vesicle formation are evaluated. The mechanism of formation of equilibrium vesicles is investigated from the standpoint of thermodynamics and from the comparison of the results with those of the micelle-forming systems. From the relatively small change of the surface density Gamma;(H) on T at a given &mcirc;, the adsorbed film is implied to be tightly packed due to the strong electrostatic attraction between the polar headgroups. The energy change associated with adsorption from the vesicular state per mole of surfactant Delta(V)(H)u is positive in the entire temperature range; thus, the curved bilayer in MLV is energetically more favorable than the planar adsorbed film. From the negative values of the entropy of vesicle formation Delta(W)(V)s, it is concluded that vesicle formation is driven by enthalpy whereas micelle formation is mostly entropy driven. Copyright 2001 Academic Press.

  15. 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.

  16. 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.

  17. Formation of geometrically complex lipid nanotube-vesicle networks of higher-order topologies

    PubMed Central

    Karlsson, Mattias; Sott, Kristin; Davidson, Maximillian; Cans, Ann-Sofie; Linderholm, Pontus; Chiu, Daniel; Orwar, Owe

    2002-01-01

    We present a microelectrofusion method for construction of fluid-state lipid bilayer networks of high geometrical complexity up to fully connected networks with genus = 3 topology. Within networks, self-organizing branching nanotube architectures could be produced where intersections spontaneously arrange themselves into three-way junctions with an angle of 120° between each nanotube. Formation of branching nanotube networks appears to follow a minimum-bending energy algorithm that solves for pathway minimization. It is also demonstrated that materials can be injected into specific containers within a network by nanotube-mediated transport of satellite vesicles having defined contents. Using a combination of microelectrofusion, spontaneous nanotube pattern formation, and satellite-vesicle injection, complex networks of containers and nanotubes can be produced for a range of applications in, for example, nanofluidics and artificial cell design. In addition, this electrofusion method allows integration of biological cells into lipid nanotube-vesicle networks. PMID:12185244

  18. SNC1, a yeast homolog of the synaptic vesicle-associated membrane protein/synaptobrevin gene family: genetic interactions with the RAS and CAP genes.

    PubMed Central

    Gerst, J E; Rodgers, L; Riggs, M; Wigler, M

    1992-01-01

    SNC1, a gene from the yeast Saccharomyces cerevisiae, encodes a homolog of vertebrate synaptic vesicle-associated membrane proteins (VAMPs) or synaptobrevins. SNC1 was isolated by its ability to suppress the loss of CAP function in S. cerevisiae strains possessing an activated allele of RAS2. CAP is a component of the RAS-responsive S. cerevisiae adenylyl cyclase complex. The N-terminal domain of CAP is required for full cellular responsiveness to activated RAS proteins. The C-terminal domain of CAP is required for normal cellular morphology and responsiveness to nutrient extremes. Multicopy plasmids expressing SNC1 suppress only the loss of the C-terminal functions of CAP and only in the presence of activated RAS2. Images PMID:1316605

  19. Modification of a Hydrophobic Layer by a Point Mutation in Syntaxin 1A Regulates the Rate of Synaptic Vesicle Fusion

    PubMed Central

    Lagow, Robert D; Bao, Hong; Cohen, Evan N; Daniels, Richard W; Zuzek, Aleksej; Williams, Wade H; Macleod, Gregory T; Sutton, R. Bryan; Zhang, Bing

    2007-01-01

    Both constitutive secretion and Ca2+-regulated exocytosis require the assembly of the soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes. At present, little is known about how the SNARE complexes mediating these two distinct pathways differ in structure. Using the Drosophila neuromuscular synapse as a model, we show that a mutation modifying a hydrophobic layer in syntaxin 1A regulates the rate of vesicle fusion. Syntaxin 1A molecules share a highly conserved threonine in the C-terminal +7 layer near the transmembrane domain. Mutation of this threonine to isoleucine results in a structural change that more closely resembles those found in syntaxins ascribed to the constitutive secretory pathway. Flies carrying the I254 mutant protein have increased levels of SNARE complexes and dramatically enhanced rate of both constitutive and evoked vesicle fusion. In contrast, overexpression of the T254 wild-type protein in neurons reduces vesicle fusion only in the I254 mutant background. These results are consistent with molecular dynamics simulations of the SNARE core complex, suggesting that T254 serves as an internal brake to dampen SNARE zippering and impede vesicle fusion, whereas I254 favors fusion by enhancing intermolecular interaction within the SNARE core complex. PMID:17341138

  20. 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

  1. Atg13 HORMA domain recruits Atg9 vesicles during autophagosome formation

    PubMed Central

    Suzuki, Sho W.; Yamamoto, Hayashi; Oikawa, Yu; Kondo-Kakuta, Chika; Kimura, Yayoi; Hirano, Hisashi; Ohsumi, Yoshinori

    2015-01-01

    During autophagosome formation, autophagosome-related (Atg) proteins are recruited hierarchically to organize the preautophagosomal structure (PAS). Atg13, which plays a central role in the initial step of PAS formation, consists of two structural regions, the N-terminal HORMA (from Hop1, Rev7, and Mad2) domain and the C-terminal disordered region. The C-terminal disordered region of Atg13, which contains the binding sites for Atg1 and Atg17, is essential for the initiation step in which the Atg1 complex is formed to serve as a scaffold for the PAS. The N-terminal HORMA domain of Atg13 is also essential for autophagy, but its molecular function has not been established. In this study, we searched for interaction partners of the Atg13 HORMA domain and found that it binds Atg9, a multispanning membrane protein that exists on specific cytoplasmic vesicles (Atg9 vesicles). After the Atg1 complex is formed, Atg9 vesicles are recruited to the PAS and become part of the autophagosomal membrane. HORMA domain mutants, which are unable to interact with Atg9, impaired the PAS localization of Atg9 vesicles and exhibited severe defects in starvation-induced autophagy. Thus, Atg9 vesicles are recruited to the PAS via the interaction with the Atg13 HORMA domain. Based on these findings, we propose that the two distinct regions of Atg13 play crucial roles in distinct steps of autophagosome formation: In the first step, Atg13 forms a scaffold for the PAS via its C-terminal disordered region, and subsequently it recruits Atg9 vesicles via its N-terminal HORMA domain. PMID:25737544

  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. Effect of equimolar salt to decyltrimethylammonium decyl sulfate on vesicle formation and surface adsorption.

    PubMed

    Villeneuve, Masumi; Kaneshina, Shoji; Aratono, Makoto

    2003-06-01

    The aqueous solution of mixture of sodium decyl sulfate (SDeS) and decyltrimethylammonium bromide (DeTAB) has been found to form equilibrium multilamellar vesicles (MLV) spontaneously. We measured the surface tension of the aqueous solution of 1:1 mixture of SDeS and DeTAB as a function of temperature T at various molalities m under atmospheric pressure. The surface density, the entropy of adsorption and the entropy of vesicle formation are evaluated and compared with those of the decyltrimethylammonium decyl sulfate (DeTADeS) aqueous solution system to investigate the role of small counterions in the mechanism of equilibrium vesicle formation. The saturated surface density Gamma (H,C ) vs T curve of the SDeS/DeTAB system sits below that of the DeTADeS system. Therefore, sodium and bromide ions are negatively adsorbed and nevertheless, they neutralize the electric charge of the decyl sulfate ion DeS(-) and the decyltrimethylammonium ion DeTA(+) to some extent to weaken the electrostatic attraction between the polar head groups in the adsorbed film. The net surfactant concentration required for vesicle formation was larger in the SDeS/DeTAB system. Hence, the electrostatic attraction between the polar head groups of the surfactant ions which is one of the major driving forces for vesicle formation is weakened by the presence of the counterions Na(+) and Br(-). Small but distinct changes in the surface density and the entropies of MLV formation of the SDeS/DeTAB system from those of the DeTADeS system were also found.

  4. SYN2 is an autism predisposing gene: loss-of-function mutations alter synaptic vesicle cycling and axon outgrowth.

    PubMed

    Corradi, Anna; Fadda, Manuela; Piton, Amélie; Patry, Lysanne; Marte, Antonella; Rossi, Pia; Cadieux-Dion, Maxime; Gauthier, Julie; Lapointe, Line; Mottron, Laurent; Valtorta, Flavia; Rouleau, Guy A; Fassio, Anna; Benfenati, Fabio; Cossette, Patrick

    2014-01-01

    An increasing number of genes predisposing to autism spectrum disorders (ASDs) has been identified, many of which are implicated in synaptic function. This 'synaptic autism pathway' notably includes disruption of SYN1 that is associated with epilepsy, autism and abnormal behavior in both human and mice models. Synapsins constitute a multigene family of neuron-specific phosphoproteins (SYN1-3) present in the majority of synapses where they are implicated in the regulation of neurotransmitter release and synaptogenesis. Synapsins I and II, the major Syn isoforms in the adult brain, display partially overlapping functions and defects in both isoforms are associated with epilepsy and autistic-like behavior in mice. In this study, we show that nonsense (A94fs199X) and missense (Y236S and G464R) mutations in SYN2 are associated with ASD in humans. The phenotype is apparent in males. Female carriers of SYN2 mutations are unaffected, suggesting that SYN2 is another example of autosomal sex-limited expression in ASD. When expressed in SYN2  knockout neurons, wild-type human Syn II fully rescues the SYN2 knockout phenotype, whereas the nonsense mutant is not expressed and the missense mutants are virtually unable to modify the SYN2 knockout phenotype. These results identify for the first time SYN2  as a novel predisposing gene for ASD and strengthen the hypothesis that a disturbance of synaptic homeostasis underlies ASD. PMID:23956174

  5. Reverse vesicle formation from the yeast glycolipid biosurfactant mannosylerythritol lipid-D.

    PubMed

    Fukuoka, Tokuma; Yanagihara, Takashi; Ito, Seya; Imura, Tomohiro; Morita, Tomotake; Sakai, Hideki; Abe, Masahiko; Kitamoto, Dai

    2012-01-01

    Mannosylerythritol lipids (MELs) are secreted by yeasts and are promising glycolipid biosurfactants. In our study on the non-aqueous phase behaviors of MEL homologues, we found that MEL-D (4-O-[2',3'-di-O-alka(e)noyl-β-D-mannopyranosyl]-(2R,3S)-erythritol) forms aggregates in decane. The microscopic observation and the X-ray scattering measurement of these aggregates revealed that they are reverse vesicles that consist of bilayers whose hydrophilic domains are located in the interior of the bilayers. In addition, MEL-D formed reverse vesicles without co-surfactants and co-solvents in various oily solutions, such as n-alkanes, cyclohexane, squalane, squalene, and silicone oils at a concentration below 10 mM. This is the first report on the reverse vesicle formation from biosurfactants. PMID:22531056

  6. 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.

  7. 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.

  8. 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. PMID:25716918

  9. Nucleation in mesoscopic systems under transient conditions: Peptide-induced pore formation in vesicles

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.; Höök, Fredrik

    2013-04-01

    Attachment of lytic peptides to the lipid membrane of virions or bacteria is often accompanied by their aggregation and pore formation, resulting eventually in membrane rupture and pathogen neutralization. The membrane rupture may occur gradually via formation of many pores or abruptly after the formation of the first pore. In academic studies, this process is observed during interaction of peptides with lipid vesicles. We present an analytical model and the corresponding Monte Carlo simulations focused on the pore formation in such situations. Specifically, we calculate the time of the first nucleation-limited pore-formation event and show the distribution of this time in the regime when the fluctuations of the number of peptides attached to a vesicle are appreciable. The results obtained are used to clarify the mechanism of the pore formation and membrane destabilization observed recently during interaction of highly active α-helical peptide with sub-100-nm lipid vesicles that mimic enveloped viruses with nanoscale membrane curvature. The model proposed and the analysis presented are generic and may be applicable to other meso- and nanosystems.

  10. 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.

  11. Salt-induced vesicle formation from single anionic surfactant SDBS and its mixture with LSB in aqueous solution.

    PubMed

    Zhai, Limin; Zhao, Mei; Sun, Dejun; Hao, Jingcheng; Zhang, Lungjun

    2005-03-31

    Vesicles can be formed spontaneously in aqueous solution of a single anionic surfactant sodium dodecyl benzenesulfonate (SDBS) just under the inducement of salt, which makes the formation of vesicle much easier and simpler. The existence of vesicles was demonstrated by TEM image using the negative-staining method. The mechanism of the formation may be attributed to the compression of salt on the electric bilayer of the surfactant headgroups, which alters the packing parameter of the surfactant. The addition of the zwitterionic surfactant lauryl sulfonate betaine (LSB) makes the vesicles more stable, expands the range of formation and vesicle size, and reduces the polydispersity of the vesicles. The vesicle region was presented in a pseudoternary diagram of SDBS/LSB/brine. The variations of vesicle size with the salinity and mixing ratios, as well as the surfactant concentration, were determined using the dynamic light scattering method. It is found that the vesicle size is independent of the surfactant concentration but subject to the salinity and the mixing ratio of the two surfactants.

  12. Spontaneous formation of giant unilamellar vesicles from microdroplets of a polyion complex by thermally induced phase separation.

    PubMed

    Oana, Hidehiro; Kishimura, Akihiro; Yonehara, Kei; Yamasaki, Yuichi; Washizu, Masao; Kataoka, Kazunori

    2009-01-01

    Water pump: Polyion complex (PIC) vesicles are spontaneously formed from PIC microdroplets, which are formed by mixing cationic and anionic polymers (see picture). The formation process can be reversibly controlled by local heating with a focused infrared laser that triggers microphase separation and subsequent water influx. The size of the resulting giant unilamellar vesicles is determined by the initial size of the PIC droplets.

  13. 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

  14. 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. PMID:24849359

  15. 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. PMID:25686250

  16. Ultrathin shell double emulsion templated giant unilamellar lipid vesicles with controlled microdomain formation.

    PubMed

    Arriaga, Laura R; Datta, Sujit S; Kim, Shin-Hyun; Amstad, Esther; Kodger, Thomas E; Monroy, Francisco; Weitz, David A

    2014-03-12

    A microfluidic approach is reported for the high-throughput, continuous production of giant unilamellar vesicles (GUVs) using water-in-oil-in-water double emulsion drops as templates. Importantly, these emulsion drops have ultrathin shells; this minimizes the amount of residual solvent that remains trapped within the GUV membrane, overcoming a major limitation of typical microfluidic approaches for GUV fabrication. This approach enables the formation of microdomains, characterized by different lipid compositions and structures within the GUV membranes. This work therefore demonstrates a straightforward and versatile approach to GUV fabrication with precise control over the GUV size, lipid composition and the formation of microdomains within the GUV membrane.

  17. 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

  18. 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

  19. Thermoregulated formation and disintegration of cationic block copolymer vesicles: fluorescence resonance energy transfer study.

    PubMed

    Maiti, Chiranjit; Dey, Debabrata; Mandal, Sarthak; Dhara, Dibakar

    2014-02-27

    Formation and disintegration of self-assembled nanostructures in response to external stimuli are important phenomena that have been widely explored for a variety of biomedical applications. In this contribution, we report the thermally triggered assembly of block copolymer molecules in aqueous solution to form vesicles (polymersomes) and their disassembly on reduction of temperature. A new thermoresponsive diblock copolymer of poly(N-isopropylacrylamide) poly((3-methacrylamidopropyl)trimethylammonium chloride) (PNIPA-b-PMAPTAC) was synthesized by reversible addition-fragmentation chain transfer technique. The solution properties and self-assembling behavior of the block copolymer molecules were studied by turbidimetry, temperature-dependent proton nuclear magnetic resonance, fluorescence spectroscopy, dynamic light scattering, and transmission electron microscopy. Fluorescence resonance energy transfer studies between coumarin-153 (C-153, donor) and rhodamine 6G (R6G, acceptor) have been performed by steady-state and picosecond-resolved fluorescence spectroscopy to probe the structural and dynamic heterogeneity of the vesicles. The occurrence of efficient energy transfer was evident from the shortening of donor lifetime in the presence of the acceptor. The capability of the vesicles to encapsulate both hydrophobic and hydrophilic molecules and release them in response to decrease in temperature makes them potentially useful as drug delivery vehicles. PMID:24490812

  20. A new method of vesicle formation by salting-out and its application to the reconstitution of sarcoplasmic reticulum.

    PubMed

    Taguchi, T; Kasai, M

    1983-04-01

    This paper describes a new method of forming membrane vesicles. It was found that the addition of salt such as KCl into a solution containing lipid (asolectin) and a non-ionic surfactant, Triton X-114, led to the formation of closed membrane vesicles. The vesicles were separated from Triton X-114 by hydrophobic interaction chromatography. Electron microscopy revealed that the mean diameter of the vesicles was 110 nm +/- 69 nm (S.D.). Measurement of osmotic volume change showed that the permeability of the vesicle was very low to salts, sugar (glucose) and amphoteric ion (glycine), but very high to glycerol, ethylene glycol and water. Vesicle formation by this 'salting-out' method is very useful for reconstitution of transport systems in biomembranes because of its advantages: completion within a short time; high yield; and the possibility of utilizing samples in non-ionic surfactant solution. When we applied the method to the reconstitution of sarcoplasmic reticulum, Ca2+-ATPase was incorporated into the reconstituted vesicles and was enzymatically active in the membrane. PMID:6830789

  1. Formation of Supported and Anchored Phospholipid Bilayers by Fusion of Unilamellar Vesicles: An AFM Study.

    NASA Astrophysics Data System (ADS)

    Reviakine, Ilya; Brisson, Alain

    2000-03-01

    The process of formation of supported phospholipid bilayers (SPBs) on the surface of mica from unilamellar vesicles prepared by sonication or extrusion was investigated by AFM. The effect of liposome size and of Ca2+ on the SPB formation was studied. Intact surface-adsorbed liposomes of various sizes could be visualized. The results obtained in this study were compared with the theoretical model of Seifert et al (1) and with the experimental results obtained by other groups (2),(3). 2-dimensional (2D) crystals formed on SPBs (4) present a surface with significantly different properties than that of mica. Phospholipid vesicles can be anchored to them via specific (ligand-receptor) interactions, forming Anchored Vesicular Layers (AVLs, by analogy with Supported Vesicular Layers, or SVLs (5)). The formation of Anchored Phospholipid Bilayers (APBs) from AVLs was also investigated (6). 1. Seifert, U. Adv. Phys. (1997), 46, 13. 2. Keller, C.A., Kasemo, B. Biophys. J. (1998), 75, 1397. 3. Reviakine and Brisson, Langmuir, in press. 4. Reviakine, I., Bergsma-Schutter, W., Brisson, A. J.Struct. Biol. (1998), 121, 356. 5. Nollert, P., Kiefer, H., Jähnig, F. Biophys. J. (1995), 69, 1447. 6. Reviakine and Brisson, Langmuir, submitted.

  2. Depression-like Behavior Induced by Nesfatin-1 in Rats: Involvement of Increased Immune Activation and Imbalance of Synaptic Vesicle Proteins

    PubMed Central

    Ge, Jin-Fang; Xu, Ya-Yun; Qin, Gan; Peng, Yao-Nan; Zhang, Chao-Feng; Liu, Xing-Rui; Liang, Li-Chuan; Wang, Zhong-Zheng; Chen, Fei-Hu

    2015-01-01

    Depression is a multicausal disorder and has been associated with metabolism regulation and immuno-inflammatory reaction. The anorectic molecule nesfatin-1 has recently been characterized as a potential mood regulator, but its precise effect on depression and the possible mechanisms remain unknown, especially when given peripherally. In the present study, nesfatin-1 was intraperitoneally injected to the rats and the depression-like behavior and activity of the hypothalamic-pituitary-adrenal (HPA) axis were evaluated. The plasma concentrations of nesfatin-1, interleukin 6 (IL-6), and C-reactive protein (CRP); and the hypothalamic expression levels of nesfatin-1, synapsin I, and synaptotagmin I mRNA were evaluated in nesfatin-1 chronically treated rats. The results showed that both acute and chronic administration of nesfatin-1 increased immobility in the forced swimming test (FST), and resulted in the hyperactivity of HPA axis, as indicated by the increase of plasma corticosterone concentration and hypothalamic expression of corticotropin-releasing hormone (CRH) mRNA. Moreover, after chronic nesfatin-1 administration, the rats exhibited decreased activity and exploratory behavior in the open field test (OFT) and increased mRNA expression of synapsin I and synaptotagmin I in the hypothalamus. Furthermore, chronic administration of nesfatin-1 elevated plasma concentrations of IL-6 and CRP, which were positively correlated with despair behavior, plasma corticosterone level, and the hypothalamic mRNA expression of synapsin I and synaptotagmin I. These results indicated that exogenous nesfatin-1 could induce the immune-inflammatory activation, which might be a central hug linking the depression-like behavior and the imbalanced mRNA expression of synaptic vesicle proteins in the hypothalamus. PMID:26617482

  3. 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.

  4. 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

  5. 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

  6. Involvement of ryanodine receptors in neurotrophin-induced hippocampal synaptic plasticity and spatial memory formation.

    PubMed

    Adasme, Tatiana; Haeger, Paola; Paula-Lima, Andrea C; Espinoza, Italo; Casas-Alarcón, M Mercedes; Carrasco, M Angélica; Hidalgo, Cecilia

    2011-02-15

    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

  7. Effect of hydrophilic groups of Ca surfactants and hydrophobic chains of C(n)DMAO on coordinated vesicle formation.

    PubMed

    Tian, Hongshan; Ding, Qi; Zhang, Juan; Song, Aixin; Hao, Jingcheng

    2010-12-21

    The effects of hydrophilic headgroups of Ca surfactants, calcium dodecylsulfate (Ca(DS)(2)), calcium dodecylsulfonate (Ca(DSA)(2)), and calcium laurate (CaL(2)) and hydrophobic chains of alkyldimethylamine oxide (C(n)DMAO, n = 12, 14, 16) on the formation of Ca(2+)-ligand coordinated vesicles was investigated in detail. On the basis of phase behavior studies, rheological properties and freeze-fracture transmission electron microscope (FF-TEM) images were measured. Quite different phase behaviors were observed in different surfactant systems. For a Ca surfactant with a highly polar group, Ca(DS)(2), vesicles were observed in all Ca(DS)(2)/C(n)DMAO (n = 12, 14, and 16) systems, whereas for Ca surfactant with lower polar group, Ca(DSA)(2), vesicles can form in Ca(DSA)(2)/C(n)DMAO systems of n = 14 and 16 but not for n = 12. For CaL(2), the surfactant with the least polar group, vesicles form only in the CaL(2)/C(16)DMAO system. The results demonstrate that in the systems formed by Ca surfactants and C(n)DMAO, the formation of vesicles is driven not only by interaction between Ca(2+) and the N → O groups of C(n)DMAO but also by electrostatic and hydrophobic interactions. Vesicles prefer to form in Ca surfactants with highly polar headgroups and C(n)DMAO with long chain length. PMID:21087007

  8. A trypsin-sensitive receptor on membrane vesicles is required for nuclear envelope formation in vitro

    PubMed Central

    1988-01-01

    The reformation of functioning organelles at the end of mitosis presents a problem in vesicle targeting. Using extracts made from Xenopus laevis frog eggs, we have studied in vitro the vesicles that reform the nuclear envelope. In the in vitro assay, nuclear envelope growth is linear with time. Furthermore, the final surface area of the nuclear envelopes formed is directly dependent upon the amount of membrane vesicles added to the assay. Egg membrane vesicles could be fractionated into two populations, only one of which was competent for nuclear envelope assembly. We found that vesicles active in nuclear envelope assembly contained markers (BiP and alpha-glucosidase II) characteristic of the endoplasmic reticulum (ER), but that the majority of ER-derived vesicles do not contribute to nuclear envelope size. This functional distinction between nuclear vesicles and ER-derived vesicles implies that nuclear vesicles are unique and possess at least one factor required for envelope assembly that is lacking in other vesicles. Consistent with this, treatment of vesicles with trypsin destroyed their ability to form a nuclear envelope; electron microscopic studies indicate that the trypsin-sensitive proteins is required for vesicles to bind to chromatin. However, the protease- sensitive component(s) is resistant to treatments that disrupt protein- protein interactions, such as high salt, EDTA, or low ionic strength solutions. We propose that an integral membrane protein, or protein tightly associated with the membrane, is critical for nuclear vesicle targeting or function. PMID:3392106

  9. 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

  10. The Strip-Hippo Pathway Regulates Synaptic Terminal Formation by Modulating Actin Organization at the Drosophila Neuromuscular Synapses.

    PubMed

    Sakuma, Chisako; Saito, Yoshie; Umehara, Tomoki; Kamimura, Keisuke; Maeda, Nobuaki; Mosca, Timothy J; Miura, Masayuki; Chihara, Takahiro

    2016-08-30

    Synapse formation requires the precise coordination of axon elongation, cytoskeletal stability, and diverse modes of cell signaling. The underlying mechanisms of this interplay, however, remain unclear. Here, we demonstrate that Strip, a component of the striatin-interacting phosphatase and kinase (STRIPAK) complex that regulates these processes, is required to ensure the proper development of synaptic boutons at the Drosophila neuromuscular junction. In doing so, Strip negatively regulates the activity of the Hippo (Hpo) pathway, an evolutionarily conserved regulator of organ size whose role in synapse formation is currently unappreciated. Strip functions genetically with Enabled, an actin assembly/elongation factor and the presumptive downstream target of Hpo signaling, to modulate local actin organization at synaptic termini. This regulation occurs independently of the transcriptional co-activator Yorkie, the canonical downstream target of the Hpo pathway. Our study identifies a previously unanticipated role of the Strip-Hippo pathway in synaptic development, linking cell signaling to actin organization. PMID:27545887

  11. 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

  12. Giant phospholipid/block copolymer hybrid vesicles: mixing behavior and domain formation.

    PubMed

    Nam, Jin; Beales, Paul A; Vanderlick, T Kyle

    2011-01-01

    Lipids and block copolymers can be individually assembled into unsupported, spherical membranes (liposomes or polymersomes), each having their own particular benefits and limitations. Here we demonstrate the preparation of microscale, hybrid "lipopolymersomes" composed of the common lipid POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine) and the commercially available copolymer PBd-b-PEO (polybutadiene-b-poly(ethylene oxide)) with the goal of incorporating the advantageous qualities of the unitary systems into mixed-membrane capsules. We investigate the lipopolymersomes using confocal fluorescence microscopy and demonstrate that these hybrid membranes are well mixed on nanoscopic length scales within the permittable compositional windows for hybrid vesicle formation. We measure the intramembrane dynamics and mechanical properties of these hybrid membranes by fluorescence recovery after photobleaching (FRAP) and micropipet aspiration, respectively. For the first time, we demonstrate the demixing of lipid-rich and polymer-rich membrane domains within the same vesicle membrane. This is achieved by the biotinylation of one of the constituent species and cross linking with the protein NeutrAvidin. The resultant domain patterning is dependent upon which component carries the biotin functionality: cross linking of the copolymer species results in domains that ripen into a single, large, copolymer-rich island, and cross linking of the lipids yields many small, "spot-like", lipid-rich domains within a copolymer-rich matrix. We discuss these morphological differences in terms of the fluidity and mechanical properties of the membrane phases and the possible resultant interdomain interactions within the membrane. These heterogeneous hybrid lipopolymersomes could find applications in fields such as targeted delivery, controlled release, and environmental detection assays where these capsules possess the characteristics of biocompatible lipid membranes combined with

  13. 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.

  14. Formation of temporal-feature maps by axonal propagation of synaptic learning.

    PubMed

    Kempter, R; Leibold, C; Wagner, H; van Hemmen, J L

    2001-03-27

    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 micros. 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

  15. Shp2 in forebrain neurons regulates synaptic plasticity, locomotion, and memory formation in mice.

    PubMed

    Kusakari, Shinya; Saitow, Fumihito; Ago, Yukio; Shibasaki, Koji; Sato-Hashimoto, Miho; Matsuzaki, Yasunori; Kotani, Takenori; Murata, Yoji; Hirai, Hirokazu; Matsuda, Toshio; Suzuki, Hidenori; Matozaki, Takashi; Ohnishi, Hiroshi

    2015-05-01

    Shp2 (Src homology 2 domain-containing protein tyrosine phosphatase 2) regulates neural cell differentiation. It is also expressed in postmitotic neurons, however, and mutations of Shp2 are associated with clinical syndromes characterized by mental retardation. Here we show that conditional-knockout (cKO) mice lacking Shp2 specifically in postmitotic forebrain neurons manifest abnormal behavior, including hyperactivity. Novelty-induced expression of immediate-early genes and activation of extracellular-signal-regulated kinase (Erk) were attenuated in the cerebral cortex and hippocampus of Shp2 cKO mice, suggestive of reduced neuronal activity. In contrast, ablation of Shp2 enhanced high-K(+)-induced Erk activation in both cultured cortical neurons and synaptosomes, whereas it inhibited that induced by brain-derived growth factor in cultured neurons. Posttetanic potentiation and paired-pulse facilitation were attenuated and enhanced, respectively, in hippocampal slices from Shp2 cKO mice. The mutant mice also manifested transient impairment of memory formation in the Morris water maze. Our data suggest that Shp2 contributes to regulation of Erk activation and synaptic plasticity in postmitotic forebrain neurons and thereby controls locomotor activity and memory formation.

  16. Shp2 in Forebrain Neurons Regulates Synaptic Plasticity, Locomotion, and Memory Formation in Mice

    PubMed Central

    Kusakari, Shinya; Saitow, Fumihito; Ago, Yukio; Shibasaki, Koji; Sato-Hashimoto, Miho; Matsuzaki, Yasunori; Kotani, Takenori; Murata, Yoji; Hirai, Hirokazu; Matsuda, Toshio; Suzuki, Hidenori

    2015-01-01

    Shp2 (Src homology 2 domain-containing protein tyrosine phosphatase 2) regulates neural cell differentiation. It is also expressed in postmitotic neurons, however, and mutations of Shp2 are associated with clinical syndromes characterized by mental retardation. Here we show that conditional-knockout (cKO) mice lacking Shp2 specifically in postmitotic forebrain neurons manifest abnormal behavior, including hyperactivity. Novelty-induced expression of immediate-early genes and activation of extracellular-signal-regulated kinase (Erk) were attenuated in the cerebral cortex and hippocampus of Shp2 cKO mice, suggestive of reduced neuronal activity. In contrast, ablation of Shp2 enhanced high-K+-induced Erk activation in both cultured cortical neurons and synaptosomes, whereas it inhibited that induced by brain-derived growth factor in cultured neurons. Posttetanic potentiation and paired-pulse facilitation were attenuated and enhanced, respectively, in hippocampal slices from Shp2 cKO mice. The mutant mice also manifested transient impairment of memory formation in the Morris water maze. Our data suggest that Shp2 contributes to regulation of Erk activation and synaptic plasticity in postmitotic forebrain neurons and thereby controls locomotor activity and memory formation. PMID:25713104

  17. Phosphate induces formation of matrix vesicles during odontoblast-initiated mineralization in vitro.

    PubMed

    Chaudhary, Sandeep C; Kuzynski, Maria; Bottini, Massimo; Beniash, Elia; Dokland, Terje; Mobley, Callie G; Yadav, Manisha C; Poliard, Anne; Kellermann, Odile; Millán, José Luis; Napierala, Dobrawa

    2016-01-01

    Mineralization is a process of deposition of calcium phosphate crystals within a fibrous extracellular matrix (ECM). In mineralizing tissues, such as dentin, bone and hypertrophic cartilage, this process is initiated by a specific population of extracellular vesicles (EV), called matrix vesicles (MV). Although it has been proposed that MV are formed by shedding of the plasma membrane, the cellular and molecular mechanisms regulating formation of mineralization-competent MV are not fully elucidated. In these studies, 17IIA11, ST2, and MC3T3-E1 osteogenic cell lines were used to determine how formation of MV is regulated during initiation of the mineralization process. In addition, the molecular composition of MV secreted by 17IIA11 cells and exosomes from blood and B16-F10 melanoma cell line was compared to identify the molecular characteristics distinguishing MV from other EV. Western blot analyses demonstrated that MV released from 17IIA11 cells are characterized by high levels of proteins engaged in calcium and phosphate regulation, but do not express the exosomal markers CD81 and HSP70. Furthermore, we uncovered that the molecular composition of MV released by 17IIA11 cells changes upon exposure to the classical inducers of osteogenic differentiation, namely ascorbic acid and phosphate. Specifically, lysosomal proteins Lamp1 and Lamp2a were only detected in MV secreted by cells stimulated with osteogenic factors. Quantitative nanoparticle tracking analyses of MV secreted by osteogenic cells determined that standard osteogenic factors stimulate MV secretion and that phosphate is the main driver of their secretion. On the molecular level, phosphate-induced MV secretion is mediated through activation of extracellular signal-regulated kinases Erk1/2 and is accompanied by re-organization of filamentous actin. In summary, we determined that mineralization-competent MV are distinct from exosomes, and we identified a new role of phosphate in the process of ECM

  18. 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.

  19. 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

  20. 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.

  1. Deciphering How Pore Formation Causes Strain-Induced Membrane Lysis of Lipid Vesicles.

    PubMed

    Jackman, Joshua A; Goh, Haw Zan; Zhdanov, Vladimir P; Knoll, Wolfgang; Cho, Nam-Joon

    2016-02-01

    Pore formation by membrane-active antimicrobial peptides is a classic strategy of pathogen inactivation through disruption of membrane biochemical gradients. It remains unknown why some membrane-active peptides also inhibit enveloped viruses, which do not depend on biochemical gradients. Here, we employ a label-free biosensing approach based on simultaneous quartz crystal microbalance-dissipation and ellipsometry measurements in order to investigate how a pore-forming, virucidal peptide destabilizes lipid vesicles in a surface-based experimental configuration. A key advantage of the approach is that it enables direct kinetic measurement of the surface-bound peptide-to-lipid (P:L) ratio. Comprehensive experiments involving different bulk peptide concentrations and biologically relevant membrane compositions support a unified model that membrane lysis occurs at or above a critical P:L ratio, which is at least several-fold greater than the value corresponding to the onset of pore formation. That is consistent with peptide-induced pores causing additional membrane strain that leads to lysis of highly curved membranes. Collectively, the work presents a new model that describes how peptide-induced pores may destabilize lipid membranes through a membrane strain-related lytic process, and this knowledge has important implications for the design and application of membrane-active peptides.

  2. Raldh2 expression in optic vesicle generates a retinoic acid signal needed for invagination of retina during optic cup formation.

    PubMed

    Mic, Felix A; Molotkov, Andrei; Molotkova, Natalia; Duester, Gregg

    2004-10-01

    Three retinaldehyde dehydrogenase genes (Raldh1, Raldh2, and Raldh3) expressed in unique spatiotemporal patterns may control synthesis of retinoic acid (RA) needed for retina development. However, previous studies indicate that retina formation still proceeds normally in Raldh1-/- mouse embryos lacking RA synthesis in the dorsal neural retina at the optic cup stage. Here, we demonstrate that Raldh2-/- embryos lacking RA synthesis in the optic vesicle exhibit a failure in retina invagination needed to develop an optic cup. This was also observed in Raldh1-/-:Raldh2-/- double mutants, which develop similarly. Both mutants retain RA activity in the lens placode associated with Raldh3 expression, but this RA activity is insufficient to induce optic cup formation. Maternal RA administration at the optic vesicle stage rescues optic cup formation in Raldh2-/- and Raldh1-/-:Raldh2-/- embryos, demonstrating that Raldh1 is not required during rescue of optic cup development. The optic cup of rescued Raldh1-/-:Raldh2-/- embryos exhibits normal RA activity and this is associated with Raldh3 expression in the retina and lens. Thus, RA signaling initiates in the optic vesicle in response to Raldh2 but can be maintained during optic cup formation by a gene other than Raldh1, most likely Raldh3. Loss of optic vesicle RA signaling does not effect expression of early determinants of retina at the optic vesicle stage (Pax6, Six3, Rx, Mitf). Our findings suggest that RA functions as one of the signals needed for invagination of the retina to generate an optic cup. PMID:15366004

  3. Postsynaptic SDC2 induces transsynaptic signaling via FGF22 for bidirectional synaptic formation

    PubMed Central

    Hu, Hsiao-Tang; Umemori, Hisashi; Hsueh, Yi-Ping

    2016-01-01

    Functional synapse formation requires tight coordination between pre- and post-synaptic termini. Previous studies have shown that postsynaptic expression of heparan sulfate proteoglycan syndecan-2 (SDC2) induces dendritic spinogenesis. Those SDC2-induced dendritic spines are frequently associated with presynaptic termini. However, how postsynaptic SDC2 accelerates maturation of corresponding presynaptic termini is unknown. Because fibroblast growth factor 22 (FGF22), a heparan sulfate binding growth factor, has been shown to act as a presynaptic organizer released from the postsynaptic site, it seems possible that postsynaptic SDC2 presents FGF22 to the presynaptic FGF receptor to promote presynaptic differentiation. Here, we show that postsynaptic SDC2 uses its ectodomain to interact with and facilitate dendritic filopodial targeting of FGF22, triggering presynaptic maturation. Since SDC2 also enhances filopodial targeting of NMDAR via interaction with the CASK-mLIN7-MINT1 adaptor complex, presynaptic maturation promoted by FGF22 further feeds back to activate NMDAR at corresponding postsynaptic sites through increased neurotransmitter release and, consequently, promotes the dendritic filopodia-spines (F-S) transition. Meanwhile, via regulation of the KIF17 motor, CaMKII (activated by the NMDAR pathway) may further facilitate FGF22 targeting to dendritic filopodia that receive presynaptic stimulation. Our study suggests a positive feedback that promotes the coordination of postsynaptic and presynaptic differentiation. PMID:27627962

  4. Synapsin Regulates Activity-Dependent Outgrowth of Synaptic Boutons at the Drosophila Neuromuscular Junction

    PubMed Central

    Vasin, Alexander; Zueva, Lidia; Torrez, Carol; Volfson, Dina; Littleton, J. Troy

    2014-01-01

    Patterned depolarization of Drosophila motor neurons can rapidly induce the outgrowth of new synaptic boutons at the larval neuromuscular junction (NMJ), providing a model system to investigate mechanisms underlying acute structural plasticity. Correlative light and electron microscopy analysis revealed that new boutons typically form near the edge of postsynaptic reticulums of presynaptic boutons. Unlike mature boutons, new varicosities have synaptic vesicles which are distributed uniformly throughout the bouton and undeveloped postsynaptic specializations. To characterize the presynaptic mechanisms mediating new synaptic growth induced by patterned activity, we investigated the formation of new boutons in NMJs lacking synapsin [Syn(−)], a synaptic protein important for vesicle clustering, neurodevelopment, and plasticity. We found that budding of new boutons at Syn(−) NMJs was significantly diminished, and that new boutons in Syn(−) preparations were smaller and had reduced synaptic vesicle density. Since synapsin is a target of protein kinase A (PKA), we assayed whether activity-dependent synaptic growth is regulated via a cAMP/PKA/synapsin pathway. We pretreated preparations with forskolin to raise cAMP levels and found this manipulation significantly enhanced activity-dependent synaptic growth in control but not Syn(−) preparations. To examine the trafficking of synapsin during synaptic growth, we generated transgenic animals expressing fluorescently tagged synapsin. Fluorescence recovery after photobleaching analysis revealed that patterned depolarization promoted synapsin movement between boutons. During new synaptic bouton formation, synapsin redistributed upon stimulation toward the sites of varicosity outgrowth. These findings support a model whereby synapsin accumulates at sites of synaptic growth and facilitates budding of new boutons via a cAMP/PKA-dependent pathway. PMID:25100589

  5. Functional characterization of retromer in GLUT4 storage vesicle formation and adipocyte differentiation.

    PubMed

    Yang, Zhe; Hong, Lee Kian; Follett, Jordan; Wabitsch, Martin; Hamilton, Nicholas A; Collins, Brett M; Bugarcic, Andrea; Teasdale, Rohan D

    2016-03-01

    Insulin-stimulated translocation of glucose transporter 4 (GLUT4) storage vesicles (GSVs), the specialized intracellular compartments within mature adipocytes, to the plasma membrane (PM) is a fundamental cellular process for maintaining glucose homeostasis. Using 2 independent adipocyte cell line models, human primary Simpson-Golabi-Behmel syndrome and mouse 3T3-L1 fibroblast cell lines, we demonstrate that the endosome-associated protein-sorting complex retromer colocalizes with GLUT4 on the GSVs by confocal microscopy in mature adipocytes. By use of both confocal microscopy and differential ultracentrifugation techniques, retromer is redistributed to the PM of mature adipocytes upon insulin stimulation. Furthermore, stable knockdown of the retromer subunit-vacuolar protein-sorting 35, or the retromer-associated protein sorting nexin 27, by lentivirus-delivered small hairpin RNA impaired the adipogenesis process when compared to nonsilence control. The knockdown of retromer decreased peroxisome proliferator activated receptor γ expression during differentiation, generating adipocytes with decreased levels of GSVs, lipid droplet accumulation, and insulin-stimulated glucose uptake. In conclusion, our study demonstrates a role for retromer in the GSV formation and adipogenesis. PMID:26581601

  6. Cell collectivity regulation within migrating cell cluster during Kupffer's vesicle formation in zebrafish

    PubMed Central

    Matsui, Takaaki; Ishikawa, Hiroshi; Bessho, Yasumasa

    2015-01-01

    Although cell adhesion is thought to fasten cells tightly, cells that adhere to each other can migrate directionally. This group behavior, called “collective cell migration,” is observed during normal development, wound healing, and cancer invasion. Loss-of-function of cell adhesion molecules in several model systems of collective cell migration results in delay or inhibition of migration of cell groups but does not lead to dissociation of the cell groups, suggesting that mechanisms of cells staying assembled as a single cell cluster, termed as “cell collectivity,” remain largely unknown. During the formation of Kupffer's vesicle (KV, an organ of laterality in zebrafish), KV progenitors form a cluster and migrate together toward the vegetal pole. Importantly, in this model system of collective cell migration, knockdown of cell adhesion molecules or signal components leads to failure of cell collectivity. In this review, we summarize recent findings in cell collectivity regulation during collective migration of KV progenitor cells and describe our current understanding of how cell collectivity is regulated during collective cell migration. PMID:26000276

  7. Functional characterization of retromer in GLUT4 storage vesicle formation and adipocyte differentiation.

    PubMed

    Yang, Zhe; Hong, Lee Kian; Follett, Jordan; Wabitsch, Martin; Hamilton, Nicholas A; Collins, Brett M; Bugarcic, Andrea; Teasdale, Rohan D

    2016-03-01

    Insulin-stimulated translocation of glucose transporter 4 (GLUT4) storage vesicles (GSVs), the specialized intracellular compartments within mature adipocytes, to the plasma membrane (PM) is a fundamental cellular process for maintaining glucose homeostasis. Using 2 independent adipocyte cell line models, human primary Simpson-Golabi-Behmel syndrome and mouse 3T3-L1 fibroblast cell lines, we demonstrate that the endosome-associated protein-sorting complex retromer colocalizes with GLUT4 on the GSVs by confocal microscopy in mature adipocytes. By use of both confocal microscopy and differential ultracentrifugation techniques, retromer is redistributed to the PM of mature adipocytes upon insulin stimulation. Furthermore, stable knockdown of the retromer subunit-vacuolar protein-sorting 35, or the retromer-associated protein sorting nexin 27, by lentivirus-delivered small hairpin RNA impaired the adipogenesis process when compared to nonsilence control. The knockdown of retromer decreased peroxisome proliferator activated receptor γ expression during differentiation, generating adipocytes with decreased levels of GSVs, lipid droplet accumulation, and insulin-stimulated glucose uptake. In conclusion, our study demonstrates a role for retromer in the GSV formation and adipogenesis.

  8. Investigation of plague lipopolysaccharide complex formation with artificial phospholipid vesicles by elastic laser radiation scattering

    NASA Astrophysics Data System (ADS)

    Gusev, V. V.; Guseva, N. P.; Tatarintsev, S. N.

    1995-01-01

    This paper describes the investigation of incorporation processes of the plague lipopolysaccharide (LPS) into artificial phospholipid vesicles (PLV) on the basis of elastic laser radiation scattering. For this purpose, the angular light scattering dependencies of PLV suspensions, containing various LPS concentrations (0 - 5 mg/ml), were measured using the polarization nephelometer. The design of the polarization nephelometer and the measurement technique are described in detail. Measuring results are compared with electron microscopy data. The most pronounced variation as a result of LPS incorporation into PLV appeared to be the light scattering integral intensity (LSII) at angles exceeding 100. It is shown that the LPS adding into the PLV suspension causes the LSII to increase by a factor 2 - 6 for a LPS concentration range from 0.5 to 5 mg/ml as compared with `empty' PLV. Proceeding from the electron microscopy data it was found that the LSII increase, in general case, is conditioned by variation of the PLV membrane refraction index and formation of PLV aggregates. It was shown that the LSII measurement for the PLV suspension containing LPS can be used as a qualitative express analysis for the LPS incorporation into PLV as well as procedure for determination of the aggregate formation stage from PLV. The LPS of the plague, which as determinants being common for various gram-negative bacteria, is of great interest from the viewpoint of creating preparations for prophylactic measures against the endotoxin infections. However, the LPS toxicity due to the lipid A presence is a disadvantage of this weak antigen. Incorporation of the LPS int bilayer phospholipid membranes leads to its lower toxicity and higher immunization ability. The immunization ability and toxicity of the LPS complexes with bilayer membranes depend essentially on the LPS quantity sorbed in the membrane, as well as on the shapes and sizes of aggregates formed by the LPS and membranes in water

  9. Self-assembled drug delivery systems 2. Cholesteryl derivatives of antiviral nucleoside analogues: synthesis, properties and the vesicle formation.

    PubMed

    Jin, Yiguang; Xin, Rui; Ai, Ping; Chen, Dawei

    2008-02-28

    Self-assembled drug delivery systems (SADDS) are defined as the self-aggregates of amphiphilic prodrugs. Prodrug, molecular self-assembly and nanotechnology are involved in SADDS manufacturing. But the knowledge of the self-assembly of amphiphilic prodrugs and the formation rules of SADDS is very limited. In this paper, five cholesteryl derivatives of antiviral nucleoside analogues were synthesized, involving antiviral acyclovir, didanosine and zidovudine, and the different acyl linkers, succinyl, adipoyl and phosphoryl. The derivatives are typical amphiphiles with nucleosides as polar heads and long-chained lipids as hydrophobic tails. The derivatives showed the similar soluble behavior, and the solubility highly depended on the types of solvents. Two forces, hydrogen bonding and hydrophobic interaction in alcohol solutions could improve the derivatives dissolving. However, the molecular self-assembly of derivatives could prefer to happen in the noncompetitive solvents including chloroform and tetrahydrofuran (THF) based on the intermolecular hydrogen bonding between nucleobase moieties, which could greatly increase their solubility. The derivatives formed nanosized vesicles based on hydrophobic interaction after injecting their THF solutions into water. The volume ratios of polar heads and hydrophobic tails of amphiphiles could determine the vesicle size, and the amphiphiles with large ratios would prefer to form small vesicles. The self-assembled vesicles would likely become SADDS. PMID:17920793

  10. Co-delivery of cell-wall-forming enzymes in the same vesicle for coordinated fungal cell wall formation.

    PubMed

    Schuster, Martin; Martin-Urdiroz, Magdalena; Higuchi, Yujiro; Hacker, Christian; Kilaru, Sreedhar; Gurr, Sarah J; Steinberg, Gero

    2016-01-01

    Fungal cells are surrounded by an extracellular cell wall. This complex matrix of proteins and polysaccharides protects against adverse stresses and determines the shape of fungal cells. The polysaccharides of the fungal wall include 1,3-β-glucan and chitin, which are synthesized by membrane-bound synthases at the growing cell tip. A hallmark of filamentous fungi is the class V chitin synthase, which carries a myosin-motor domain. In the corn smut fungus Ustilago maydis, the myosin-chitin synthase Mcs1 moves to the plasma membrane in secretory vesicles, being delivered by kinesin-1 and myosin-5. The myosin domain of Mcs1 enhances polar secretion by tethering vesicles at the site of exocytosis. It remains elusive, however, how other cell-wall-forming enzymes are delivered and how their activity is coordinated post secretion. Here, we show that the U. maydis class VII chitin synthase and 1,3-β-glucan synthase travel in Mcs1-containing vesicles, and that their apical secretion depends on Mcs1. Once in the plasma membrane, anchorage requires enzyme activity, which suggests co-synthesis of chitin and 1,3-β-glucan polysaccharides at sites of exocytosis. Thus, delivery of cell-wall-forming enzymes in Mcs1 vesicles ensures local foci of fungal cell wall formation. PMID:27563844

  11. Co-delivery of cell-wall-forming enzymes in the same vesicle for coordinated fungal cell wall formation.

    PubMed

    Schuster, Martin; Martin-Urdiroz, Magdalena; Higuchi, Yujiro; Hacker, Christian; Kilaru, Sreedhar; Gurr, Sarah J; Steinberg, Gero

    2016-01-01

    Fungal cells are surrounded by an extracellular cell wall. This complex matrix of proteins and polysaccharides protects against adverse stresses and determines the shape of fungal cells. The polysaccharides of the fungal wall include 1,3-β-glucan and chitin, which are synthesized by membrane-bound synthases at the growing cell tip. A hallmark of filamentous fungi is the class V chitin synthase, which carries a myosin-motor domain. In the corn smut fungus Ustilago maydis, the myosin-chitin synthase Mcs1 moves to the plasma membrane in secretory vesicles, being delivered by kinesin-1 and myosin-5. The myosin domain of Mcs1 enhances polar secretion by tethering vesicles at the site of exocytosis. It remains elusive, however, how other cell-wall-forming enzymes are delivered and how their activity is coordinated post secretion. Here, we show that the U. maydis class VII chitin synthase and 1,3-β-glucan synthase travel in Mcs1-containing vesicles, and that their apical secretion depends on Mcs1. Once in the plasma membrane, anchorage requires enzyme activity, which suggests co-synthesis of chitin and 1,3-β-glucan polysaccharides at sites of exocytosis. Thus, delivery of cell-wall-forming enzymes in Mcs1 vesicles ensures local foci of fungal cell wall formation.

  12. Self-assembled drug delivery systems 2. Cholesteryl derivatives of antiviral nucleoside analogues: synthesis, properties and the vesicle formation.

    PubMed

    Jin, Yiguang; Xin, Rui; Ai, Ping; Chen, Dawei

    2008-02-28

    Self-assembled drug delivery systems (SADDS) are defined as the self-aggregates of amphiphilic prodrugs. Prodrug, molecular self-assembly and nanotechnology are involved in SADDS manufacturing. But the knowledge of the self-assembly of amphiphilic prodrugs and the formation rules of SADDS is very limited. In this paper, five cholesteryl derivatives of antiviral nucleoside analogues were synthesized, involving antiviral acyclovir, didanosine and zidovudine, and the different acyl linkers, succinyl, adipoyl and phosphoryl. The derivatives are typical amphiphiles with nucleosides as polar heads and long-chained lipids as hydrophobic tails. The derivatives showed the similar soluble behavior, and the solubility highly depended on the types of solvents. Two forces, hydrogen bonding and hydrophobic interaction in alcohol solutions could improve the derivatives dissolving. However, the molecular self-assembly of derivatives could prefer to happen in the noncompetitive solvents including chloroform and tetrahydrofuran (THF) based on the intermolecular hydrogen bonding between nucleobase moieties, which could greatly increase their solubility. The derivatives formed nanosized vesicles based on hydrophobic interaction after injecting their THF solutions into water. The volume ratios of polar heads and hydrophobic tails of amphiphiles could determine the vesicle size, and the amphiphiles with large ratios would prefer to form small vesicles. The self-assembled vesicles would likely become SADDS.

  13. Propeptide of aminopeptidase 1 protein mediates aggregation and vesicle formation in cytoplasm-to-vacuole targeting pathway.

    PubMed

    Morales Quinones, Mariana; Winston, Jared T; Stromhaug, Per E

    2012-03-23

    Misfolded protein aggregation causes disease and aging; autophagy counteracts this by eliminating damaged components, enabling cells to survive starvation. The cytoplasm-to-vacuole targeting pathway in yeast encompasses the aggregation of the premature form of aminopeptidase 1 (prApe1) in cytosol and its sequestration by autophagic proteins into a vesicle for vacuolar transport. We show that the propeptide of Ape1 is important for aggregation and vesicle formation and that it is sufficient for binding to prApe1 and Atg19. Defective aggregation disrupts vacuolar transport, suggesting that aggregate shape is important in vesicle formation, whereas Atg19 binding is not sufficient for vacuolar transport. Aggregation involves hydrophobicity, whereas Atg19 binding requires additional electrostatic interactions. Ape1 dodecamerization may cluster propeptides into trimeric structures, with sufficient affinity to form propeptide hexamers by binding to other dodecamers, causing aggregation. We show that Ape1 aggregates bind Atg19 and Atg8 in vitro; this could be used as a scaffold for an in vitro assay of autophagosome formation to elucidate the mechanisms of autophagy.

  14. Regulation of synaptic connectivity: levels of Fasciclin II influence synaptic growth in the Drosophila CNS.

    PubMed

    Baines, Richard A; Seugnet, Laurent; Thompson, Annemarie; Salvaterra, Paul M; Bate, Michael

    2002-08-01

    Much of our understanding of synaptogenesis comes from studies that deal with the development of the neuromuscular junction (NMJ). Although well studied, it is not clear how far the NMJ represents an adequate model for the formation of synapses within the CNS. Here we investigate the role of Fasciclin II (Fas II) in the development of synapses between identified motor neurons and cholinergic interneurons in the CNS of Drosophila. Fas II is a neural cell adhesion molecule homolog that is involved in both target selection and synaptic plasticity at the NMJ in Drosophila. In this study, we show that levels of Fas II are critical determinants of synapse formation and growth in the CNS. The initial establishment of synaptic contacts between these identified neurons is seemingly independent of Fas II. The subsequent proliferation of these synaptic connections that occurs postembryonically is, in contrast, significantly retarded by the absence of Fas II. Although the initial formation of synaptic connectivity between these neurons is seemingly independent of Fas II, we show that their formation is, nevertheless, significantly affected by manipulations that alter the relative balance of Fas II in the presynaptic and postsynaptic neurons. Increasing expression of Fas II in either the presynaptic or postsynaptic neurons, during embryogenesis, is sufficient to disrupt the normal level of synaptic connectivity that occurs between these neurons. This effect of Fas II is isoform specific and, moreover, phenocopies the disruption to synaptic connectivity observed previously after tetanus toxin light chain-dependent blockade of evoked synaptic vesicle release in these neurons. PMID:12151538

  15. Reelin and apoE actions on signal transduction, synaptic function and memory formation.

    PubMed

    Rogers, Justin T; Weeber, Edwin J

    2008-08-01

    Low-density-lipoprotein receptors (LDLRs) are an evolutionarily ancient surface protein family with the ability to activate a diversity of extracellular signals across the cellular membrane in the adult central nervous system (CNS). Their intimate roles in modulating synaptic plasticity and their necessity in hippocampal-dependent learning and memory have only recently come to light. Two known LDLR ligands, specifically apolipoprotein E (apoE) and reelin, have been the most widely investigated in this regard. Most of our understanding of synaptic plasticity comes from investigation of both pre- and postsynaptic alterations. Therefore, it is interesting to note that neurons and glia that do not contribute to the synaptic junction in question can secrete signaling molecules that affect synaptic plasticity. Notably, reelin and apoE have been shown to modulate hippocampal long-term potentiation in general, and affect NMDA receptor and AMPA receptor regulation specifically. Furthermore, these receptors and signaling molecules have significant roles in neuronal degenerative diseases such as Alzheimer's disease. The recent production of recombinant proteins, knockout and transgenic mice for receptors and ligands and the development of human ApoE targeted replacement mice have significantly expanded our understanding of the roles LDLRs and their ligands have in certain disease states and the accompanying initiation of specific signaling pathways. This review describes the role LDLRs, apoE and reelin have in the regulation of hippocampal synaptic plasticity.

  16. Similar oxysterols may lead to opposite effects on synaptic transmission: Olesoxime versus 5α-cholestan-3-one at the frog neuromuscular junction.

    PubMed

    Kasimov, M R; Zakyrjanova, G F; Giniatullin, A R; Zefirov, A L; Petrov, A M

    2016-07-01

    Cholesterol oxidation products frequently have a high biological activity. In the present study, we have used microelectrode recording of end plate currents and FM-based optical detection of synaptic vesicle exo-endocytosis to investigate the effects of two structurally similar oxysterols, olesoxime (cholest-4-en-3-one, oxime) and 5ɑ-cholestan-3-one (5ɑCh3), on neurotransmission at the frog neuromuscular junction. Olesoxime is an exogenous, potentially neuroprotective, substance and 5ɑCh3 is an intermediate product in cholesterol metabolism, which is elevated in the case of cerebrotendinous xanthomatosis. We found that olesoxime slightly increased evoked neurotransmitter release in response to a single stimulus and significantly reduced synaptic depression during high frequency activity. The last effect was due to an increase in both the number of synaptic vesicles involved in exo-endocytosis and the rate of synaptic vesicle recycling. In contrast, 5ɑCh3 reduced evoked neurotransmitter release during the low- and high frequency synaptic activities. The depressant action of 5ɑCh3 was associated with a reduction in the number of synaptic vesicles participating in exo- and endocytosis during high frequency stimulation, without a change in rate of the synaptic vesicle recycling. Of note, olesoxime increased the staining of synaptic membranes with the B-subunit of cholera toxin and the formation of fluorescent ganglioside GM1 clusters, and decreased the fluorescence of 22-NBD-cholesterol, while 5ɑCh3 had the opposite effects, suggesting that the two oxysterols have different effects on lipid raft stability. Taken together, these data show that these two structurally similar oxysterols induce marked different changes in neuromuscular transmission which are related with the alteration in synaptic vesicle cycle. PMID:27102612

  17. Tubular Membrane Formation of Binary Giant Unilamellar Vesicles Composed of Cylinder and Inverse-Cone-Shaped Lipids

    PubMed Central

    Sakuma, Yuka; Taniguchi, Takashi; Kawakatsu, Toshihiro; Imai, Masayuki

    2013-01-01

    We have succeeded in controlling tubular membrane formations in binary giant unilamellar vesicles (GUVs) using a simple temperature changing between the homogeneous one-phase region and the two-phase coexistence region. The binary GUV is composed of inverse-cone (bulky hydrocarbon chains and a small headgroup) and cylinder-shaped lipids. When the temperature was set in the two-phase coexistence region, the binary GUV had a spherical shape with solidlike domains. By increasing the temperature to the homogeneous one-phase region, the excess area created by the chain melting of the lipid produced tubes inside the GUV. The tubes had a radius on the micrometer scale and were stable in the one-phase region. When we again decreased the temperature to the two-phase coexisting region, the tubes regressed and the GUVs recovered their phase-separated spherical shape. We infer that the tubular formation was based on the mechanical balance of the vesicle membrane (spontaneous tension) coupled with the asymmetric distribution of the inverse-cone-shaped lipids between the inner and outer leaflets of the vesicle (lipid sorting). PMID:24209852

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

    NASA Astrophysics Data System (ADS)

    Noguchi, Hiroshi

    2015-12-01

    The shape transformations of fluid membranes induced by curved protein rods are studied using meshless membrane simulations. The rod assembly at low rod density induces a flat membrane tube and oblate vesicle. It is found that the polyhedral shapes are stabilized at high rod densities. The discrete shape transition between triangular and buckled discoidal tubes is obtained and their curvature energies are analyzed by a simple geometric model. For vesicles, triangular hosohedron and elliptic-disk shapes are formed in equilibrium, whereas tetrahedral and triangular prism shapes are obtained as metastable states.

  19. Innervation regulates synaptic ribbons in lateral line mechanosensory hair cells.

    PubMed

    Suli, Arminda; Pujol, Remy; Cunningham, Dale E; Hailey, Dale W; Prendergast, Andrew; Rubel, Edwin W; Raible, David W

    2016-06-01

    Failure to form proper synapses in mechanosensory hair cells, the sensory cells responsible for hearing and balance, leads to deafness and balance disorders. Ribbons are electron-dense structures that tether synaptic vesicles to the presynaptic zone of mechanosensory hair cells where they are juxtaposed with the post-synaptic endings of afferent fibers. They are initially formed throughout the cytoplasm, and, as cells mature, ribbons translocate to the basolateral membrane of hair cells to form functional synapses. We have examined the effect of post-synaptic elements on ribbon formation and maintenance in the zebrafish lateral line system by observing mutants that lack hair cell innervation, wild-type larvae whose nerves have been transected and ribbons in regenerating hair cells. Our results demonstrate that innervation is not required for initial ribbon formation but suggest that it is crucial for regulating the number, size and localization of ribbons in maturing hair cells, and for ribbon maintenance at the mature synapse.

  20. Innervation regulates synaptic ribbons in lateral line mechanosensory hair cells.

    PubMed

    Suli, Arminda; Pujol, Remy; Cunningham, Dale E; Hailey, Dale W; Prendergast, Andrew; Rubel, Edwin W; Raible, David W

    2016-06-01

    Failure to form proper synapses in mechanosensory hair cells, the sensory cells responsible for hearing and balance, leads to deafness and balance disorders. Ribbons are electron-dense structures that tether synaptic vesicles to the presynaptic zone of mechanosensory hair cells where they are juxtaposed with the post-synaptic endings of afferent fibers. They are initially formed throughout the cytoplasm, and, as cells mature, ribbons translocate to the basolateral membrane of hair cells to form functional synapses. We have examined the effect of post-synaptic elements on ribbon formation and maintenance in the zebrafish lateral line system by observing mutants that lack hair cell innervation, wild-type larvae whose nerves have been transected and ribbons in regenerating hair cells. Our results demonstrate that innervation is not required for initial ribbon formation but suggest that it is crucial for regulating the number, size and localization of ribbons in maturing hair cells, and for ribbon maintenance at the mature synapse. PMID:27103160

  1. Direct visualization of trans-synaptic neurexin-neuroligin interactions during synapse formation.

    PubMed

    Tsetsenis, Theodoros; Boucard, Antony A; Araç, Demet; Brunger, Axel T; Südhof, Thomas C

    2014-11-01

    Neurexins and neuroligins are synaptic cell-adhesion molecules that are essential for normal synapse specification and function and are thought to bind to each other trans-synaptically, but such interactions have not been demonstrated directly. Here, we generated neurexin-1β and neuroligin-1 and neuroligin-2 fusion proteins containing complementary "split" GFP fragments positioned such that binding of neurexin-1β to neuroligin-1 or neuroligin-2 allowed GFP reconstitution without dramatically changing their binding affinities. GFP fluorescence was only reconstituted from split-GFP-modified neurexin-1β and neuroligin-1 if and after neurexin-1β bound to its neuroligin partner; reassociation of the split-GFP components with each other did not mediate binding. Using trans-cellular reconstitution of GFP fluorescence from split-GFP-modified neurexin-1β and neuroligins as an assay, we demonstrate that trans-synaptic neurexin/neuroligin binding indeed occurred when mouse hippocampal neurons formed synapses onto non-neuronal COS-7 cells expressing neuroligins or when mouse hippocampal neurons formed synapses with each other. This visualization of synapses by neurexin/neuroligin binding prompted us to refer to this approach as "SynView." Our data demonstrate that neurexin-1β forms a trans-synaptic complex with neuroligin-1 and neuroligin-2 and that this interaction can be used to label synapses in a specific fashion in vivo.

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

    PubMed

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

    2015-05-12

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

  3. Cryopreservation of germinal vesicle stage porcine oocytes based on intracellular ice formation assessment.

    PubMed

    Yang, C Y; Chen, M C; Lee, P T; Lin, T T

    2012-01-01

    This study aimed at evaluating the feasibility of slow freezing for cryopreservation of germinal vesicle (GV) stage porcine oocytes. In this study, intracellular ice formation (IIF) characteristics of GV porcine oocytes were investigated by using a thermoelectric cooling (TEC) cryomicroscope system. This cryomicroscope system used a thermoelectric cooling (TEC) chip in its cold stage as a heat sink and employed a PID control algorithm to achieve accurate temperature control. The temperature was controlled to a range between 70 degree C and -55 degree C with an accuracy of +/- 0.5 degree C. Five constant cooling rates of 24, 12, 6, 3 and 1.5 degree C/min were tested in experiments in freezing GV porcine oocytes from 20 degree C to -50 degree C in an NCSU-23 medium plus 2.0 M DMSO. The IIF temperature of each individual oocyte was recorded and cumulative IIF probabilities were calculated for each cooling rate. The total cumulative probabilities of IIF temperature distribution were 100 percent, 100 percent, 50.0 percent, 54.3 percent and 58.6 percent at cooling rates of 24, 12, 6, 3 and 1.5 degree C/min, respectively. A Weibull distribution model was found to adequately describe the distribution of IIF temperatures of GV porcine oocytes for the cooling rates tested (R2 = 0.858 +/- 0.09). The IIF experimental results indicate that cooling rates of 6, 3 and 1.5°C/min could be considered as possible cryopreservation protocols. Further experiments were performed to examine the feasibility of using these protocols to cryopreserve GV porcine oocytes. After 44 h of in-vitro maturation in NCSU-23, the survival of thawed oocytes was checked. Porcine oocytes developed from the GV stage to the MII stage by using Hoechst 33258 staining, followed by Lacmoid staining as a secondary check. Normalized survival rates of 37.7 +/- 4.6 percent, 45.0 4.4 percent and 45.4 +/- 5.9 percent were obtained for GV oocytes frozen at 1.5, 3 and 6 degree C/min, respectively. The experimental

  4. Ternary phase behaviour and vesicle formation of a sodium N-lauroylsarcosinate hydrate/1-decanol/water system

    PubMed Central

    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. PMID:22355590

  5. Diacylglycerol-Rich Domain Formation in Giant Stearoyl-Oleoyl Phosphatidylcholine Vesicles Driven by Phospholipase C Activity

    PubMed Central

    Riske, Karin A.; Döbereiner, Hans-Günther

    2003-01-01

    We have studied the effect of phospholipase C from Bacillus cereus and Clostridium perfringens (α-toxin) on giant stearoyl-oleoyl phosphatidylcholine (SOPC) vesicles. Enzyme activity leads to a binary mixture of SOPC and the diacylglycerol SOG, which phase separates into a SOPC-rich bilayer phase and a SOG-rich isotropic bulk-like domain embedded within the membrane, as seen directly by phase contrast microscopy. After prolonged enzymatic attack, all bilayer membranes are transformed into an isotropic pure SOG phase as characterized by fluorescence microscopy, differential scanning calorimetry, fluorescence anisotropy measurements, and small angle x-ray scattering. These domains may have biological relevance, serving as storage compartments for hydrophobic molecules and/or catalyzing cellular signaling events at their boundaries. Furthermore, in the early stages of asymmetric enzymatic attack to the external monolayer of giant vesicles, we observe a transient coupling of the second-messenger diacylglycerol to membrane spontaneous curvature, which decreases due to enzyme activity, before domain formation and final vesicle collapse occurs. PMID:14507699

  6. The Transmembrane Domain Peptide of Vesicular Stomatitis Virus Promotes Both Intermediate and Pore Formation during PEG-Mediated Vesicle Fusion

    PubMed Central

    Sengupta, Tanusree; Chakraborty, Hirak; Lentz, Barry R.

    2014-01-01

    We propose mechanisms by which the transmembrane domain of vesicular stomatitis virus (VSV-TMD) promotes both initiation of fusion and formation of a fusion pore. Time courses of polyethyleneglycol (PEG)-mediated fusion of 25 nm small unilamellar vesicles composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), bovine brain sphingomyelin, and cholesterol (35:30:15:20 molar ratio) were recorded at pH 7.4 at five different temperatures (from 17°C to 37°C) and compared with time courses obtained with the same vesicles containing the fusion-active TMD of the G protein of VSV. Multiple time courses were fitted globally to a one-intermediate ensemble kinetic model to estimate the rate constants for conversion of the aggregated state to an intermediate hemifused state (k1, stalk, or I1) that rapidly transits to an unstable intermediate (I2 state) that converts to a final fusion pore state with a combined rate k3. The probabilities of lipid mixing, contents mixing, and contents leakage in the three states were also obtained from this analysis. The activation thermodynamics for each step were consistent with previously published models of lipid rearrangements during intermediate and pore formation. The influences of VSV-TMD, hexadecane, and VSV-TMD + hexadecane on the kinetics, activation thermodynamics, and membrane structure support the hypothesis that these two agents do not catalyze fusion by a common mechanism, except possibly at the lowest temperatures examined. VSV-TMD primarily catalyzed initial intermediate formation, although it substantially increased the probability of contents mixing in the intermediate state. Our results support the hypothesis that the catalytic influence of VSV-TMD on the initial-intermediate- and pore-forming steps of PEG-mediated fusion derives from its ability to impose a positive intrinsic curvature and thereby stress small unilamellar vesicle outer leaflets as well as the periphery of intermediate

  7. The role of cell adhesion molecules in visual circuit formation: From neurite outgrowth to maps and synaptic specificity

    PubMed Central

    Missaire, Mégane

    2015-01-01

    ABSTRACT The formation of visual circuitry is a multistep process that involves cell–cell interactions based on a range of molecular mechanisms. The correct implementation of individual events, including axon outgrowth and guidance, the formation of the topographic map, or the synaptic targeting of specific cellular subtypes, are prerequisites for a fully functional visual system that is able to appropriately process the information captured by the eyes. Cell adhesion molecules (CAMs) with their adhesive properties and their high functional diversity have been identified as key actors in several of these fundamental processes. Because of their growth‐promoting properties, CAMs play an important role in neuritogenesis. Furthermore, they are necessary to control additional neurite development, regulating dendritic spacing and axon pathfinding. Finally, trans‐synaptic interactions of CAMs ensure cell type‐specific connectivity as a basis for the establishment of circuits processing distinct visual features. Recent discoveries implicating CAMs in novel mechanisms have led to a better general understanding of neural circuit formation, but also revealed an increasing complexity of their function. This review aims at describing the different levels of action for CAMs to shape neural connectivity, with a special focus on the visual system. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 569–583, 2015 PMID:25649254

  8. Coacervate formation from natural glycolipid: one acetyl group on the headgroup triggers coacervate-to-vesicle transition.

    PubMed

    Imura, T; Yanagishita, H; Kitamoto, D

    2004-09-01

    Coacervate (L3 phase) formation of the single component "natural" glycoliped biosurfactant, MEL-A, was observed for the first time by using an optical microscope, a confocal laser scanning microscope (CLSM), and a freeze-fracture electron microscope (FF-TEM). It was also found that only a slight decrease in spontaneous curvature resulting from the absence of one acetyl group on the headgroup induced a drastic morphological change in the 3D self-assembled structure from coacervates (L3 phase) to ordered vesicles (Lalpha phase).

  9. Mind Bomb-2 Regulates Hippocampus-dependent Memory Formation and Synaptic Plasticity.

    PubMed

    Kim, Somi; Kim, TaeHyun; Lee, Hye-Ryeon; Kong, Young-Yun; Kaang, Bong-Kiun

    2015-11-01

    Notch signaling is a key regulator of neuronal fate during embryonic development, but its function in the adult brain is still largely unknown. Mind bomb-2 (Mib2) is an essential positive regulator of the Notch pathway, which acts in the Notch signal-sending cells. Therefore, genetic deletion of Mib2 in the mouse brain might help understand Notch signaling-mediated cell-cell interactions between neurons and their physiological function. Here we show that deletion of Mib2 in the mouse brain results in impaired hippocampal spatial memory and contextual fear memory. Accordingly, we found impaired hippocampal synaptic plasticity in Mib2 knock-out (KO) mice; however, basal synaptic transmission did not change at the Schaffer collateral-CA1 synapses. Using western blot analysis, we found that the level of cleaved Notch1 was lower in Mib2 KO mice than in wild type (WT) littermates after mild foot shock. Taken together, these data suggest that Mib2 plays a critical role in synaptic plasticity and spatial memory through the Notch signaling pathway.

  10. Mind Bomb-2 Regulates Hippocampus-dependent Memory Formation and Synaptic Plasticity

    PubMed Central

    Kim, Somi; Kim, TaeHyun; Lee, Hye-Ryeon; Kong, Young-Yun

    2015-01-01

    Notch signaling is a key regulator of neuronal fate during embryonic development, but its function in the adult brain is still largely unknown. Mind bomb-2 (Mib2) is an essential positive regulator of the Notch pathway, which acts in the Notch signal-sending cells. Therefore, genetic deletion of Mib2 in the mouse brain might help understand Notch signaling-mediated cell-cell interactions between neurons and their physiological function. Here we show that deletion of Mib2 in the mouse brain results in impaired hippocampal spatial memory and contextual fear memory. Accordingly, we found impaired hippocampal synaptic plasticity in Mib2 knock-out (KO) mice; however, basal synaptic transmission did not change at the Schaffer collateral-CA1 synapses. Using western blot analysis, we found that the level of cleaved Notch1 was lower in Mib2 KO mice than in wild type (WT) littermates after mild foot shock. Taken together, these data suggest that Mib2 plays a critical role in synaptic plasticity and spatial memory through the Notch signaling pathway. PMID:26557018

  11. Phase behavior of semifluorinated catanionic mixtures: head group dependence and spontaneous formation of vesicles.

    PubMed

    Blanco, Elena; Olsson, Ulf; Ruso, Juan M; Schulz, Pablo C; Prieto, Gerardo; Sarmiento, Félix

    2009-03-15

    Hexadecyltrimethylammonium bromide (C(16)TAB)-sodium perfluorooctanoate (C(8)FONa) and hexadecylpyridynium bromide (C(16)PyB)-C(8)FONa catanionic semifluorinated mixtures have been studied by conductivity, dynamic light scattering (DLS), cryo-transmission electron microscopy (cryo-TEM) and polarizing microscopy. The regular solution theory, applicable for a limited fluorinated molar ratio, does not predict long-range electrostatic interactions. The results are consistent with the fact that in the hydrogenated-rich region the interaction is attractive in both catanionic mixtures. The systems containing pyridinium headgroups were of the stronger interaction. A transition from micelles was found in both mixtures as a function of fluorinated molar ratio. Special attention was devoted to the effect of the head group in the system properties. The information related with the mean vesicle radius measured by DLS was compared with the vesicle size distribution as well as the elastic properties of the bilayer measured with cryo-TEM.

  12. Estradiol acts via estrogen receptors alpha and beta on pathways important for synaptic plasticity in the mouse hippocampal formation

    PubMed Central

    Spencer-Segal, Joanna L.; Tsuda, Mumeko C.; Mattei, Larissa; Waters, Elizabeth M.; Romeo, Russell D.; Milner, Teresa A.; McEwen, Bruce S.; Ogawa, Sonoko

    2012-01-01

    Estradiol affects hippocampal-dependent spatial memory and underlying structural and electrical synaptic plasticity in female mice and rats. Using estrogen receptor (ER) alpha and beta knockout mice and wild-type littermates, we investigated the role of ERs in estradiol effects on multiple pathways important for hippocampal plasticity and learning. Six hours of estradiol administration increased immunoreactivity for phosphorylated Akt throughout the hippocampal formation, while 48 hours of estradiol increased immunoreactivity for phosphorylated TrkB receptor. Estradiol effects on phosphorylated Akt and TrkB immunoreactivities were abolished in ER alpha and ER beta knockout mice. Estradiol also had distinct effects on immunoreactivity for PSD-95 and BDNF mRNA in ER alpha and beta knockout mice. Thus, estradiol acts through both ERs alpha and beta in several subregions of the hippocampal formation. The different effects of estradiol at 6 and 48 hours indicate that several mechanisms of estrogen receptor signaling contribute to this female hormone’s influence on hippocampal synaptic plasticity. By further delineating these mechanisms, we will better understand and predict the effects of endogenous and exogenous ovarian steroids on mood, cognition, and other hippocampal-dependent behaviors. PMID:22133892

  13. Glycolytic Enzymes Localize to Synapses under Energy Stress to Support Synaptic Function.

    PubMed

    Jang, SoRi; Nelson, Jessica C; Bend, Eric G; Rodríguez-Laureano, Lucelenie; Tueros, Felipe G; Cartagenova, Luis; Underwood, Katherine; Jorgensen, Erik M; Colón-Ramos, Daniel A

    2016-04-20

    Changes in neuronal activity create local and transient changes in energy demands at synapses. Here we discover a metabolic compartment that forms in vivo near synapses to meet local energy demands and support synaptic function in Caenorhabditis elegans neurons. Under conditions of energy stress, glycolytic enzymes redistribute from a diffuse localization in the cytoplasm to a punctate localization adjacent to synapses. Glycolytic enzymes colocalize, suggesting the ad hoc formation of a glycolysis compartment, or a "glycolytic metabolon," that can maintain local levels of ATP. Local formation of the glycolytic metabolon is dependent on presynaptic scaffolding proteins, and disruption of the glycolytic metabolon blocks the synaptic vesicle cycle, impairs synaptic recovery, and affects locomotion. Our studies indicate that under energy stress conditions, energy demands in C. elegans synapses are met locally through the assembly of a glycolytic metabolon to sustain synaptic function and behavior. VIDEO ABSTRACT.

  14. Aqueous-phase behavior and vesicle formation of natural glycolipid biosurfactant, mannosylerythritol lipid-B.

    PubMed

    Worakitkanchanakul, Wannasiri; Imura, Tomohiro; Fukuoka, Tokuma; Morita, Tomotake; Sakai, Hideki; Abe, Masahiko; Rujiravanit, Ratana; Chavadej, Sumaeth; Minamikawa, Hiroyuki; Kitamoto, Dai

    2008-08-01

    Mannosylerythritol lipids (MELs) are one of the most promising glycolipid biosurfactants produced by yeast strains of the genus Pseudozyma. In this study, the aqueous-phase behavior of a new monoacetyl MEL derivative, 1-O-beta-(2',3'-di-O-alka(e)noyl-6'-O-acetyl-d-mannopyranosyl)-d-erythritol (MEL-B), was investigated using polarized optical microscopy, small-angle X-ray scattering (SAXS), confocal laser scanning microscopy (CLSM), and differential scanning calorimetry (DSC). The present MEL-B was found to self-assemble into a lamellar (L(alpha)) phase over remarkably wide concentration and temperature ranges. According to SAXS measurement, the interlayer spacing (d) was estimated to be almost constant (about 4.7 nm) at the low MEL-B concentration (60 wt.%) region, the d-spacing gradually decreased to 3.1 nm with an increase in the MEL-B concentration. The thermal stability of the liquid crystalline phase was investigated by DSC measurement. The obtained L(alpha) phase was found to be stable up to 95 degrees C below a MEL-B concentration of 85 wt.%; then, the melting temperature of the liquid crystalline phase dramatically decreased with an increase in MEL-B concentration (above 85 wt.%). Furthermore, we found relatively large vesicles (1-5 microm) at the low MEL-B concentration using CLSM observation. The trapped volume of the obtained MEL-B vesicle was estimated to be about 0.42 microL/mumol by glucose dialysis method. These results suggest that the natural glycolipid biosurfactant, the newly found MEL-B, would be useful in various fields of applications as an L(alpha) phase- and/or vesicle-forming lipid. PMID:18456469

  15. Translational control of synaptic plasticity.

    PubMed

    Richter, Joel D

    2010-12-01

    Synapses, points of contact between axons and dendrites, are conduits for the flow of information in the circuitry of the central nervous system. The strength of synaptic transmission reflects the interconnectedness of the axons and dendrites at synapses; synaptic strength in turn is modified by the frequency with which the synapses are stimulated. This modulation of synaptic strength, or synaptic plasticity, probably forms the cellular basis for learning and memory. RNA metabolism, particularly translational control at or near the synapse, is one process that controls long-lasting synaptic plasticity and, by extension, memory formation and consolidation. In the present paper, I review some salient features of translational control of synaptic plasticity.

  16. Vesicle Photonics

    SciTech Connect

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

    2013-04-03

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

  17. Voluntary running depreciates the requirement of Ca2+-stimulated cAMP signaling in synaptic potentiation and memory formation.

    PubMed

    Zheng, Fei; Zhang, Ming; Ding, Qi; Sethna, Ferzin; Yan, Lily; Moon, Changjong; Yang, Miyoung; Wang, Hongbing

    2016-08-01

    Mental health and cognitive functions are influenced by both genetic and environmental factors. Although having active lifestyle with physical exercise improves learning and memory, how it interacts with the specific key molecular regulators of synaptic plasticity is largely unknown. Here, we examined the effects of voluntary running on long-term potentiation (LTP) and memory formation in mice lacking type 1 adenylyl cyclase (AC1), a neurospecific synaptic enzyme that contributes to Ca(2+)-stimulated cAMP production. Following 1 mo of voluntary running-wheel exercise, the impaired LTP and object recognition memory in AC1 knockout (KO) mice were significantly attenuated. Running up-regulated exon II mRNA level of BDNF (brain-derived neurotrophic factor), though it failed to increase exon I and IV mRNAs in the hippocampus of AC1 KO mice. Intrahippocampal infusion of recombinant BDNF was sufficient to rescue LTP and object recognition memory defects in AC1 KO mice. Therefore, voluntary running and exogenous BDNF application overcome the defective Ca(2+)-stimulated cAMP signaling. Our results also demonstrate that alteration in Ca(2+)-stimulated cAMP can affect the molecular outcome of physical exercise.

  18. Voluntary running depreciates the requirement of Ca2+-stimulated cAMP signaling in synaptic potentiation and memory formation.

    PubMed

    Zheng, Fei; Zhang, Ming; Ding, Qi; Sethna, Ferzin; Yan, Lily; Moon, Changjong; Yang, Miyoung; Wang, Hongbing

    2016-08-01

    Mental health and cognitive functions are influenced by both genetic and environmental factors. Although having active lifestyle with physical exercise improves learning and memory, how it interacts with the specific key molecular regulators of synaptic plasticity is largely unknown. Here, we examined the effects of voluntary running on long-term potentiation (LTP) and memory formation in mice lacking type 1 adenylyl cyclase (AC1), a neurospecific synaptic enzyme that contributes to Ca(2+)-stimulated cAMP production. Following 1 mo of voluntary running-wheel exercise, the impaired LTP and object recognition memory in AC1 knockout (KO) mice were significantly attenuated. Running up-regulated exon II mRNA level of BDNF (brain-derived neurotrophic factor), though it failed to increase exon I and IV mRNAs in the hippocampus of AC1 KO mice. Intrahippocampal infusion of recombinant BDNF was sufficient to rescue LTP and object recognition memory defects in AC1 KO mice. Therefore, voluntary running and exogenous BDNF application overcome the defective Ca(2+)-stimulated cAMP signaling. Our results also demonstrate that alteration in Ca(2+)-stimulated cAMP can affect the molecular outcome of physical exercise. PMID:27421897

  19. Effect of toluene on Pseudomonas stutzeri ST-9 morphology - plasmolysis, cell size, and formation of outer membrane vesicles.

    PubMed

    Michael, Esti; Nitzan, Yeshayahu; Langzam, Yakov; Luboshits, Galia; Cahan, Rivka

    2016-08-01

    Isolated toluene-degrading Pseudomonas stutzeri ST-9 bacteria were grown in a minimal medium containing toluene (100 mg·L(-1)) (MMT) or glucose (MMG) as the sole carbon source, with specific growth rates of 0.019 h(-1) and 0.042 h(-1), respectively. Scanning (SEM) as well as transmission (TEM) electron microscope analyses showed that the bacterial cells grown to mid-log phase in the presence of toluene possess a plasmolysis space. TEM analysis revealed that bacterial cells that were grown in MMT were surrounded by an additional "material" with small vesicles in between. Membrane integrity was analyzed by leakage of 260 nm absorbing material and demonstrated only 7% and 8% leakage from cultures grown in MMT compared with MMG. X-ray microanalysis showed a 4.3-fold increase in Mg and a 3-fold increase in P in cells grown in MMT compared with cells grown in MMG. Fluorescence-activated cell sorting (FACS) analysis indicated that the permeability of the membrane to propidium iodide was 12.6% and 19.6% when the cultures were grown in MMG and MMT, respectively. The bacterial cell length increased by 8.5% ± 0.1% and 17% ± 2%, as measured using SEM images and FACS analysis, respectively. The results obtained in this research show that the presence of toluene led to morphology changes, such as plasmolysis, cell size, and formation of outer membrane vesicles. However, it does not cause significant damage to membrane integrity.

  20. Spontaneous formation of interfacial lipid-protein monolayers during adsorption from vesicles.

    PubMed Central

    Nag, K; Perez-Gil, J; Cruz, A; Rich, N H; Keough, K M

    1996-01-01

    Spread and adsorbed monolayers of lipid-protein mixtures have served as models for biomembranes and pulmonary surfactant, but their similarity was unclear. Epifluorescence microscopy of monolayers spontaneously adsorbed from vesicles of dipalmitoylphosphatidylcholine or dipalmitoylphosphatidylcholine plus surfactant protein C (SP-C) showed gas, liquid expanded, and liquid condensed (LC) domains. The shapes and distribution of LC domains in the adsorbed and solvent-spread monolayers were quite similar. Labeled SP-C adsorbed into the air-water interface in the company of the lipids. In both forms of monolayers, SP-C occupied the fluid phase and reduced the size and amount of the LC domains. The properties suggest that these adsorbed and spread monolayers are analogous to one another. Images FIGURE 1 FIGURE 2 FIGURE 3 PMID:8874011

  1. In vitro approach to the mechanics of lipid membrane area regulation: vesicle absorption and tube formation

    NASA Astrophysics Data System (ADS)

    Staykova, Margarita; Holmes, Douglas; Read, Clarke; Stone, Howard A.

    2011-03-01

    We have designed an experimental approach that allows us to study the response of supported lipid bilayers to cycles of biaxial expansion and compression. We observed that the bilayer effectively adjusts its area during dilatational or compressive strains in order to reduce its tension. For example, if there is a sufficient lipid reservoir in the form of attached vesicles, then a lipid bilayer may accommodate strains tens of times larger than the critical strain for rupture by expanding its area. Additionally, upon compression the bilayer reduces its area by expelling lipid tubes out of its plane. These observations offer new insights into how cells regulate their surface area in response to various mechanical stimuli, i.e. during physiological volume changes, locomotion, cyclic expansion and compression of the uro- and the alveolar- epithelium, etc.

  2. Prohormone processing in the trans-Golgi network: endoproteolytic cleavage of prosomatostatin and formation of nascent secretory vesicles in permeabilized cells

    PubMed Central

    1993-01-01

    Many peptide hormones are synthesized as larger precursors which undergo endoproteolytic cleavage at paired basic residues to generate a bioactive molecule. Morphological evidence from several laboratories has implicated either the TGN or immature secretory granules as the site of prohormone cleavage. To identify the site where prohormone cleavage is initiated, we have used retrovirally infected rat anterior pituitary GH3 cells which express high levels of prosomatostatin (proSRIF) (Stoller, T. J., and D. Shields. J. Cell Biol. 1988. 107:2087- 2095). By incubating these cells at 20 degrees C, a temperature that prevents exit from the Golgi apparatus, proSRIF accumulated quantitatively in the TGN and no proteolytic processing was evident; processing resumed upon shifting the cells back to 37 degrees C. After the 20 degrees C block, the cells were mechanically permeabilized and pro-SRIF processing determined. Cleavage of proSRIF to the mature hormone was approximately 35-50% efficient, required incubation at 37 degrees C and ATP hydrolysis, but was independent of GTP or cytosol. The in vitro ATP-dependent proSRIF processing was inhibited by inclusion of chloroquine, a weak base, CCCP, a protonophore, or by preincubating the permeabilized cells with low concentrations of N- ethylmaleimide, an inhibitor of vacuolar-type ATP-dependent proton pumps. These data suggest that: (a) proSRIF cleavage is initiated in the TGN, and (b) this reaction requires an acidic pH which is facilitated by a Golgi-associated vacuolar-type ATPase. A characteristic feature of polypeptide hormone-producing cells is their ability to store the mature hormone in dense core secretory granules. To investigate the mechanism of protein sorting to secretory granules, the budding of nascent secretory vesicles from the TGN was determined. No vesicle formation occurred at 20 degrees C; in contrast, at 37 degrees C, the budding of secretory vesicles was approximately 40% efficient and was dependent on ATP

  3. Insights into structural and regulatory roles of Sec16 in COPII vesicle formation at ER exit sites

    PubMed Central

    Yorimitsu, Tomohiro; Sato, Ken

    2012-01-01

    COPII-coated buds are formed at endoplasmic reticulum exit sites (ERES) to mediate ER-to-Golgi transport. Sec16 is an essential factor in ERES formation, as well as in COPII-mediated traffic in vivo. Sec16 interacts with multiple COPII proteins, although the functional significance of these interactions remains unknown. Here we present evidence that full-length Sec16 plays an important role in regulating Sar1 GTPase activity at the late steps of COPII vesicle formation. We show that Sec16 interacts with Sec23 and Sar1 through its C-terminal conserved region and hinders the ability of Sec31 to stimulate Sec23 GAP activity toward Sar1. We also find that purified Sec16 alone can self-assemble into homo-oligomeric complexes on a planar lipid membrane. These features ensure prolonged COPII coat association within a preformed Sec16 cluster, which may lead to the formation of ERES. Our results indicate a mechanistic relationship between COPII coat assembly and ERES formation. PMID:22675024

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

    PubMed

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

    2016-02-26

    Synaptic transmission is mediated by the release of neurotransmitters, which involves exo-endocytotic cycling of synaptic vesicles. To maintain synaptic function, synaptic vesicles are refilled with thousands of neurotransmitter molecules within seconds after endocytosis, using the energy provided by an electrochemical proton gradient. However, it is unclear how transmitter molecules carrying different net charges can be efficiently sequestered while maintaining charge neutrality and osmotic balance. We used single-vesicle imaging to monitor pH and electrical gradients and directly showed different uptake mechanisms for glutamate and γ-aminobutyric acid (GABA) operating in parallel. In contrast to glutamate, GABA was exchanged for protons, with no other ions participating in the transport cycle. Thus, only a few components are needed to guarantee reliable vesicle filling with different neurotransmitters. PMID:26912364

  5. Investigation of vesicle-capsular plague antigen complex formation by elastic laser radiation scattering

    NASA Astrophysics Data System (ADS)

    Guseva, N. P.; Maximova, Irina S.; Romanov, Sergey V.; Shubochkin, L. P.; Tatarintsev, Sergey N.

    1991-05-01

    Recently a great deal of attention has been given to the investigation artificial lipid liposomes, due to their application as "containers" for directed transport of biologically active compounds into particular cells, organs and tissues for prophylaxis and therapy of infectious diseases. The use of traditional methods of liposome investigation, such as sedimentation, electrophoresis and chromatography is impeded by low liposome resistivity to different deformations. In conjunction with this, optical methods of laser light scattering are promising as they allow nondisturbing, precise and quick investigations. This paper describes the investigation of vesicle systems prepared from egg lecithin of Serva Corporation and their complexes with the capsular antigen of the plague microbe. The capsular antigen Fl was obtained from EV plague microbe grown at 37° C on Huttinger agar. Fl was isolated by gel-filtration on ASA-22 followed by freeze drying of the preparation. Angular dependences of polarized radiation scattering were measured for several liposome suspension samples in a saline solution before and after the interaction with the plague microbe capsular antigen. The aim of the investigation was to analyze the nature of mutual antigen arrangement in a liposome and to develop methods for measuring its inclusion percentage.

  6. Lipid Peroxides Promote Large Rafts: Effects of Excitation of Probes in Fluorescence Microscopy and Electrochemical Reactions during Vesicle Formation

    PubMed Central

    Ayuyan, Artem G.; Cohen, Fredric S.

    2006-01-01

    Raft formation and enlargement was investigated in liposomes and supported bilayers prepared from sphingomyelin (SM), cholesterol, and unsaturated phospholipids; NBD-DPPE and rhodamine-(DOPE) were employed as fluorescent probes. Rafts were created by lowering temperature. Maintaining 20 mol % SM, fluorescence microscopy showed that, in the absence of photooxidation, large rafts did not form in giant unilamellar vesicles (GUVs) containing 20 or more mol % cholesterol. But if photooxidation was allowed to proceed, large rafts were readily observed. In population, cuvette experiments, small rafts formed without photooxidation at high cholesterol concentrations. Thus, photooxidation was the cause of raft enlargement during microscopy experiments. Because photooxidation results in peroxidation at lipid double bonds, photosensitization experiments were performed to explicitly produce peroxides of SM and an unsaturated phospholipid. GUVs of high cholesterol content containing the breakdown products of SM-peroxide, but not phospholipid-peroxide, resulted in large rafts after lowering temperature. In addition, GUV production by electroswelling can result in peroxides that cause large raft formation. The use of titanium electrodes eliminates this problem. In conclusion, lipid peroxides and their breakdown products are the cause of large raft formation in GUVs containing biological levels of cholesterol. It is critical that experiments investigating rafts in bilayer membranes avoid the production of peroxides. PMID:16815906

  7. Effect of toluene on Pseudomonas stutzeri ST-9 morphology - plasmolysis, cell size, and formation of outer membrane vesicles.

    PubMed

    Michael, Esti; Nitzan, Yeshayahu; Langzam, Yakov; Luboshits, Galia; Cahan, Rivka

    2016-08-01

    Isolated toluene-degrading Pseudomonas stutzeri ST-9 bacteria were grown in a minimal medium containing toluene (100 mg·L(-1)) (MMT) or glucose (MMG) as the sole carbon source, with specific growth rates of 0.019 h(-1) and 0.042 h(-1), respectively. Scanning (SEM) as well as transmission (TEM) electron microscope analyses showed that the bacterial cells grown to mid-log phase in the presence of toluene possess a plasmolysis space. TEM analysis revealed that bacterial cells that were grown in MMT were surrounded by an additional "material" with small vesicles in between. Membrane integrity was analyzed by leakage of 260 nm absorbing material and demonstrated only 7% and 8% leakage from cultures grown in MMT compared with MMG. X-ray microanalysis showed a 4.3-fold increase in Mg and a 3-fold increase in P in cells grown in MMT compared with cells grown in MMG. Fluorescence-activated cell sorting (FACS) analysis indicated that the permeability of the membrane to propidium iodide was 12.6% and 19.6% when the cultures were grown in MMG and MMT, respectively. The bacterial cell length increased by 8.5% ± 0.1% and 17% ± 2%, as measured using SEM images and FACS analysis, respectively. The results obtained in this research show that the presence of toluene led to morphology changes, such as plasmolysis, cell size, and formation of outer membrane vesicles. However, it does not cause significant damage to membrane integrity. PMID:27256870

  8. Defensive slime formation in Pacific hagfish requires Ca2+- and aquaporin-mediated swelling of released mucin vesicles.

    PubMed

    Herr, Julia E; Clifford, Alexander M; Goss, Greg G; Fudge, Douglas S

    2014-07-01

    Hagfishes defend themselves from fish predators via the rapid deployment of a fibrous slime that adheres to and clogs gills. The slime transforms from a thick glandular exudate to a fully hydrated product in a fraction of a second through a process that involves the swelling and rupture of numerous mucin vesicles. Here we demonstrate that the vesicle membrane plays an important role in regulating the swelling of mucin granules, and provide evidence that the membrane contains proteins that facilitate the movement of ions and water molecules. By exposing isolated mucin vesicles to varying combinations of inorganic ions, organic compounds and membrane channel inhibitors, we found that the majority of hagfish mucin vesicles require Ca(2+) to rupture. We also show that Ca(2+)-dependent rupture can be pharmacologically inhibited, which suggests a role for Ca(2+)-activated membrane transporters. We demonstrate that the aquaporin inhibitor mercuric chloride reduces the rate of vesicle swelling by an order of magnitude, which suggests that aquaporins facilitate the influx of water during vesicle deployment. Molecular evidence of two aquaporin homologues expressed in the slime glands further supports this idea. We propose a model of hagfish slime mucin vesicle rupture that involves Ca(2+)-activated transporters and aquaporins, and suggest that the presence of these proteins is an adaptation for increasing the speed of vesicle rupture and, consequently, the speed of the sliming response of hagfishes.

  9. Structure formation of lipid membranes: Membrane self-assembly and vesicle opening-up to octopus-like micelles

    NASA Astrophysics Data System (ADS)

    Noguchi, Hiroshi

    2013-02-01

    We briefly review our recent studies on self-assembly and vesicle rupture of lipid membranes using coarse-grained molecular simulations. For single component membranes, lipid molecules self-assemble from random gas states to vesicles via disk-shaped clusters. Clusters aggregate into larger clusters, and subsequently the large disks close into vesicles. The size of vesicles are determined by kinetics than by thermodynamics. When a vesicle composed of lipid and detergent types of molecules is ruptured, a disk-shaped micelle called bicelle can be formed. When both surfactants have negligibly low critical micelle concentration, it is found that bicelles connected with worm-like micelles are also formed depending on the surfactant ratio and spontaneous curvature of the membrane monolayer.

  10. Thrombospondin 1 promotes synaptic formation in bone marrow-derived neuron-like cells.

    PubMed

    Huang, Yun; Lu, Mingnan; Guo, Weitao; Zeng, Rong; Wang, Bin; Wang, Huaibo

    2013-04-01

    In this study, a combination of growth factors was used to induce bone marrow mesenchymal stem cells differentiation into neuron-like cells, in a broader attempt to observe the role of thrombospondin 1 in synapse formation. Results showed that there was no significant difference in the differentiation rate of neuron-like cells between bone marrow mesenchymal stem cells with thrombospondin induction and those without. However, the cell shape was more complex and the neurites were dendritic, with unipolar, bipolar or multipolar morphologies, after induction with thrombospondin 1. The induced cells were similar in morphology to normal neurites. Immunohistochemical staining showed that the number of positive cells for postsynaptic density protein 95 and synaptophysin 1 protein was significantly increased after induction with thrombospondin 1. These findings indicate that thrombospondin 1 promotes synapse formation in neuron-like cells that are differentiated from bone marrow mesenchymal stem cells.

  11. Avidin-biotin interactions at vesicle surfaces: adsorption and binding, cross-bridge formation, and lateral interactions.

    PubMed

    Noppl-Simson, D A; Needham, D

    1996-03-01

    Densely packed domains of membrane proteins are important structures in cellular processes that involve ligand-receptor binding, receptor-mediated adhesion, and macromolecule aggregation. We have used the biotin-avidin interaction at lipid vesicle surfaces to mimic these processes, including the influence of a surface grafted polymer, polyethyleneglycol (PEG). Single vesicles were manipulated by micropipette in solutions of fluorescently labeled avidin to measure the rate and give an estimate of the amount of avidin binding to a biotinylated vesicle as a function of surface biotin concentration and surface-grafted PEG as PEG-lipid. The rate of avidin adsorption was found to be four times less with 2 mol% PEG750 than for the unmodified surface, and 10 mol% PEG completely inhibited binding of avidin to biotin for a 2-min incubation. Using two micropipettes, an avidin-coated vesicle was presented to a biotinylated vesicle. In this vesicle-vesicle adhesion test, the accumulation of avidin in the contact zone was observed, again by using fluorescent avidin. More importantly, by controlling the vesicle membrane tension, this adhesion test provided a direct measure of the spreading pressure of the biotin-avidin-biotin cross-bridges confined in the contact zone. Assuming ideality, this spreading pressure gives the concentration of avidin cross-bridges in the contact zone. The rate of cross-bridge accumulation was consistent with the diffusion of the lipid-linked "receptors" into the contact zone. Once adherent, the membranes failed in tension before they could be peeled apart. PEG750 did not influence the mechanical equilibrium because it was not compressed in the contact zone, but it did perform an important function by eliminating all nonspecific adhesion. This vesicle-vesicle adhesion experiment, with a lower tension limit of 0.01 dyn/cm, now provides a new and useful method with which to measure the spreading pressures and therefore colligative properties of a range of

  12. The central role of heat shock factor 1 in synaptic fidelity and memory consolidation.

    PubMed

    Hooper, Philip L; Durham, Heather D; Török, Zsolt; Hooper, Paul L; Crul, Tim; Vígh, László

    2016-09-01

    Networks of neuronal synapses are the fundamental basis for making and retaining memory. Reduced synapse number and quality correlates with loss of memory in dementia. Heat shock factor 1 (HSF1), the major transcription factor regulating expression of heat shock genes, plays a central role in proteostasis, in establishing and sustaining synaptic fidelity and function, and in memory consolidation. Support for this thesis is based on these observations: (1) heat shock induces improvements in synapse integrity and memory consolidation; (2) synaptic depolarization activates HSF1; (3) activation of HSF1 alone (independent of the canonical heat shock response) augments formation of essential synaptic elements-neuroligands, vesicle transport, synaptic scaffolding proteins, lipid rafts, synaptic spines, and axodendritic synapses; (4) HSF1 coalesces and activates memory receptors in the post-synaptic dendritic spine; (5) huntingtin or α-synuclein accumulation lowers HSF1 while HSF1 lowers huntingtin and α-synuclein aggregation-a potential vicious cycle; and (6) HSF1 agonists (including physical activity) can improve cognitive function in dementia models. Thus, via direct gene expression of synaptic elements, production of HSPs that assure high protein fidelity, and activation of other neuroprotective signaling pathways, HSF1 agonists could provide breakthrough therapy for dementia-associated disease.

  13. The central role of heat shock factor 1 in synaptic fidelity and memory consolidation.

    PubMed

    Hooper, Philip L; Durham, Heather D; Török, Zsolt; Hooper, Paul L; Crul, Tim; Vígh, László

    2016-09-01

    Networks of neuronal synapses are the fundamental basis for making and retaining memory. Reduced synapse number and quality correlates with loss of memory in dementia. Heat shock factor 1 (HSF1), the major transcription factor regulating expression of heat shock genes, plays a central role in proteostasis, in establishing and sustaining synaptic fidelity and function, and in memory consolidation. Support for this thesis is based on these observations: (1) heat shock induces improvements in synapse integrity and memory consolidation; (2) synaptic depolarization activates HSF1; (3) activation of HSF1 alone (independent of the canonical heat shock response) augments formation of essential synaptic elements-neuroligands, vesicle transport, synaptic scaffolding proteins, lipid rafts, synaptic spines, and axodendritic synapses; (4) HSF1 coalesces and activates memory receptors in the post-synaptic dendritic spine; (5) huntingtin or α-synuclein accumulation lowers HSF1 while HSF1 lowers huntingtin and α-synuclein aggregation-a potential vicious cycle; and (6) HSF1 agonists (including physical activity) can improve cognitive function in dementia models. Thus, via direct gene expression of synaptic elements, production of HSPs that assure high protein fidelity, and activation of other neuroprotective signaling pathways, HSF1 agonists could provide breakthrough therapy for dementia-associated disease. PMID:27283588

  14. Membrane Vesicle Formation as a Multiple-Stress Response Mechanism Enhances Pseudomonas putida DOT-T1E Cell Surface Hydrophobicity and Biofilm Formation

    PubMed Central

    Baumgarten, Thomas; Sperling, Stefanie; Seifert, Jana; von Bergen, Martin; Steiniger, Frank; Wick, Lukas Y.

    2012-01-01

    Among the adaptive responses of bacteria to rapid changes in environmental conditions, those of the cell envelope are known to be the most crucial. Therefore, several mechanisms with which bacteria change their cell surface and membranes in the presence of different environmental stresses have been elucidated. Among these mechanisms, the release of outer membrane vesicles (MV) in Gram-negative bacteria has attracted particular research interest because of its involvement in pathogenic processes, such as that of Pseudomonas aeruginosa biofilm formation in cystic fibrosis lungs. In this study, we investigated the role of MV formation as an adaptive response of Pseudomonas putida DOT-T1E to several environmental stress factors and correlated it to the formation of biofilms. In the presence of toxic concentrations of long-chain alcohols, under osmotic stress caused by NaCl, in the presence of EDTA, and after heat shock, cells of this strain released MV within 10 min in the presence of a stressor. The MV formed showed similar size and charge properties, as well as comparable compositions of proteins and fatty acids. MV release caused a significant increase in cell surface hydrophobicity, and an enhanced tendency to form biofilms was demonstrated in this study. Therefore, the release of MV as a stress response could be put in a physiological context. PMID:22752175

  15. Point-to-Plane Nonhomogeneous Electric-Field-Induced Simultaneous Formation of Giant Unilamellar Vesicles (GUVs) and Lipid Tubes.

    PubMed

    Zhu, Chuntao; Zhang, Ying; Wang, Yinan; Li, Qingchuan; Mu, Wei; Han, Xiaojun

    2016-02-24

    It is well-known that homogeneous electric fields can be used to generate giant unilamellar vesicles (GUVs). Herein we report an interesting phenomenon of formation of GUVs and lipid tubes simultaneously using a nonhomogeneous electric field generated by point-to-plane electrodes. The underlying mechanism was analyzed using finite element analysis. The two forces play main roles, that is, the pulling force (F) to drag GUVs into lipid tubes induced by fluid flow, and the critical force (Fc) to prevent GUVs from deforming into lipid tubes induced by electric fields. In the center area underneath the needle electrode, the GUVs were found because F is less than Fc in that region, whereas in the edge area the lipid tubes were obtained because F is larger than Fc. The diffusion coefficient of lipid in the tubes was found to be 4.45 μm(2)  s(-1) using a fluorescence recovery after photobleaching (FRAP) technique. The method demonstrated here is superior to conventional GUV or lipid tube fabrication methods, and has great potential in cell mimic or hollow material fabrication using GUVs and tubes as templates. PMID:26756162

  16. Formation of Polyion Complex (PIC) Micelles and Vesicles with Anionic pH-Responsive Unimer Micelles and Cationic Diblock Copolymers in Water.

    PubMed

    Ohno, Sayaka; Ishihara, Kazuhiko; Yusa, Shin-Ichi

    2016-04-26

    A random copolymer (p(A/MaU)) of sodium 2-(acrylamido)-2-methylpropanesulfonate (AMPS) and sodium 11-methacrylamidoundecanate (MaU) was prepared via conventional radical polymerization, which formed a unimer micelle under acidic conditions due to intramolecular hydrophobic interactions between the pendant undecanoic acid groups. Under basic conditions, unimer micelles were opened up to an expanded chain conformation by electrostatic repulsion between the pendant sulfonate and undecanoate anions. A cationic diblock copolymer (P163M99) consisting of poly(3-(methacrylamido)propyl)trimethylammonium chloride (PMAPTAC) and hydrophilic polybetaine, 2-(methacryloyloxy)ethylphosphorylcholine (MPC), blocks was prepared via controlled radical polymerization. Mixing of p(A/MaU) and P163M99 in 0.1 M aqueous NaCl under acidic conditions resulted in the formation of spherical polyion complex (PIC) micelles and vesicles, depending on polymer concentration before mixing. Shapes of the PIC micelles and vesicles changed under basic conditions due to collapse of the charge balance between p(A/MaU) and P163M99. The PIC vesicles can incorporate nonionic hydrophilic guest molecules, and the PIC micelles and vesicles can accept hydrophobic guest molecules in the hydrophobic core formed from p(A/MaU).

  17. Synaptic Cell Adhesion Molecules in Alzheimer's Disease

    PubMed Central

    Leshchyns'ka, Iryna

    2016-01-01

    Alzheimer's disease (AD) is a neurodegenerative brain disorder associated with the loss of synapses between neurons in the brain. Synaptic cell adhesion molecules are cell surface glycoproteins which are expressed at the synaptic plasma membranes of neurons. These proteins play key roles in formation and maintenance of synapses and regulation of synaptic plasticity. Genetic studies and biochemical analysis of the human brain tissue, cerebrospinal fluid, and sera from AD patients indicate that levels and function of synaptic cell adhesion molecules are affected in AD. Synaptic cell adhesion molecules interact with Aβ, a peptide accumulating in AD brains, which affects their expression and synaptic localization. Synaptic cell adhesion molecules also regulate the production of Aβ via interaction with the key enzymes involved in Aβ formation. Aβ-dependent changes in synaptic adhesion affect the function and integrity of synapses suggesting that alterations in synaptic adhesion play key roles in the disruption of neuronal networks in AD. PMID:27242933

  18. Quercetin Targets Cysteine String Protein (CSPα) and Impairs Synaptic Transmission

    PubMed Central

    Xu, Fenglian; Proft, Juliane; Gibbs, Sarah; Winkfein, Bob; Johnson, Jadah N.; Syed, Naweed; Braun, Janice E. A.

    2010-01-01

    Background Cysteine string protein (CSPα) is a synaptic vesicle protein that displays unique anti-neurodegenerative properties. CSPα is a member of the conserved J protein family, also called the Hsp40 (heat shock protein of 40 kDa) protein family, whose importance in protein folding has been recognized for many years. Deletion of the CSPα in mice results in knockout mice that are normal for the first 2–3 weeks of life followed by an unexplained presynaptic neurodegeneration and premature death. How CSPα prevents neurodegeneration is currently not known. As a neuroprotective synaptic vesicle protein, CSPα represents a promising therapeutic target for the prevention of neurodegenerative disorders. Methodology/Principal Findings Here, we demonstrate that the flavonoid quercetin promotes formation of stable CSPα-CSPα dimers and that quercetin-induced dimerization is dependent on the unique cysteine string region. Furthermore, in primary cultures of Lymnaea neurons, quercetin induction of CSPα dimers correlates with an inhibition of synapse formation and synaptic transmission suggesting that quercetin interfers with CSPα function. Quercetin's action on CSPα is concentration dependent and does not promote dimerization of other synaptic proteins or other J protein family members and reduces the assembly of CSPα:Hsc70 units (70kDa heat shock cognate protein). Conclusions/Significance Quercetin is a plant derived flavonoid and popular nutritional supplement proposed to prevent memory loss and altitude sickness among other ailments, although its precise mechanism(s) of action has been unclear. In view of the therapeutic promise of upregulation of CSPα and the undesired consequences of CSPα dysfunction, our data establish an essential proof of principle that pharmaceutical agents can selectively target the neuroprotective J protein CSPα. PMID:20548785

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

    PubMed

    Schikorski, Thomas

    2014-04-01

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

  20. Gas vesicles.

    PubMed Central

    Walsby, A E

    1994-01-01

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

  1. Avidin-biotin interactions at vesicle surfaces: adsorption and binding, cross-bridge formation, and lateral interactions.

    PubMed Central

    Noppl-Simson, D A; Needham, D

    1996-01-01

    Densely packed domains of membrane proteins are important structures in cellular processes that involve ligand-receptor binding, receptor-mediated adhesion, and macromolecule aggregation. We have used the biotin-avidin interaction at lipid vesicle surfaces to mimic these processes, including the influence of a surface grafted polymer, polyethyleneglycol (PEG). Single vesicles were manipulated by micropipette in solutions of fluorescently labeled avidin to measure the rate and give an estimate of the amount of avidin binding to a biotinylated vesicle as a function of surface biotin concentration and surface-grafted PEG as PEG-lipid. The rate of avidin adsorption was found to be four times less with 2 mol% PEG750 than for the unmodified surface, and 10 mol% PEG completely inhibited binding of avidin to biotin for a 2-min incubation. Using two micropipettes, an avidin-coated vesicle was presented to a biotinylated vesicle. In this vesicle-vesicle adhesion test, the accumulation of avidin in the contact zone was observed, again by using fluorescent avidin. More importantly, by controlling the vesicle membrane tension, this adhesion test provided a direct measure of the spreading pressure of the biotin-avidin-biotin cross-bridges confined in the contact zone. Assuming ideality, this spreading pressure gives the concentration of avidin cross-bridges in the contact zone. The rate of cross-bridge accumulation was consistent with the diffusion of the lipid-linked "receptors" into the contact zone. Once adherent, the membranes failed in tension before they could be peeled apart. PEG750 did not influence the mechanical equilibrium because it was not compressed in the contact zone, but it did perform an important function by eliminating all nonspecific adhesion. This vesicle-vesicle adhesion experiment, with a lower tension limit of 0.01 dyn/cm, now provides a new and useful method with which to measure the spreading pressures and therefore colligative properties of a range of

  2. Pearling of lipid vesicles induced by nanoparticles.

    PubMed

    Yu, Yan; Granick, Steve

    2009-10-14

    We show that cationic nanoparticles encapsulated within vesicles of phosphocholine lipid can induce pearling. The dynamic process occurs as two stages: formation of tubular protrusions followed by pearling instability. The breakup into individual vesicles can be controlled by nanoparticle concentration.

  3. The real catecholamine content of secretory vesicles in the CNS revealed by electrochemical cytometry

    PubMed Central

    Omiatek, Donna M.; Bressler, Amanda J.; Cans, Ann-Sofie; Andrews, Anne M.; Heien, Michael L.; Ewing, Andrew G.

    2013-01-01

    Resolution of synaptic vesicle neurotransmitter content has mostly been limited to the study of stimulated release in cultured cell systems, and it has been controversial as to whether synaptic vesicle transmitter levels are saturated in vivo. We use electrochemical cytometry to count dopamine molecules in individual synaptic vesicles in populations directly sampled from brain tissue. Vesicles from the striatum yield an average of 33,000 dopamine molecules per vesicle, an amount considerably greater than typically measured during quantal release at cultured neurons. Vesicular content was markedly increased by L-DOPA or decreased by reserpine in a time-dependent manner in response to in vivo administration of drugs known to alter dopamine release. We investigated the effects of the psychostimulant amphetamine on vesicle content, finding that vesicular transmitter is rapidly depleted by 50% following in vivo administration, supporting the “weak base hypothesis” that amphetamine reduces synaptic vesicle transmitter and quantal size. PMID:23486177

  4. Podoplanin is a component of extracellular vesicles that reprograms cell-derived exosomal proteins and modulates lymphatic vessel formation

    PubMed Central

    Andrés, Germán; Gopal, Shashi K.; Martín-Villar, Ester; Renart, Jaime; Simpson, Richard J.; Quintanilla, Miguel

    2016-01-01

    Podoplanin (PDPN) is a transmembrane glycoprotein that plays crucial roles in embryonic development, the immune response, and malignant progression. Here, we report that cells ectopically or endogenously expressing PDPN release extracellular vesicles (EVs) that contain PDPN mRNA and protein. PDPN incorporates into membrane shed microvesicles (MVs) and endosomal-derived exosomes (EXOs), where it was found to colocalize with the canonical EV marker CD63 by immunoelectron microscopy. We have previously found that expression of PDPN in MDCK cells induces an epithelial-mesenchymal transition (EMT). Proteomic profiling of MDCK-PDPN cells compared to control cells shows that PDPN-induced EMT is associated with upregulation of oncogenic proteins and diminished expression of tumor suppressors. Proteomic analysis of exosomes reveals that MDCK-PDPN EXOs were enriched in protein cargos involved in cell adhesion, cytoskeletal remodeling, signal transduction and, importantly, intracellular trafficking and EV biogenesis. Indeed, expression of PDPN in MDCK cells stimulated both EXO and MV production, while knockdown of endogenous PDPN in human HN5 squamous carcinoma cells reduced EXO production and inhibited tumorigenesis. EXOs released from MDCK-PDPN and control cells both stimulated in vitro angiogenesis, but only EXOs containing PDPN were shown to promote lymphatic vessel formation. This effect was mediated by PDPN on the surface of EXOs, as demonstrated by a neutralizing specific monoclonal antibody. These results contribute to our understanding of PDPN-induced EMT in association to tumor progression, and suggest an important role for PDPN in EV biogenesis and/or release and for PDPN-EXOs in modulating lymphangiogenesis. PMID:26893367

  5. Morphology transition of raft-model membrane induced by osmotic pressure: Formation of double-layered vesicle similar to an endo- and/or exocytosis

    NASA Astrophysics Data System (ADS)

    Onai, Teruaki; Hirai, Mitsuhiro

    2010-10-01

    The effect of osmotic pressure on the structure of large uni-lamellar vesicle (LUV) of the lipid mixtures of monosialoganglioside (GM1)-cholesterol-dioleoyl-phosphatidylcholine (DOPC) was studies by using wide-angle X-ray scattering (WAXS) method. The molar ratios of the mixtures were 0.1/0.1/1, 0/0.1/1, and 0/0/1. The ternary lipid mixture is a model of lipid rafts. The value of osmotic pressure was varied from 0 to 4.16×105 N/m2 by adding the polyvinylpyrrolidone (PVP) in the range from 0 to 25 % w/v. In the case of the mixtures without GM1, the rise of the osmotic pressure just enhances the multi-lamellar stacking with deceasing the inter-lamellar spacing. On the other hand, the mixture containing GM1 shows the structural transition from a uni-lamellar vesicle to a double-layered vesicle (a liposome including a smaller one inside) by the rise of osmotic pressure. In this morphology transition the total surface area of the double-layered vesicle is mostly as same as that of the LUV at the initial state. The polar head region of GM1 is bulky and highly hydrophilic due to the oligosaccharide chain containing a sialic acid residue. Then, the present results suggest that the existence of GM1 in the outer-leaflet of the LUV is essentially important for such a double-layered vesicle formation. Alternatively, a phenomenon similar to an endo- and/or exocytosis in cells can be caused simply by a variation of osmotic pressure.

  6. A Nonconventional Model of Protocell-like Vesicles: Anionic Clay Surface-Mediated Formation from a Single-Tailed Amphiphile.

    PubMed

    Du, Na; Song, Ruiying; Li, Haiping; Song, Shue; Zhang, Renjie; Hou, Wanguo

    2015-11-24

    We report a novel model system of precursor cellular membranes, self-assembled from micellar solution of a common anionic single-tailed amphiphile (STA), including sodium dodecyl sulfate (SDS) and sodium dodecylbenzenesulfonate (SDBS). The self-assembly process was mediated with solid surfaces of Mg2Al-CO3 hydrotalcite-like compound (HTlc), an anionic clay, in the absence of cosurfactants or any additives. The resultant STA vesicles were characterized using negative-staining and cryogenic transmission electron microscopies, as well as dynamic light scattering and steady state fluorescence techniques. Interestingly, the obtained STA vesicles displayed good stability even after the removal of the anionic clay surface (ACS), and a self-reproduction phenomenon was observed for the "preformed" STA vesicles when mixing with corresponding STA micellar solutions. More importantly, the micelle-to-vesicle transition for SDS could be still arisen in high-salinity artificial seawater under the ACS mediation. Instead of conventional fatty acid scenario, our finding provides another novel possible model for protocell-like vesicles, which are easily formed under the plausible prebiotic conditions. PMID:26524569

  7. Rab11 modulates α-synuclein-mediated defects in synaptic transmission and behaviour

    PubMed Central

    Breda, Carlo; Nugent, Marie L.; Estranero, Jasper G.; Kyriacou, Charalambos P.; Outeiro, Tiago F.; Steinert, Joern R.; Giorgini, Flaviano

    2015-01-01

    A central pathological hallmark of Parkinson's disease (PD) is the presence of proteinaceous depositions known as Lewy bodies, which consist largely of the protein α-synuclein (aSyn). Mutations, multiplications and polymorphisms in the gene encoding aSyn are associated with familial forms of PD and susceptibility to idiopathic PD. Alterations in aSyn impair neuronal vesicle formation/transport, and likely contribute to PD pathogenesis by neuronal dysfunction and degeneration. aSyn is functionally associated with several Rab family GTPases, which perform various roles in vesicle trafficking. Here, we explore the role of the endosomal recycling factor Rab11 in the pathogenesis of PD using Drosophila models of aSyn toxicity. We find that aSyn induces synaptic potentiation at the larval neuromuscular junction by increasing synaptic vesicle (SV) size, and that these alterations are reversed by Rab11 overexpression. Furthermore, Rab11 decreases aSyn aggregation and ameliorates several aSyn-dependent phenotypes in both larvae and adult fruit flies, including locomotor activity, degeneration of dopaminergic neurons and shortened lifespan. This work emphasizes the importance of Rab11 in the modulation of SV size and consequent enhancement of synaptic function. Our results suggest that targeting Rab11 activity could have a therapeutic value in PD. PMID:25305083

  8. Pulling on adhered vesicles

    NASA Astrophysics Data System (ADS)

    Smith, Ana-Suncana; Goennenwein, Stefanie; Lorz, Barbara; Seifert, Udo; Sackmann, Erich

    2004-03-01

    A theoretical model describing pulling of vesicles adhered in a contact potential has been developed. Two different regimes have been recognized. For weak to middle-strength adhesive potentials, locally stable shapes are found in a range of applied forces, separated from the free shape by an energy barrier. The phase diagram contains regions with either a unique bound shape or an additional meta-stable shape. Upon pulling, these shapes unbind discontinuously since the vesicle disengage from the surface while still possessing a finite adhesion area (Smith 2003a). In a strong adhesion regime, a competition between adhesion and tether formation is observed. A critical onset force is identified where a tether spontaneously appears as a part of a second order shape transition. Further growth of a tether is followed by a detachment process which terminates at a finite force when a vesicle continuously unbinds from the substrate (Smith 2003b). Both critical forces, as well as all shape parameters, are calculated as a function of the reduced volume and the strength of adhesive potential. Analogous experimental study has been performed where a vertical magnetic tweezers are used in combination with micro-interferometric and confocal techniques to reproduce the same symmetry as in the theoretical investigation. Giant vesicles are bound to the substrate by numerous specific bonds formed between ligands and receptors incorporated into the vesicle and the substrate, respectively. Application of a constant force is inducing a new thermodynamic equilibrium of the system where the vesicle is partially unbound from the substrate (Goennenwein 2003). The shapes of vesicles are compared prior and during application of the force. Very good agreement is obtained, particularly in the middle-strength adhesion regime (Smith 2003c). References: 1. A.-S. Smith, E. Sackmann, U. Seifert: Effects of a pulling force on the shape of a bound vesicle, Europhys. Lett., 64, 2 (2003). 2. A.-S. Smith

  9. Morphological docking of secretory vesicles

    PubMed Central

    2010-01-01

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

  10. The Arabidopsis Endosomal Sorting Complex Required for Transport III Regulates Internal Vesicle Formation of the Prevacuolar Compartment and Is Required for Plant Development1[C][W][OPEN

    PubMed Central

    Cai, Yi; Zhuang, Xiaohong; Gao, Caiji; Wang, Xiangfeng; Jiang, Liwen

    2014-01-01

    We have established an efficient transient expression system with several vacuolar reporters to study the roles of endosomal sorting complex required for transport (ESCRT)-III subunits in regulating the formation of intraluminal vesicles of prevacuolar compartments (PVCs)/multivesicular bodies (MVBs) in plant cells. By measuring the distributions of reporters on/within the membrane of PVC/MVB or tonoplast, we have identified dominant negative mutants of ESCRT-III subunits that affect membrane protein degradation from both secretory and endocytic pathways. In addition, induced expression of these mutants resulted in reduction in luminal vesicles of PVC/MVB, along with increased detection of membrane-attaching vesicles inside the PVC/MVB. Transgenic Arabidopsis (Arabidopsis thaliana) plants with induced expression of ESCRT-III dominant negative mutants also displayed severe cotyledon developmental defects with reduced cell size, loss of the central vacuole, and abnormal chloroplast development in mesophyll cells, pointing out an essential role of the ESCRT-III complex in postembryonic development in plants. Finally, membrane dissociation of ESCRT-III components is important for their biological functions and is regulated by direct interaction among Vacuolar Protein Sorting-Associated Protein20-1 (VPS20.1), Sucrose Nonfermenting7-1, VPS2.1, and the adenosine triphosphatase VPS4/SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT1. PMID:24812106

  11. Coated vesicles contain a phosphatidylinositol kinase.

    PubMed

    Campbell, C R; Fishman, J B; Fine, R E

    1985-09-15

    When coated vesicles (CVs) are incubated with [gamma-32P]ATP, radioactivity is rapidly incorporated into a compound identified by thin layer chromatography as phosphatidylinositol 4-phosphate. This activity has been identified in CVs isolated from bovine brain as well as from rat liver and chick embryo skeletal muscle. Phosphatidylinositol (PI) kinase is not separated from CVs during agarose electrophoresis, which produces CVs of greater than 95% purity, indicating that the activity present does not derive from contamination. The specific activity of these highly purified CVs was demonstrated to be approximately twice that of synaptic plasma membranes, further ruling out contamination from this source. The PI kinase remains associated with the vesicle upon removal of clathrin and its associated proteins and is solubilized by nonionic detergents, suggesting it is an integral membrane protein. We have been unable to demonstrate the formation of significant amounts of phosphatidylinositol 4,5-bisphosphate in any of our CV preparations. In the presence of exogenous PI, activity is stimulated, with maximal phosphorylation occurring at 0.1 mM. The enzyme appears to be maximally stimulated by 200 mM MgCl2 and 1 mM ATP and is most active at pH 7.25. Calculations indicate that, under optimal conditions, approximately 25 molecules of PIP are produced per CV within 60 s, suggesting that these structures may play an important role in cellular PI metabolism. PMID:2863269

  12. Electrohydrodynamics Of Multicomponent Vesicles

    NASA Astrophysics Data System (ADS)

    Gera, Prerna; Salac, David

    2015-11-01

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

  13. Permeation of bacterial cells, permeation of cytoplasmic and artificial membrane vesicles, and channel formation on lipid bilayers by peptide antibiotic AS-48.

    PubMed Central

    Gálvez, A; Maqueda, M; Martínez-Bueno, M; Valdivia, E

    1991-01-01

    Peptide AS-48 induces ion permeation, which is accompanied by the collapse of the cytoplasmic membrane potential, in sensitive bacteria. Active transport by cytoplasmic membrane vesicles is also impaired by AS-48. At low concentrations, this peptide also causes permeability of liposomes to low-molecular-weight compounds without a requirement for a membrane potential. Higher antibiotic concentrations induce severe disorganization, which is visualized under electron microscopy as aggregation and formation of multilamellar structures. Electrical measurements suggest that AS-48 can form channels in lipid bilayers. Images PMID:1702784

  14. The Formation and Chronology of the PAT 91501 Impact-Melt L-Chondrite with Vesicle-Metal-Sulfide Assemblages

    NASA Technical Reports Server (NTRS)

    Benedix, G. K.; Ketcham, R. A.; Wilson, L.; McCoy, T. J.; Bogard, D. D.; Garrison, D. H.; Herzog, G. F.; Xue, S.; Klein, J.; Middleton, R.

    2007-01-01

    The L chondrite Patuxent Range (PAT) 41 91501 is an 8.5-kg unshocked, homogeneous, igneous-textured impact melt that cooled slowly compared to other meteoritic impact melts in a crater floor melt sheet or sub-crater dike. We conducted mineralogical and tomographic studies of previously unstudied mm- to cm-sized metal-sulfide-vesicle assemblages and chronologic studies of the silicate host. Metal-sulfide clasts constitute about 1 vol.%, comprise zoned taenite, troilite and pentlandite, and exhibit a consistent orientation between metal and sulfide and of metal-sulfide contacts. Vesicles make up approximately 2 vol.% and exhibit a similar orientation of long axes. Ar-39-Ar-40 measurements date the time of impact at 4.461 +/- 0.008 Gyr B.P. Cosmogenic noble gases and Be-10 and Al-2l activities suggest a pre-atmospheric radius of 40-60 cm and a cosmic ray exposure age of 25-29 Myr, similar to ages of a cluster of L chondrites. PAT 91501 dates the oldest known impact on the L chondrite parent body. The dominant vesicle-forming gas was S2 (approximately 15-20 ppm), which formed in equilibrium with impact-melted sulfides. The meteorite formed in an impact melt dike beneath a crater, as did other impact melted L chondrites, such as Chico. Cooling and solidification occurred over approximately 2 hours. During this time, approximately 90% of metal and sulfide segregated from the local melt. Remaining metal and sulfide grains oriented themselves in the local gravitational field, a feature nearly unique among meteorites. Many of these metal sulfide grains adhered to vesicles to form aggregates that may have been close to neutrally buoyant. These aggregates would have been carried upward with the residual melt, inhibiting further buoyancy-driven segregation. Although similar processes operated individually in other chondritic impact melts, their interaction produced the unique assemblage observed in PAT 91501.

  15. Bis(propyl)-cognitin Prevents β-amyloid-induced Memory Deficits as Well as Synaptic Formation and Plasticity Impairments via the Activation of PI3-K Pathway.

    PubMed

    Jiang, Liting; Huang, Meng; Xu, Shujun; Wang, Yu; An, Pengyuan; Feng, Chenxi; Chen, Xiaowei; Wei, Xiaofei; Han, Yifan; Wang, Qinwen

    2016-08-01

    Bis(propyl)-cognitin (B3C), derived from tacrine linked with three methylene (-CH2-) groups, is a dimerized molecule interacting multiple targets. During the past several years, it has been reported as a promising therapeutic drug for Alzheimer's disease (AD) and other neurodegenerative disorders. However, the therapeutic mechanism of B3C for AD needs further demonstration. Based on a combination of behavioral tests, electrophysiological technique, immunocytochemistry, and live cell imaging, we studied the effects and the underlying mechanism of B3C on the impairments of cognitive function, synapse formation, and synaptic plasticity induced by soluble amyloid-β protein (Aβ) oligomers. Our study showed that spatial learning and memory in a Morris water maze task and recognition memory in a novel object recognition task were significantly decreased in the AD model mice created by hippocampal injection of Aβ. Chronic administration of B3C for 21 days prevented the memory impairments of the AD model mice in a dose-dependent manner. Live cell imaging study showed that 2-h pretreatment of B3C prevented the decrease in the number of filopodia and synapses induced by Aβ (0.5 μM) in a dose-dependent manner. Besides, electrophysiological recording data showed that the inhibition of long-term potentiation (LTP) induced by Aβ1-42 oligomers in the dentate gyrus (DG) of hippocampus was prevented by B3C in a dose-dependent manner. Furthermore, we found that the neuroprotective effect of B3C against Aβ-oligomer-induced impairments of synaptic formation and plasticity could be partially blocked by a specific phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 (50 μM). Therefore, these results indicate that B3C can prevent Aβ-oligomer-induced cognitive deficits, synaptic formation impairments, and synaptic plasticity impairments in a concentration-dependent manner. These effects of B3C are partially mediated via the PI3-K pathway. This study provides novel insights

  16. More Docked Vesicles and Larger Active Zones at Basket Cell-to-Granule Cell Synapses in a Rat Model of Temporal Lobe Epilepsy

    PubMed Central

    Yamawaki, Ruth; Thind, Khushdev

    2016-01-01

    Temporal lobe epilepsy is a common and challenging clinical problem, and its pathophysiological mechanisms remain unclear. One possibility is insufficient inhibition in the hippocampal formation where seizures tend to initiate. Normally, hippocampal basket cells provide strong and reliable synaptic inhibition at principal cell somata. In a rat model of temporal lobe epilepsy, basket cell-to-granule cell (BC→GC) synaptic transmission is more likely to fail, but the underlying cause is unknown. At some synapses, probability of release correlates with bouton size, active zone area, and number of docked vesicles. The present study tested the hypothesis that impaired GABAergic transmission at BC→GC synapses is attributable to ultrastructural changes. Boutons making axosomatic symmetric synapses in the granule cell layer were reconstructed from serial electron micrographs. BC→GC boutons were predicted to be smaller in volume, have fewer and smaller active zones, and contain fewer vesicles, including fewer docked vesicles. Results revealed the opposite. Compared with controls, epileptic pilocarpine-treated rats displayed boutons with over twice the average volume, active zone area, total vesicles, and docked vesicles and with more vesicles closer to active zones. Larger active zones in epileptic rats are consistent with previous reports of larger amplitude miniature IPSCs and larger BC→GC quantal size. Results of this study indicate that transmission failures at BC→GC synapses in epileptic pilocarpine-treated rats are not attributable to smaller boutons or fewer docked vesicles. Instead, processes following vesicle docking, including priming, Ca2+ entry, or Ca2+ coupling with exocytosis, might be responsible. SIGNIFICANCE STATEMENT One in 26 people develops epilepsy, and temporal lobe epilepsy is a common form. Up to one-third of patients are resistant to currently available treatments. This study tested a potential underlying mechanism for previously reported

  17. The formation and chronology of the PAT 91501 impact-melt L chondrite with vesicle metal sulfide assemblages

    NASA Astrophysics Data System (ADS)

    Benedix, G. K.; Ketcham, R. A.; Wilson, L.; McCoy, T. J.; Bogard, D. D.; Garrison, D. H.; Herzog, G. F.; Xue, S.; Klein, J.; Middleton, R.

    2008-05-01

    The L chondrite Patuxent Range (PAT) 91501 is an 8.5-kg unshocked, homogeneous, igneous-textured impact melt that cooled slowly compared to other meteoritic impact melts in a crater floor melt sheet or sub-crater dike [Mittlefehldt D. W. and Lindstrom M. M. (2001) Petrology and geochemistry of Patuxent Range 91501 and Lewis Cliff 88663. Meteoritics Planet. Sci. 36, 439-457]. We conducted mineralogical and tomographic studies of previously unstudied mm- to cm-sized metal-sulfide-vesicle assemblages and chronologic studies of the silicate host. Metal-sulfide clasts constitute about 1 vol.%, comprise zoned taenite, troilite, and pentlandite, and exhibit a consistent orientation between metal and sulfide and of metal-sulfide contacts. Vesicles make up ˜2 vol.% and exhibit a similar orientation of long axes. 39Ar- 40Ar measurements probably date the time of impact at 4.461 ± 0.008 Gyr B.P. Cosmogenic noble gases and 10Be and 26Al activities suggest a pre-atmospheric radius of 40-60 cm and a cosmic ray exposure age of 25-29 Myr, similar to ages of a cluster of L chondrites. PAT 91501 dates the oldest known impact on the L chondrite parent body. The dominant vesicle-forming gas was S 2 (˜15-20 ppm), which formed in equilibrium with impact-melted sulfides. The meteorite formed in an impact melt dike beneath a crater, as did other impact melted L chondrites, such as Chico. Cooling and solidification occurred over ˜2 h. During this time, ˜90% of metal and sulfide segregated from the local melt. Remaining metal and sulfide grains oriented themselves in the local gravitational field, a feature nearly unique among meteorites. Many of these metal-sulfide grains adhered to vesicles to form aggregates that may have been close to neutrally buoyant. These aggregates would have been carried upward with the residual melt, inhibiting further buoyancy-driven segregation. Although similar processes operated individually in other chondritic impact melts, their interaction produced

  18. The resilient synapse: insights from genetic interference of synaptic cell adhesion molecules.

    PubMed

    Piechotta, Kerstin; Dudanova, Irina; Missler, Markus

    2006-11-01

    Synaptic cell adhesion molecules (SCAMs) are mostly membrane-anchored molecules with extracellular domains that extend into the synaptic cleft. Prototypical SCAMs interact with homologous or heterologous molecules on the surface of adjacent cells, ensuring the precise apposition of pre- and postsynaptic elements. More recent definitions of SCAMs often include molecules involved in axon pathfinding, cell recognition and synaptic differentiation events, making SCAMs functionally and molecularly a highly diverse group. In this review, we summarize the proposed in vivo functions of a large variety of SCAMs. We mainly focus on results obtained from analyses of genetic model organisms, mostly mouse knockout mutants, lacking expression of the respective candidate genes. In contrast to the substantial effect yielded by some knockouts of molecules involved in synaptic vesicle release, no SCAM mutant has been reported thus far that shows a prominently altered structure of the majority of synapses or even lacks synapses altogether. This surprising resilience of synaptic structure might be explained by a high redundancy between different SCAMs, by the assumption that the crucial molecular players in synapse structure have yet to be discovered or by a grand variability in the mechanisms of synapse formation that underlies the diversity of synapses. Whatever the final answer turns out to be, the genetic dissection of the SCAM superfamilies has led to a much better understanding of the different steps required to form, differentiate and modify a synapse.

  19. Kinetic analysis of mineral formation during in vitro modeling of matrix vesicle mineralization: effect of annexin A5, phosphatidylserine, and type II collagen.

    PubMed

    Genge, Brian R; Wu, Licia N Y; Wuthier, Roy E

    2007-08-15

    Matrix vesicles (MVs) are involved in de novo mineral formation by nearly all vertebrate tissues. The driving force for MV mineralization is a nucleational core composed of three principal constituents: (i) amorphous calcium phosphate (ACP), complexed in part with phosphatidylserine (PS) to form (ii) calcium-phosphate-lipid complexes (CPLX), and (iii) annexin A5 (AnxA5), the principal lipid-dependent Ca(2+)-binding protein in MVs. We describe methods for reconstituting the nucleational core using a biomimetic approach and for analyzing the kinetics of its induction of mineral formation. The method is based on light scattering by the nascent crystallites at 340 nm and monitors mineral formation at regular intervals without disturbing the system using an automated plate reader. It yields precise replicate values that typically agree within less than 5%. As with MVs, mineral formation by the synthetic complex follows a sigmoidal pattern; following a quiescent induction period, rapid formation ensues for a limited time, followed by a distinct decline in rate that continues to slow, ultimately reaching a maximal asymptotic value. Key to quantization of mineral formation is the use of first-derivative analysis, which defines the induction time, the rate and the amount of initial mineral formation. Furthermore, using a five-parameter logistic curve-fitting algorithm, the maximal amount of mineral formation can be predicted accurately. Using these methods, we document the dramatic finding that AnxA5 synergistically activates PS-CPLX, transforming it from a very weak nucleator of mineral formation to a potent one. The methods presented should enable systematic study of the effects of numerous other factors thought to contribute to mineral formation.

  20. A genetic screen for synaptic transmission mutants mapping to the right arm of chromosome 3 in Drosophila.

    PubMed Central

    Babcock, Michael C; Stowers, R Steven; Leither, Jennifer; Goodman, Corey S; Pallanck, Leo J

    2003-01-01

    Neuronal function depends upon the proper formation of synaptic connections and rapid communication at these sites, primarily through the regulated exocytosis of chemical neurotransmitters. Recent biochemical and genomic studies have identified a large number of candidate molecules that may function in these processes. To complement these studies, we are pursuing a genetic approach to identify genes affecting synaptic transmission in the Drosophila visual system. Our screening approach involves a recently described genetic method allowing efficient production of mosaic flies whose eyes are entirely homozygous for a mutagenized chromosome arm. From a screen of 42,500 mutagenized flies, 32 mutations on chromosome 3R that confer synaptic transmission defects in the visual system were recovered. These mutations represent 14 complementation groups, of which at least 9 also appear to perform functional roles outside of the eye. Three of these complementation groups disrupt photoreceptor axonal projection, whereas the remaining complementation groups confer presynaptic defects in synaptic transmission without detectably altering photoreceptor structure. Mapping and complementation testing with candidate mutations revealed new alleles of the neuronal fate determinant svp and the synaptic vesicle trafficking component lap among the collection of mutants recovered in this screen. Given the tools available for investigation of synaptic function in Drosophila, these mutants represent a valuable resource for future analysis of synapse development and function. PMID:14504225

  1. Ultrastructural and functional fate of recycled vesicles in hippocampal synapses.

    PubMed

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

    2015-01-01

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

  2. Relationship between vesicle size and steric hindrance influences vesicle rupture on solid supports.

    PubMed

    Jackman, Joshua A; Kim, Min Chul; Zhdanov, Vladimir P; Cho, Nam-Joon

    2016-01-28

    Phospholipid assemblies on solid supports mimic the cell membrane, and provide a platform to study membrane biology. Among the different types of model membranes, the planar bilayer is a two-dimensional lipid bilayer sheet that can be formed by the adsorption and spontaneous rupture of vesicles. The formation process is influenced by the interactions between vesicles and the solid support as well as between vesicles. On silicon oxide, which is a commonly used solid support, vesicles typically adsorb until reaching a critical coverage and then spontaneous rupture begins. Although it is generally understood that spontaneous rupture leads to planar bilayer formation, oversaturation of vesicles at the critical coverage can hinder the whole process due to a steric factor. To date, the role of this factor has been scrutinized only in relation to temperature, and the influence of additional parameters remains to be elucidated. In this work, we have investigated how vesicle size and corresponding steric constraints influence the kinetics of vesicle adsorption and rupture and, more specifically, how the state of adsorbed vesicles after fusion depends on the vesicle size. Using quartz crystal microbalance-dissipation (QCM-D) and fluorescence recovery after photobleaching (FRAP), we characterized the adsorption kinetics of vesicles onto silicon oxide and the lateral mobility of solid-supported lipid assemblies. While the vesicle adsorption kinetics were diffusion-limited up to the onset of vesicle rupture, the extent of rupture depended on vesicle size and it was observed that larger vesicles are more prone to steric effects than smaller vesicles. We discuss this finding in terms of the structural transformation from adsorbed vesicles to a planar bilayer, including how the interplay of thermodynamic, kinetic and steric factors can affect vesicle rupture on solid supports. PMID:26739602

  3. Relationship between vesicle size and steric hindrance influences vesicle rupture on solid supports.

    PubMed

    Jackman, Joshua A; Kim, Min Chul; Zhdanov, Vladimir P; Cho, Nam-Joon

    2016-01-28

    Phospholipid assemblies on solid supports mimic the cell membrane, and provide a platform to study membrane biology. Among the different types of model membranes, the planar bilayer is a two-dimensional lipid bilayer sheet that can be formed by the adsorption and spontaneous rupture of vesicles. The formation process is influenced by the interactions between vesicles and the solid support as well as between vesicles. On silicon oxide, which is a commonly used solid support, vesicles typically adsorb until reaching a critical coverage and then spontaneous rupture begins. Although it is generally understood that spontaneous rupture leads to planar bilayer formation, oversaturation of vesicles at the critical coverage can hinder the whole process due to a steric factor. To date, the role of this factor has been scrutinized only in relation to temperature, and the influence of additional parameters remains to be elucidated. In this work, we have investigated how vesicle size and corresponding steric constraints influence the kinetics of vesicle adsorption and rupture and, more specifically, how the state of adsorbed vesicles after fusion depends on the vesicle size. Using quartz crystal microbalance-dissipation (QCM-D) and fluorescence recovery after photobleaching (FRAP), we characterized the adsorption kinetics of vesicles onto silicon oxide and the lateral mobility of solid-supported lipid assemblies. While the vesicle adsorption kinetics were diffusion-limited up to the onset of vesicle rupture, the extent of rupture depended on vesicle size and it was observed that larger vesicles are more prone to steric effects than smaller vesicles. We discuss this finding in terms of the structural transformation from adsorbed vesicles to a planar bilayer, including how the interplay of thermodynamic, kinetic and steric factors can affect vesicle rupture on solid supports.

  4. Long-term avoidance memory formation is associated with a transient increase in mushroom body synaptic complexes in leaf-cutting ants

    PubMed Central

    Falibene, Agustina; Roces, Flavio; Rössler, Wolfgang

    2015-01-01

    Long-term behavioral changes related to learning and experience have been shown to be associated with structural remodeling in the brain. Leaf-cutting ants learn to avoid previously preferred plants after they have proved harmful for their symbiotic fungus, a process that involves long-term olfactory memory. We studied the dynamics of brain microarchitectural changes after long-term olfactory memory formation following avoidance learning in Acromyrmex ambiguus. After performing experiments to control for possible neuronal changes related to age and body size, we quantified synaptic complexes (microglomeruli, MG) in olfactory regions of the mushroom bodies (MBs) at different times after learning. Long-term avoidance memory formation was associated with a transient change in MG densities. Two days after learning, MG density was higher than before learning. At days 4 and 15 after learning—when ants still showed plant avoidance—MG densities had decreased to the initial state. The structural reorganization of MG triggered by long-term avoidance memory formation clearly differed from changes promoted by pure exposure to and collection of novel plants with distinct odors. Sensory exposure by the simultaneous collection of several, instead of one, non-harmful plant species resulted in a decrease in MG densities in the olfactory lip. We hypothesize that while sensory exposure leads to MG pruning in the MB olfactory lip, the formation of long-term avoidance memory involves an initial growth of new MG followed by subsequent pruning. PMID:25904854

  5. Neonatal Treatment with a Pegylated Leptin Antagonist Induces Sexually Dimorphic Effects on Neurones and Glial Cells, and on Markers of Synaptic Plasticity in the Developing Rat Hippocampal Formation.

    PubMed

    López-Gallardo, M; Antón-Fernández, A; Llorente, R; Mela, V; Llorente-Berzal, A; Prada, C; Viveros, M P

    2015-08-01

    The present study aimed to better understand the role of the neonatal leptin surge, which peaks on postnatal day (PND)9-10, on the development of the hippocampal formation. Accordingly, male and female rats were administered with a pegylated leptin antagonist on PND9 and the expression of neurones, glial cells and diverse markers of synaptic plasticity was then analysed by immunohistochemistry in the hippocampal formation. Antagonism of the actions of leptin at this specific postnatal stage altered the number of glial fibrillary acidic protein positive cells, and also affected type 1 cannabinoid receptors, synaptophysin and brain-derived neurotrophic factor (BDNF), with the latter effect being sexually dimorphic. The results indicate that the physiological leptin surge occurring around PND 9-10 is critical for hippocampal formation development and that the dynamics of leptin activity might be different in males and females. The data obtained also suggest that some but not all the previously reported effects of maternal deprivation on hippocampal formation development (which markedly reduces leptin levels at PND 9-10) might be mediated by leptin deficiency in these animals.

  6. The Atg1 Kinase Complex Is Involved in the Regulation of Protein Recruitment to Initiate Sequestering Vesicle Formation for Nonspecific Autophagy in Saccharomyces cerevisiae

    PubMed Central

    Cheong, Heesun; Nair, Usha; Geng, Jiefei

    2008-01-01

    Autophagy is the major degradative process for recycling cytoplasmic constituents and eliminating unnecessary organelles in eukaryotic cells. Most autophagy-related (Atg) proteins are recruited to the phagophore assembly site (PAS), a proposed site for vesicle formation during either nonspecific or specific types of autophagy. Therefore, appropriate recruitment of Atg proteins to this site is critical for their function in autophagy. Atg11 facilitates PAS recruitment for the cytoplasm-to-vacuole targeting pathway, which is a specific, autophagy-like process that occurs under vegetative conditions. In contrast, it is not known how Atg proteins are recruited to the PAS, nor which components are involved in PAS formation under nonspecific autophagy-inducing, starvation conditions. Here, we studied PAS assembly during nonspecific autophagy, using an atg11Δ mutant background to eliminate the PAS formation that occurs during vegetative growth. We found that protein complexes containing the Atg1 kinase have two roles for PAS formation during nonspecific autophagy. The Atg1 C terminus mediates an interaction with Atg13 and Atg17, facilitating a structural role of Atg1 that is needed to efficiently organize an initial step of PAS assembly, whereas Atg1 kinase activity affects the dynamics of protein movement at the PAS involved in Atg protein cycling. PMID:18077553

  7. EDITORIAL: Synaptic electronics Synaptic electronics

    NASA Astrophysics Data System (ADS)

    Demming, Anna; Gimzewski, James K.; Vuillaume, Dominique

    2013-09-01

    Conventional computers excel in logic and accurate scientific calculations but make hard work of open ended problems that human brains handle easily. Even von Neumann—the mathematician and polymath who first developed the programming architecture that forms the basis of today's computers—was already looking to the brain for future developments before his death in 1957 [1]. Neuromorphic computing uses approaches that better mimic the working of the human brain. Recent developments in nanotechnology are now providing structures with very accommodating properties for neuromorphic approaches. This special issue, with guest editors James K Gimzewski and Dominique Vuillaume, is devoted to research at the serendipitous interface between the two disciplines. 'Synaptic electronics', looks at artificial devices with connections that demonstrate behaviour similar to synapses in the nervous system allowing a new and more powerful approach to computing. Synapses and connecting neurons respond differently to incident signals depending on the history of signals previously experienced, ultimately leading to short term and long term memory behaviour. The basic characteristics of a synapse can be replicated with around ten simple transistors. However with the human brain having around 1011 neurons and 1015 synapses, artificial neurons and synapses from basic transistors are unlikely to accommodate the scalability required. The discovery of nanoscale elements that function as 'memristors' has provided a key tool for the implementation of synaptic connections [2]. Leon Chua first developed the concept of the 'The memristor—the missing circuit element' in 1971 [3]. In this special issue he presents a tutorial describing how memristor research has fed into our understanding of synaptic behaviour and how they can be applied in information processing [4]. He also describes, 'The new principle of local activity, which uncovers a minuscule life-enabling "Goldilocks zone", dubbed the

  8. EDITORIAL: Synaptic electronics Synaptic electronics

    NASA Astrophysics Data System (ADS)

    Demming, Anna; Gimzewski, James K.; Vuillaume, Dominique

    2013-09-01

    Conventional computers excel in logic and accurate scientific calculations but make hard work of open ended problems that human brains handle easily. Even von Neumann—the mathematician and polymath who first developed the programming architecture that forms the basis of today's computers—was already looking to the brain for future developments before his death in 1957 [1]. Neuromorphic computing uses approaches that better mimic the working of the human brain. Recent developments in nanotechnology are now providing structures with very accommodating properties for neuromorphic approaches. This special issue, with guest editors James K Gimzewski and Dominique Vuillaume, is devoted to research at the serendipitous interface between the two disciplines. 'Synaptic electronics', looks at artificial devices with connections that demonstrate behaviour similar to synapses in the nervous system allowing a new and more powerful approach to computing. Synapses and connecting neurons respond differently to incident signals depending on the history of signals previously experienced, ultimately leading to short term and long term memory behaviour. The basic characteristics of a synapse can be replicated with around ten simple transistors. However with the human brain having around 1011 neurons and 1015 synapses, artificial neurons and synapses from basic transistors are unlikely to accommodate the scalability required. The discovery of nanoscale elements that function as 'memristors' has provided a key tool for the implementation of synaptic connections [2]. Leon Chua first developed the concept of the 'The memristor—the missing circuit element' in 1971 [3]. In this special issue he presents a tutorial describing how memristor research has fed into our understanding of synaptic behaviour and how they can be applied in information processing [4]. He also describes, 'The new principle of local activity, which uncovers a minuscule life-enabling "Goldilocks zone", dubbed the

  9. Imaging synaptic zinc: promises and perils.

    PubMed

    Kay, Alan R

    2006-04-01

    It is well established that some excitatory nerve terminals have high concentrations of Zn(2+) in their synaptic vesicles. For some time, it has been believed that synaptic Zn(2+) is released during neurotransmission and acts as a neuromodulator. Fluorescent Zn(2+) indicators that do not penetrate membranes offer the prospect of rendering the release of Zn(2+) visible. Here, I take a critical look at fluorimetric imaging experiments devised to determine whether Zn(2+) is released and show that they are particularly susceptible to artifacts. Moreover, I will argue that recent experiments suggest that, rather than being released, Zn(2+) is presented to the extracellular space firmly coordinated to presynaptic macromolecules.

  10. γ-Aminobutyric Acid Type A (GABAA) Receptor Subunits Play a Direct Structural Role in Synaptic Contact Formation via Their N-terminal Extracellular Domains*

    PubMed Central

    Brown, Laura E.; Nicholson, Martin W.; Arama, Jessica E.; Thomson, Alex M.

    2016-01-01

    The establishment of cell-cell contacts between presynaptic GABAergic neurons and their postsynaptic targets initiates the process of GABAergic synapse formation. GABAA receptors (GABAARs), the main postsynaptic receptors for GABA, have been recently demonstrated to act as synaptogenic proteins that can single-handedly induce the formation and functional maturation of inhibitory synapses. To establish how the subunit composition of GABAARs influences their ability to induce synaptogenesis, a co-culture model system incorporating GABAergic medium spiny neurons and the HEK293 cells, stably expressing different combinations of receptor subunits, was developed. Analyses of HEK293 cell innervation by medium spiny neuron axons using immunocytochemistry, activity-dependent labeling, and electrophysiology have indicated that the γ2 subunit is required for the formation of active synapses and that its effects are influenced by the type of α/β subunits incorporated into the functional receptor. To further characterize this process, the large N-terminal extracellular domains (ECDs) of α1, α2, β2, and γ2 subunits were purified using the baculovirus/Sf9 cell system. When these proteins were applied to the co-cultures of MSNs and α1/β2/γ2-expressing HEK293 cells, the α1, β2, or γ2 ECD each caused a significant reduction in contact formation, in contrast to the α2 ECD, which had no effect. Together, our experiments indicate that the structural role of GABAARs in synaptic contact formation is determined by their subunit composition, with the N-terminal ECDs of each of the subunits directly participating in interactions between the presynaptic and postsynaptic elements, suggesting the these interactions are multivalent and specific. PMID:27129275

  11. Reduced release probability prevents vesicle depletion and transmission failure at dynamin mutant synapses.

    PubMed

    Lou, Xuelin; Fan, Fan; Messa, Mirko; Raimondi, Andrea; Wu, Yumei; Looger, Loren L; Ferguson, Shawn M; De Camilli, Pietro

    2012-02-21

    Endocytic recycling of synaptic vesicles after exocytosis is critical for nervous system function. At synapses of cultured neurons that lack the two "neuronal" dynamins, dynamin 1 and 3, smaller excitatory postsynaptic currents are observed due to an impairment of the fission reaction of endocytosis that results in an accumulation of arrested clathrin-coated pits and a greatly reduced synaptic vesicle number. Surprisingly, despite a smaller readily releasable vesicle pool and fewer docked vesicles, a strong facilitation, which correlated with lower vesicle release probability, was observed upon action potential stimulation at such synapses. Furthermore, although network activity in mutant cultures was lower, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity was unexpectedly increased, consistent with the previous report of an enhanced state of synapsin 1 phosphorylation at CaMKII-dependent sites in such neurons. These changes were partially reversed by overnight silencing of synaptic activity with tetrodotoxin, a treatment that allows progression of arrested endocytic pits to synaptic vesicles. Facilitation was also counteracted by CaMKII inhibition. These findings reveal a mechanism aimed at preventing synaptic transmission failure due to vesicle depletion when recycling vesicle traffic is backed up by a defect in dynamin-dependent endocytosis and provide new insight into the coupling between endocytosis and exocytosis.

  12. Concentration of carbon dioxide, interstitial pH and synaptic transmission in hippocampal formation of the rat.

    PubMed

    Balestrino, M; Somjen, G G

    1988-02-01

    1. Interstitial pH (pHo) was measured with ion-selective microelectrodes in the fascia dentata of rats anaesthetized with urethane, while CO2 levels were controlled by varying pulmonary ventilation and CO2 content of inspired air. In the CA1 sector of hippocampal tissue slices in vitro pHo was similarly measured and altered by varying CO2 in the gas phase, or by adding HCl or NaOH to the artificial cerebrospinal fluid (ACSF) of the bath, or by changing the concentration of HCO3-. 2. Orthodromically evoked compound action potentials ('population spikes') were depressed in hypercapnia and increased in hypocapnia. In the fascia dentata of intact brains the population spike of the granule cells varied on average by more than 40% of control amplitude for each 0.1 change of pHo. In the CA1 zone of tissue slices in vitro, the change of population spike amplitude was approximately 30% per pH change of 0.1 caused by altered CO2 or HCO3- concentration, but only about 15% per pH change of 0.1 when HCl or NaOH were administered. 3. In anaesthetized rats the focal synaptic potential (FEPSP) evoked by a given stimulus intensity was weakly influenced by varying [CO2]; in tissue slices weak effects on FEPSP were inconsistent. In hippocampus both in situ and in vitro the population spike triggered by a given magnitude of FEPSP increased in hypocapnia and decreased in hypercapnia. This suggests that the main effect of CO2 is on the electric excitability of postsynaptic cells, with minor or no effect on transmitter release and on the interaction of the transmitter with its receptors. 4. Hypercapnia of anaesthetized rats was usually associated with a slight increase of [K+]o in the fascia dentata. Tissue [Ca2+]o changed little and not consistently. Neither of these two ions, nor concomitant changes of blood pressure or tissue partial pressure of oxygen, (Pt, O2), could account for the effects of pH on neuronal excitability. 5. The results show that increasing the extracellular

  13. Regulation of Synaptic Extracellular Matrix Composition Is Critical for Proper Synapse Morphology

    PubMed Central

    Kurshan, Peri T.; Phan, Allan Q.; Wang, George J.; Crane, Matthew M.; Lu, Hang

    2014-01-01

    Synapses are surrounded by a layer of extracellular matrix (ECM), which is instrumental for their development and maintenance. ECM composition is dynamically controlled by proteases, but how the precise composition of the ECM affects synaptic morphology is largely unknown. Through an unbiased forward genetic screen, we found that Caenorhabditis elegans gon-1, a conserved extracellular ADAMTS protease, is required for maintaining proper synaptic morphology at the neuromuscular junction. In gon-1 mutants, once synapse formation is complete, motor neuron presynaptic varicosities develop into large bulbous protrusions that contain synaptic vesicles and active zone proteins. A concomitant overgrowth of postsynaptic muscle membrane is found in close apposition to presynaptic axonal protrusions. Mutations in the muscle-specific, actin-severing protein cofilin (unc-60) suppress the axon phenotype, suggesting that muscle outgrowth is necessary for presynaptic protrusions. gon-1 mutants can also be suppressed by loss of the ECM components collagen IV (EMB-9) and fibulin (FBL-1). We propose that GON-1 regulates a developmental switch out of an initial “pro-growth” phase during which muscle arms grow out and form synapses with motor neuron axons. We postulate that this switch involves degradation or reorganization of collagen IV (EMB-9), whereas FBL-1 opposes GON-1 by stabilizing EMB-9. Our results describe a mechanism for regulating synaptic ECM composition and reveal the importance of precise ECM composition for neuronal morphology and synapse integrity. PMID:25232106

  14. Synaptic AMPA Receptor Plasticity and Behavior

    PubMed Central

    Kessels, Helmut W.; Malinow, Roberto

    2014-01-01

    The ability to change behavior likely depends on the selective strengthening and weakening of brain synapses. The cellular models of synaptic plasticity, long-term potentiation (LTP) and depression (LTD) of synaptic strength, can be expressed by the synaptic insertion or removal of AMPA receptors (AMPARs), respectively. We here present an overview of studies that have used animal models to show that such AMPAR trafficking underlies several experience-driven phenomena—from neuronal circuit formation to the modification of behavior. We argue that monitoring and manipulating synaptic AMPAR trafficking represents an attractive means to study cognitive function and dysfunction in animal models. PMID:19217372

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

    Suckow, Arthur T.; Craige, Branch; Faundez, Victor; Cain, William J.

    2010-01-01

    Pancreatic islet β-cells contain synaptic-like microvesicles (SLMVs). The origin, trafficking, and role of these SLMVs are poorly understood. In neurons, synaptic vesicle (SV) biogenesis is mediated by two different cytosolic adaptor protein complexes, a ubiquitous AP-2 complex and the neuron-specific AP-3B complex. Mice lacking AP-3B subunits exhibit impaired GABAergic (inhibitory) neurotransmission and reduced neuronal vesicular GABA transporter (VGAT) content. Since β-cell maturation and exocytotic function seem to parallel that of the inhibitory synapse, we predicted that AP-3B-associated vesicles would be present in β-cells. Here, we test the hypothesis that AP-3B is expressed in islets and mediates β-cell SLMV biogenesis. A secondary aim was to test whether the sedimentation properties of INS-1 β-cell microvesicles are identical to those of bona fide SLMVs isolated from PC12 cells. Our results show that the two neuron-specific AP-3 subunits β3B and μ3B are expressed in β-cells, the first time these proteins have been found to be expressed outside the nervous system. We found that β-cell SLMVs share the same sedimentation properties as PC12 SLMVs and contain SV proteins that sort specifically to AP-3B-associated vesicles in the brain. Brefeldin A, a drug that interferes with AP-3-mediated SV biogenesis, inhibits the delivery of AP-3 cargoes to β-cell SLMVs. Consistent with a role for AP-3 in the biogenesis of GABAergic SLMV in β-cells, INS-1 cell VGAT content decreases upon inhibition of AP-3 δ-subunit expression. Our findings suggest that β-cells and neurons share molecules and mechanisms important for mediating the neuron-specific membrane trafficking pathways that underlie synaptic vesicle formation. PMID:20442321

  20. Extracellular-vesicle type of volume transmission and tunnelling-nanotube type of wiring transmission add a new dimension to brain neuro-glial networks.

    PubMed

    Agnati, Luigi F; Fuxe, Kjell

    2014-09-26

    Two major types of intercellular communication are found in the central nervous system (CNS), namely wiring transmission (WT; point-to-point communication via private channels, e.g. synaptic transmission) and volume transmission (VT; communication in the extracellular fluid and in the cerebrospinal fluid). Volume and synaptic transmission become integrated because their chemical signals activate different types of interacting receptors in heteroreceptor complexes located synaptically and extrasynaptically in the plasma membrane. In VT, we focus on the role of the extracellular-vesicle type of VT, and in WT, on the potential role of the tunnelling-nanotube (TNT) type of WT. The so-called exosomes appear to be the major vesicular carrier for intercellular communication but the larger microvesicles also participate. Extracellular vesicles are released from cultured cortical neurons and different types of glial cells and modulate the signalling of the neuronal-glial networks of the CNS. This type of VT has pathological relevance, and epigenetic mechanisms may participate in the modulation of extracellular-vesicle-mediated VT. Gerdes and co-workers proposed the existence of a novel type of WT based on TNTs, which are straight transcellular channels leading to the formation in vitro of syncytial cellular networks found also in neuronal and glial cultures. PMID:25135966

  1. Nanoplasmonic ruler to measure lipid vesicle deformation.

    PubMed

    Jackman, Joshua A; Špačková, Barbora; Linardy, Eric; Kim, Min Chul; Yoon, Bo Kyeong; Homola, Jiří; Cho, Nam-Joon

    2016-01-01

    A nanoplasmonic ruler method is presented in order to measure the deformation of adsorbed, nm-scale lipid vesicles on solid supports. It is demonstrated that single adsorbed vesicles undergo greater deformation on silicon oxide over titanium oxide, offering direct experimental evidence to support membrane tension-based theoretical models of supported lipid bilayer formation.

  2. Synaptic Activity Regulates the Abundance and Binding of Complexin

    PubMed Central

    Wragg, Rachel T.; Gouzer, Géraldine; Bai, Jihong; Arianna, Gianluca; Ryan, Timothy A.; Dittman, Jeremy S.

    2015-01-01

    Nervous system function relies on precise chemical communication between neurons at specialized junctions known as synapses. Complexin (CPX) is one of a small number of cytoplasmic proteins that are indispensable in controlling neurotransmitter release through SNARE and synaptic vesicle interactions. However, the mechanisms that recruit and stabilize CPX are poorly understood. The mobility of CPX tagged with photoactivatable green fluorescent protein (pGFP) was quantified in vivo using Caenorhabditis elegans. Although pGFP escaped the synapse within seconds, CPX-pGFP displayed both fast and slow decay components, requiring minutes for complete exchange of the synaptic pool. The longer synaptic residence time of CPX arose from both synaptic vesicle and SNARE interactions, and surprisingly, CPX mobility depended on synaptic activity. Moreover, mouse CPX-GFP reversibly dispersed out of hippocampal presynaptic terminals during stimulation, and blockade of vesicle fusion prevented CPX dispersion. Hence, synaptic CPX can rapidly redistribute and this exchange is influenced by neuronal activity, potentially contributing to use-dependent plasticity. PMID:25809246

  3. Mutations in domain I interhelical loops affect the rate of pore formation by the Bacillus thuringiensis Cry1Aa toxin in insect midgut brush border membrane vesicles.

    PubMed

    Lebel, Geneviève; Vachon, Vincent; Préfontaine, Gabrielle; Girard, Frédéric; Masson, Luke; Juteau, Marc; Bah, Aliou; Larouche, Geneviève; Vincent, Charles; Laprade, Raynald; Schwartz, Jean-Louis

    2009-06-01

    Pore formation in the apical membrane of the midgut epithelial cells of susceptible insects constitutes a key step in the mode of action of Bacillus thuringiensis insecticidal toxins. In order to study the mechanism of toxin insertion into the membrane, at least one residue in each of the pore-forming-domain (domain I) interhelical loops of Cry1Aa was replaced individually by cysteine, an amino acid which is normally absent from the activated Cry1Aa toxin, using site-directed mutagenesis. The toxicity of most mutants to Manduca sexta neonate larvae was comparable to that of Cry1Aa. The ability of each of the activated mutant toxins to permeabilize M. sexta midgut brush border membrane vesicles was examined with an osmotic swelling assay. Following a 1-h preincubation, all mutants except the V150C mutant were able to form pores at pH 7.5, although the W182C mutant had a weaker activity than the other toxins. Increasing the pH to 10.5, a procedure which introduces a negative charge on the thiol group of the cysteine residues, caused a significant reduction in the pore-forming abilities of most mutants without affecting those of Cry1Aa or the I88C, T122C, Y153C, or S252C mutant. The rate of pore formation was significantly lower for the F50C, Q151C, Y153C, W182C, and S252C mutants than for Cry1Aa at pH 7.5. At the higher pH, all mutants formed pores significantly more slowly than Cry1Aa, except the I88C mutant, which formed pores significantly faster, and the T122C mutant. These results indicate that domain I interhelical loop residues play an important role in the conformational changes leading to toxin insertion and pore formation.

  4. Vesicle recycling at ribbon synapses in the finely branched axon terminals of mouse retinal bipolar neurons

    PubMed Central

    LoGiudice, Lisamarie; Sterling, Peter; Matthews, Gary

    2009-01-01

    In retinal bipolar neurons, synaptic ribbons mark the presence of exocytotic active zones in the synaptic terminal. It is unknown, however, where compensatory vesicle retrieval is localized in this cell type and by what mechanism(s) excess membrane is recaptured. To determine whether endocytosis is localized or diffuse in mouse bipolar neurons, we imaged FM4-64 to track vesicles in cells whose synaptic ribbons were tagged with a fluorescent peptide. In synaptic terminals, vesicle retrieval occurred at discrete sites that were spatially consistent over multiple stimuli, indicative of endocytotic “hot spots.” Retrieval sites were spatially correlated with fluorescently labeled synaptic ribbons. EM analysis of bipolar cell terminals after photoconversion of internalized FM dye revealed that almost all of the dye was contained within vesicles ~30 nm in diameter. Clathrin-coated vesicles were observed budding from the plasma membrane and within the cytosol, and application of dynasore, a dynamin inhibitor, arrested membrane retrieval just after the budding stage. We conclude that synaptic vesicles in the fine branches of mouse bipolar axon terminals are retrieved locally near active zones, at least in part via a clathrin-mediated pathway. PMID:19778591

  5. IL-1 receptor accessory protein-like 1 associated with mental retardation and autism mediates synapse formation by trans-synaptic interaction with protein tyrosine phosphatase δ.

    PubMed

    Yoshida, Tomoyuki; Yasumura, Misato; Uemura, Takeshi; Lee, Sung-Jin; Ra, Moonjin; Taguchi, Ryo; Iwakura, Yoichiro; Mishina, Masayoshi

    2011-09-21

    Mental retardation (MR) and autism are highly heterogeneous neurodevelopmental disorders. IL-1-receptor accessory protein-like 1 (IL1RAPL1) is responsible for nonsyndromic MR and is associated with autism. Thus, the elucidation of the functional role of IL1RAPL1 will contribute to our understanding of the pathogenesis of these mental disorders. Here, we showed that knockdown of endogenous IL1RAPL1 in cultured cortical neurons suppressed the accumulation of punctate staining signals for active zone protein Bassoon and decreased the number of dendritic protrusions. Consistently, the expression of IL1RAPL1 in cultured neurons stimulated the accumulation of Bassoon and spinogenesis. The extracellular domain (ECD) of IL1RAPL1 was required and sufficient for the presynaptic differentiation-inducing activity, while both the ECD and cytoplasmic domain were essential for the spinogenic activity. Notably, the synaptogenic activity of IL1RAPL1 was specific for excitatory synapses. Furthermore, we identified presynaptic protein tyrosine phosphatase (PTP) δ as a major IL1RAPL1-ECD interacting protein by affinity chromatography. IL1RAPL1 interacted selectively with certain forms of PTPδ splice variants carrying mini-exon peptides in Ig-like domains. The synaptogenic activity of IL1RAPL1 was abolished in primary neurons from PTPδ knock-out mice. IL1RAPL1 showed robust synaptogenic activity in vivo when transfected into the cortical neurons of wild-type mice but not in PTPδ knock-out mice. These results suggest that IL1RAPL1 mediates synapse formation through trans-synaptic interaction with PTPδ. Our findings raise an intriguing possibility that the impairment of synapse formation may underlie certain forms of MR and autism as a common pathogenic pathway shared by these mental disorders.

  6. ADAR-mediated RNA editing suppresses sleep by acting as a brake on glutamatergic synaptic plasticity

    PubMed Central

    Robinson, J. E.; Paluch, J.; Dickman, D. K.; Joiner, W. J.

    2016-01-01

    It has been postulated that synaptic potentiation during waking is offset by a homoeostatic reduction in net synaptic strength during sleep. However, molecular mechanisms to support such a process are lacking. Here we demonstrate that deficiencies in the RNA-editing gene Adar increase sleep due to synaptic dysfunction in glutamatergic neurons in Drosophila. Specifically, the vesicular glutamate transporter is upregulated, leading to over-activation of NMDA receptors, and the reserve pool of glutamatergic synaptic vesicles is selectively expanded in Adar mutants. Collectively these changes lead to sustained neurotransmitter release under conditions that would otherwise result in synaptic depression. We propose that a shift in the balance from synaptic depression towards synaptic potentiation in sleep-promoting neurons underlies the increased sleep pressure of Adar-deficient animals. Our findings provide a plausible molecular mechanism linking sleep and synaptic plasticity. PMID:26813350

  7. ADAR-mediated RNA editing suppresses sleep by acting as a brake on glutamatergic synaptic plasticity.

    PubMed

    Robinson, J E; Paluch, J; Dickman, D K; Joiner, W J

    2016-01-01

    It has been postulated that synaptic potentiation during waking is offset by a homoeostatic reduction in net synaptic strength during sleep. However, molecular mechanisms to support such a process are lacking. Here we demonstrate that deficiencies in the RNA-editing gene Adar increase sleep due to synaptic dysfunction in glutamatergic neurons in Drosophila. Specifically, the vesicular glutamate transporter is upregulated, leading to over-activation of NMDA receptors, and the reserve pool of glutamatergic synaptic vesicles is selectively expanded in Adar mutants. Collectively these changes lead to sustained neurotransmitter release under conditions that would otherwise result in synaptic depression. We propose that a shift in the balance from synaptic depression towards synaptic potentiation in sleep-promoting neurons underlies the increased sleep pressure of Adar-deficient animals. Our findings provide a plausible molecular mechanism linking sleep and synaptic plasticity.

  8. Fungicidal effect of piscidin on Candida albicans: pore formation in lipid vesicles and activity in fungal membranes.

    PubMed

    Sung, Woo Sang; Lee, Juneyoung; Lee, Dong Gun

    2008-10-01

    Piscidin, a 22-residue cationic peptide isolated from the mast cells of hybrid striped bass, has potent antimicrobial activities. In the present study, we investigated the fungicidal activity and mode of action of piscidins. Fungicidal and hemolytic assays were examined in order to assess their potency and toxicity, respectively. The results showed that fungicidal and hemolytic activities were higher for piscidin 1 (P1) than piscidin 3 (P3). Additionally, the abilities to permeabilize the model phospholipids membranes were also higher for P1 than P3, which were consistent with the biological activities of P1 and P3. These results suggest that the biological action of the peptides may be carried out through the lipid membrane. To understand the fungicidal properties of P1, we focused on a membrane-active mechanism of the peptide by in vivo testing against Candida albicans as the model organism. Flow cytometric analysis by using bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC(4)(3)] and protoplast regeneration experiments showed that P1 caused fungal membrane damage. Furthermore, fluorescence analysis, using 1,6-diphenyl-1,3,5-hexatriene, revealed that these peptides created pores in fungal membranes. Thus, the present study demonstrated that piscidins exert their fungicidal effects by disrupting fungal membrane through pore formation.

  9. Transformation of oil droplets into giant vesicles.

    PubMed

    Sheng, Li; Kurihara, Kensuke

    2016-06-14

    We propose a protocell model in which compartments are constructed via a new process involving the formation of robust vesicles using an autocatalytic, self-reproducing oil droplet system as a 'scaffold'. PMID:27152371

  10. Extracellular vesicles: Exosomes, microvesicles, and friends

    PubMed Central

    Stoorvogel, Willem

    2013-01-01

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

  11. Primary hippocampal neurons, which lack four crucial extracellular matrix molecules, display abnormalities of synaptic structure and function and severe deficits in perineuronal net formation.

    PubMed

    Geissler, Maren; Gottschling, Christine; Aguado, Ainhara; Rauch, Uwe; Wetzel, Christian H; Hatt, Hanns; Faissner, Andreas

    2013-05-01

    The extracellular matrix (ECM) of the brain plays crucial roles during the development, maturation, and regeneration of the CNS. In a subpopulation of neurons, the ECM condenses to superstructures called perineuronal nets (PNNs) that surround synapses. Camillo Golgi described PNNs a century ago, yet their biological functions remain elusive. Here, we studied a mouse mutant that lacks four ECM components highly enriched in the developing brain: the glycoproteins tenascin-C and tenascin-R and the chondroitin sulfate proteoglycans brevican and neurocan. Primary embryonic hippocampal neurons and astrocytes were cultivated using a cell insert system that allows for co-culture of distinct cell populations in the absence of direct membrane contacts. The wild-type and knock-out cells were combined in the four possible permutations. Using this approach, neurons cultivated in the presence of mutant astrocytes displayed a transient increase of synapses after 2 weeks. However, after a period of 3 weeks or longer, synapse formation and stabilization were compromised when either neuron or astrocyte cell populations or both were of mutant origin. The development of PNN structures was observed, but their size was substantially reduced on knock-out neurons. The synaptic activity of both wild-type and knock-out neurons was monitored using whole-cell patch clamping. The salient observation was a reduced frequency of IPSCs and EPSCs, whereas the amplitudes were not modified. Remarkably, the knock-out neuron phenotypes could not be rescued by wild-type astrocytes. We conclude that the elimination of four ECM genes compromises neuronal function.

  12. Engineered Asymmetric Synthetic Vesicles

    NASA Astrophysics Data System (ADS)

    Lu, Li; Chiarot, Paul

    2013-11-01

    Synthetic vesicles are small, fluid-filled spheres that are enclosed by a bilayer of lipid molecules. They can be used as models for investigating membrane biology and as delivery vehicles for pharmaceuticals. In practice, it is difficult to simultaneously control membrane asymmetry, unilamellarity, vesicle size, vesicle-to-vesicle uniformity, and luminal content. Membrane asymmetry, where each leaflet of the bilayer is composed of different lipids, is of particular importance as it is a feature of most natural membranes. In this study, we leverage microfluidic technology to build asymmetric vesicles at high-throughput. We use the precise flow control offered by microfluidic devices to make highly uniform emulsions, with controlled internal content, that serve as templates to build the synthetic vesicles. Flow focusing, dielectrophoretic steering, and interfacial lipid self-assembly are critical procedures performed on-chip to produce the vesicles. Fluorescent and confocal microscopy are used to evaluate the vesicle characteristics.

  13. Caenorhabditis elegans rab-3 mutant synapses exhibit impaired function and are partially depleted of vesicles.

    PubMed

    Nonet, M L; Staunton, J E; Kilgard, M P; Fergestad, T; Hartwieg, E; Horvitz, H R; Jorgensen, E M; Meyer, B J

    1997-11-01

    Rab molecules regulate vesicular trafficking in many different exocytic and endocytic transport pathways in eukaryotic cells. In neurons, rab3 has been proposed to play a crucial role in regulating synaptic vesicle release. To elucidate the role of rab3 in synaptic transmission, we isolated and characterized Caenorhabditis elegans rab-3 mutants. Similar to the mouse rab3A mutants, these mutants survived and exhibited only mild behavioral abnormalities. In contrast to the mouse mutants, synaptic transmission was perturbed in these animals. Extracellular electrophysiological recordings revealed that synaptic transmission in the pharyngeal nervous system was impaired. Furthermore, rab-3 animals were resistant to the acetylcholinesterase inhibitor aldicarb, suggesting that cholinergic transmission was generally depressed. Last, synaptic vesicle populations were redistributed in rab-3 mutants. In motor neurons, vesicle populations at synapses were depleted to 40% of normal levels, whereas in intersynaptic regions of the axon, vesicle populations were elevated. On the basis of the morphological defects at neuromuscular junctions, we postulate that RAB-3 may regulate recruitment of vesicles to the active zone or sequestration of vesicles near release sites.

  14. Use-dependent inhibition of synaptic transmission by the secretion of intravesicularly accumulated antipsychotic drugs.

    PubMed

    Tischbirek, Carsten H; Wenzel, Eva M; Zheng, Fang; Huth, Tobias; Amato, Davide; Trapp, Stefan; Denker, Annette; Welzel, Oliver; Lueke, Katharina; Svetlitchny, Alexei; Rauh, Manfred; Deusser, Janina; Schwab, Annemarie; Rizzoli, Silvio O; Henkel, Andreas W; Müller, Christian P; Alzheimer, Christian; Kornhuber, Johannes; Groemer, Teja W

    2012-06-01

    Antipsychotic drugs are effective for the treatment of schizophrenia. However, the functional consequences and subcellular sites of their accumulation in nervous tissue have remained elusive. Here, we investigated the role of the weak-base antipsychotics haloperidol, chlorpromazine, clozapine, and risperidone in synaptic vesicle recycling. Using multiple live-cell microscopic approaches and electron microscopy of rat hippocampal neurons as well as in vivo microdialysis experiments in chronically treated rats, we demonstrate the accumulation of the antipsychotic drugs in synaptic vesicles and their release upon neuronal activity, leading to a significant increase in extracellular drug concentrations. The secreted drugs exerted an autoinhibitory effect on vesicular exocytosis, which was promoted by the inhibition of voltage-gated sodium channels and depended on the stimulation intensity. Taken together, these results indicate that accumulated antipsychotic drugs recycle with synaptic vesicles and have a use-dependent, autoinhibitory effect on synaptic transmission. PMID:22681688

  15. Bassoon speeds vesicle reloading at a central excitatory synapse.

    PubMed

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

    2010-11-18

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

  16. UNC-31/CAPS docks and primes dense core vesicles in C. elegans neurons.

    PubMed

    Lin, Xian-Guang; Ming, Min; Chen, Mao-Rong; Niu, Wei-Pin; Zhang, Yong-Deng; Liu, Bei; Jiu, Ya-Ming; Yu, Jun-Wei; Xu, Tao; Wu, Zheng-Xing

    2010-07-01

    UNC-31 or its mammalian homologue, Ca(2+)-dependent activator protein for secretion (CAPS), is indispensable for exocytosis of dense core vesicle (DCV) and synaptic vesicle (SV). From N- to the C-terminus, UNC-31 contains putative functional domains, including dynactin 1 binding domain (DBD), C2, PH, (M)UNC-13 homology domain (MHD) and DCV binding domain (DCVBD), the last four we examined in this study. We employed UNC-31 null mutant C. elegans worms to examine whether UNC-31 functions could be rescued by ectopic expression of full length UNC-31 vs each of these four domain-deleted mutants. Full length UNC-31 cDNA rescued the phenotypes of C. elegans null mutants in response to Ca(2+)-elevation in ALA neurons. Surprisingly, MHD deletion also rescued UNC-31 exocytotic function in part because the relatively high Ca(2+) level (pre-flash Ca(2+) was 450 nM) used in the capacitance study could bypass the MHD defect. Nonetheless, the three other domain-truncation cDNAs had almost no rescue on Ca(2+) evoked secretion. Importantly, this genetic null mutant rescue strategy enabled physiological studies at levels of whole organism to single cells, such as locomotion assay, pharmacological study of neurotransmission at neuromuscular junction, in vivo neuropeptide release measurement and analysis of vesicular docking. Our results suggest that each of these UNC-31 domains support distinct sequential molecular actions of UNC-31 in vesicular exocytosis, including steps in vesicle tethering and docking that bridge vesicle with plasma membrane, and subsequently priming vesicle by initiating the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) core complex. PMID:20515653

  17. Proteomics of extracellular vesicles: Exosomes and ectosomes.

    PubMed

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

    2015-01-01

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

  18. A trans-synaptic nanocolumn aligns neurotransmitter release to receptors.

    PubMed

    Tang, Ai-Hui; Chen, Haiwen; Li, Tuo P; Metzbower, Sarah R; MacGillavry, Harold D; Blanpied, Thomas A

    2016-08-11

    Synaptic transmission is maintained by a delicate, sub-synaptic molecular architecture, and even mild alterations in synapse structure drive functional changes during experience-dependent plasticity and pathological disorders. Key to this architecture is how the distribution of presynaptic vesicle fusion sites corresponds to the position of receptors in the postsynaptic density. However, while it has long been recognized that this spatial relationship modulates synaptic strength, it has not been precisely described, owing in part to the limited resolution of light microscopy. Using localization microscopy, here we show that key proteins mediating vesicle priming and fusion are mutually co-enriched within nanometre-scale subregions of the presynaptic active zone. Through development of a new method to map vesicle fusion positions within single synapses in cultured rat hippocampal neurons, we find that action-potential-evoked fusion is guided by this protein gradient and occurs preferentially in confined areas with higher local density of Rab3-interacting molecule (RIM) within the active zones. These presynaptic RIM nanoclusters closely align with concentrated postsynaptic receptors and scaffolding proteins, suggesting the existence of a trans-synaptic molecular 'nanocolumn'. Thus, we propose that the nanoarchitecture of the active zone directs action-potential-evoked vesicle fusion to occur preferentially at sites directly opposing postsynaptic receptor-scaffold ensembles. Remarkably, NMDA receptor activation triggered distinct phases of plasticity in which postsynaptic reorganization was followed by trans-synaptic nanoscale realignment. This architecture suggests a simple organizational principle of central nervous system synapses to maintain and modulate synaptic efficiency. PMID:27462810

  19. Imaging synaptic density in the living human brain.

    PubMed

    Finnema, Sjoerd J; Nabulsi, Nabeel B; Eid, Tore; Detyniecki, Kamil; Lin, Shu-Fei; Chen, Ming-Kai; Dhaher, Roni; Matuskey, David; Baum, Evan; Holden, Daniel; Spencer, Dennis D; Mercier, Joël; Hannestad, Jonas; Huang, Yiyun; Carson, Richard E

    2016-07-20

    Chemical synapses are the predominant neuron-to-neuron contact in the central nervous system. Presynaptic boutons of neurons contain hundreds of vesicles filled with neurotransmitters, the diffusible signaling chemicals. Changes in the number of synapses are associated with numerous brain disorders, including Alzheimer's disease and epilepsy. However, all current approaches for measuring synaptic density in humans require brain tissue from autopsy or surgical resection. We report the use of the synaptic vesicle glycoprotein 2A (SV2A) radioligand [(11)C]UCB-J combined with positron emission tomography (PET) to quantify synaptic density in the living human brain. Validation studies in a baboon confirmed that SV2A is an alternative synaptic density marker to synaptophysin. First-in-human PET studies demonstrated that [(11)C]UCB-J had excellent imaging properties. Finally, we confirmed that PET imaging of SV2A was sensitive to synaptic loss in patients with temporal lobe epilepsy. Thus, [(11)C]UCB-J PET imaging is a promising approach for in vivo quantification of synaptic density with several potential applications in diagnosis and therapeutic monitoring of neurological and psychiatric disorders. PMID:27440727

  20. Functional regions of the presynaptic cytomatrix protein bassoon: significance for synaptic targeting and cytomatrix anchoring.

    PubMed

    Dresbach, Thomas; Hempelmann, Anne; Spilker, Christina; tom Dieck, Susanne; Altrock, Wilko D; Zuschratter, Werner; Garner, Craig C; Gundelfinger, Eckart D

    2003-06-01

    Exocytosis of neurotransmitter from synaptic vesicles is restricted to specialized sites of the presynaptic plasma membrane called active zones. A complex cytomatrix of proteins exclusively assembled at active zones, the CAZ, is thought to form a molecular scaffold that organizes neurotransmitter release sites. Here, we have analyzed synaptic targeting and cytomatrix association of Bassoon, a major scaffolding protein of the CAZ. By combining immunocytochemistry and transfection of cultured hippocampal neurons, we show that the central portion of Bassoon is crucially involved in synaptic targeting and CAZ association. An N-terminal region harbors a distinct capacity for N-myristoylation-dependent targeting to synaptic vesicle clusters, but is not incorporated into the CAZ. Our data provide the first experimental evidence for the existence of distinct functional regions in Bassoon and suggest that a centrally located CAZ targeting function may be complemented by an N-terminal capacity for targeting to membrane-bounded synaptic organelles.

  1. Voluntary Running Depreciates the Requirement of Ca[superscript 2+]-Stimulated cAMP Signaling in Synaptic Potentiation and Memory Formation

    ERIC Educational Resources Information Center

    Zheng, Fei; Zhang, Ming; Ding, Qi; Sethna, Ferzin; Yan, Lily; Moon, Changjong; Yang, Miyoung; Wang, Hongbing

    2016-01-01

    Mental health and cognitive functions are influenced by both genetic and environmental factors. Although having active lifestyle with physical exercise improves learning and memory, how it interacts with the specific key molecular regulators of synaptic plasticity is largely unknown. Here, we examined the effects of voluntary running on long-term…

  2. Expression of VAMP-2-like protein in kidney collecting duct intracellular vesicles. Colocalization with Aquaporin-2 water channels.

    PubMed Central

    Nielsen, S; Marples, D; Birn, H; Mohtashami, M; Dalby, N O; Trimble, M; Knepper, M

    1995-01-01

    Body water balance is controlled by vasopressin, which regulates Aquaporin-2 (AQP2) water channels in kidney collecting duct cells by vesicular trafficking between intracellular vesicles and the plasma membrane. To examine the molecular apparatus involved in vesicle trafficking and vasopressin regulation of AQP2 in collecting duct cells, we tested if targeting proteins expressed in the synaptic vesicles, namely vesicle-associated membrane proteins 1 and 2 (VAMP1 and 2), are expressed in kidney collecting duct. Immunoblotting revealed specific labeling of VAMP2 (18-kD band) but not VAMP1 in membrane fractions prepared from kidney inner medulla. Controls using preadsorbed antibody or preimmune serum were negative. Bands of identical molecular size were detected in immunoblots of brain membrane vesicles and purified synaptic vesicles. VAMP2 in kidney membranes was cleaved by tetanus toxin, revealing a tetanus toxin-sensitive VAMP homologue. Similarly, tetanus toxin cleaved VAMP2 in synaptic vesicles. In kidney inner medulla, VAMP2 was predominantly expressed in the membrane fraction enriched for intracellular vesicles, with little or no VAMP2 in the plasma membrane enriched fraction. This was confirmed by immunocytochemistry using semithin cryosections, which showed mainly vesicular labeling in collecting duct principal cells, with no labeling of intercalated cells. VAMP2 immunolabeling colocalized with AQP2 labeling in intracellular vesicles, as determined by immunoelectron microscopy after double immunolabeling of isolated vesicles. Quantitative analysis of 1,310 vesicles revealed a highly significant association of both AQP2 and VAMP2 in the same vesicles (P < 0.0001). Furthermore, the presence of AQP2 in vesicles immunoisolated with anti-VAMP2 antibodies was confirmed by immunoblotting. In conclusion, VAMP2, a component of the neuronal SNARE complex, is expressed in vesicles carrying AQP2, suggesting a role in vasopressin-regulated vesicle trafficking of AQP2

  3. Miniature Neurotransmission Regulates Drosophila Synaptic Structural Maturation

    PubMed Central

    Choi, Ben Jiwon; Imlach, Wendy L.; Jiao, Wei; Wolfram, Verena; Wu, Ying; Grbic, Mark; Cela, Carolina; Baines, Richard A.; Nitabach, Michael N.; McCabe, Brian D.

    2014-01-01

    Summary Miniature neurotransmission is the transsynaptic process where single synaptic vesicles spontaneously released from presynaptic neurons induce miniature postsynaptic potentials. Since their discovery over 60 years ago, miniature events have been found at every chemical synapse studied. However, the in vivo necessity for these small-amplitude events has remained enigmatic. Here, we show that miniature neurotransmission is required for the normal structural maturation of Drosophila glutamatergic synapses in a developmental role that is not shared by evoked neurotransmission. Conversely, we find that increasing miniature events is sufficient to induce synaptic terminal growth. We show that miniature neurotransmission acts locally at terminals to regulate synapse maturation via a Trio guanine nucleotide exchange factor (GEF) and Rac1 GTPase molecular signaling pathway. Our results establish that miniature neurotransmission, a universal but often-overlooked feature of synapses, has unique and essential functions in vivo. PMID:24811381

  4. Docking of Secretory Vesicles Is Syntaxin Dependent

    PubMed Central

    de Wit, Heidi; Cornelisse, L. Niels; Toonen, Ruud F.G.; Verhage, Matthijs

    2006-01-01

    Secretory vesicles dock at the plasma membrane before they undergo fusion. Molecular docking mechanisms are poorly defined but believed to be independent of SNARE proteins. Here, we challenged this hypothesis by acute deletion of the target SNARE, syntaxin, in vertebrate neurons and neuroendocrine cells. Deletion resulted in fusion arrest in both systems. No docking defects were observed in synapses, in line with previous observations. However, a drastic reduction in morphologically docked secretory vesicles was observed in chromaffin cells. Syntaxin-deficient chromaffin cells showed a small reduction in total and plasma membrane staining for the docking factor Munc18-1, which appears insufficient to explain the drastic reduction in docking. The sub-membrane cortical actin network was unaffected by syntaxin deletion. These observations expose a docking role for syntaxin in the neuroendocrine system. Additional layers of regulation may have evolved to make syntaxin redundant for docking in highly specialized systems like synaptic active zones. PMID:17205130

  5. Protein composition of immunoprecipitated synaptic ribbons

    PubMed Central

    Kantardzhieva, A.; Peppi, M.; Lane, W.S.; Sewell, W.F.

    2012-01-01

    The synaptic ribbon is an electron-dense structure found in hair cells and photoreceptors. The ribbon is surrounded by neurotransmitter-filled vesicles and considered to play a role in vesicle release. We generated an objective, quantitative analysis of the protein composition of the ribbon complex using a mass spectrometry-based proteomics analysis. Our use of affinity-purified ribbons and control IgG immunoprecipitations ensure that the identified proteins are indeed associated with the ribbon complex. The use of mouse tissue, where the proteome is complete, generated a comprehensive analysis of the candidates. We identified 30 proteins (comprising 56 isoforms and subunits) associated with the ribbon complex. The ribbon complex primarily comprises proteins found in conventional synapses, which we categorized into 6 functional groups: vesicle handling (38.5%), scaffold (7.3%), cytoskeletal molecules (20.6%), phosphorylation enzymes (10.6%), molecular chaperones (8.2%), and transmembrane proteins from the presynaptic membrane firmly attached to the ribbon (11.3%). The 3 CtBP isoforms represent the major protein in the ribbon whether calculated by molar amount (30%) or by mass (20%). The relatively high quantity of phosphorylation enzymes suggests a very active and regulated structure. The ribbon appears to comprise a concentrated cluster of proteins dealing with vesicle creation, retention and distribution, and consequent exocytosis. PMID:22103298

  6. Nanotubes from gelly vesicles

    NASA Astrophysics Data System (ADS)

    Kremer, S.; Campillo, C.; Pepin-Donat, B.; Viallat, A.; Brochard-Wyart, F.

    2008-05-01

    Hydrodynamic extrusions of tethers from giant unilamellar vesicles (GUV) enclosing a poly-N-isopropylacrylamide (polyNIPAM) gel are studied. The collapse of the gel upon heating induces a deswelling of the GUV, showing that the membrane is linked to the polymer network. The gelly vesicle is attached to a micro-rod and submitted to a flow (velocity U). Above a threshold velocity (U>Uc) a tether is extruded and reaches a stationary length L∞simeτ0U in a characteristic time τ0. The vesicle behaves like an entropic spring with a tether length L∞ proportional to the Stokes friction force. Compared to viscous "sol" vesicles, gelly vesicle are much stiffer: L∞ and τ0 being hundred times smaller. We conclude that the mobility of lipids is reduced, only a small portion of the vesicle area being free to flow into the tube.

  7. Activity-Dependent Calpain Activation Plays a Critical Role in Synaptic Facilitation and Post-Tetanic Potentiation

    ERIC Educational Resources Information Center

    Khoutorsky, Arkady; Spira, Micha E.

    2009-01-01

    Synaptic facilitation and post-tetanic potentiation (PTP) are believed to necessitate active regeneration of the release machinery and supply of synaptic vesicles to a ready-releasable site. The prevailing hypothesis assumes that synapsins play pivotal roles in these processes. Using a cholinergic synapse formed between cultured "Aplysia" neurons…

  8. Optical Manipulation of Vesicles for Optofluidic Applications

    SciTech Connect

    Vasdekis, Andreas E.; Scott, E. A.; O'Neil, C. P.; Psaltis, D.; Hubbell, J. A.

    2013-09-12

    In this report, we review our recent results in the optical micromanipulation of vesicles. Traditionally, vesicle manipulation has been possible by employing photon momentum and optical trapping, giving rise to unique observations of vesicle shape changes and soft matter mechanics. Contrary to these attempts, we employ photon energy rather than momentum, by sensitizing vesicles with an oxidizing moiety. The later converts incident photons to reactive oxygen species, which in turn attack and compromise the stability of the vesicle membrane. Both coherent and incoherent radiation was employed. Polymersome re-organization into smaller diameter vesicles was possible by focusing the excitation beam in the vicinity of the polymersomes. Extended vesicle illumination with a collimated beam lead to their complete destabilization and micelle formation. Single particle analysis revealed that payload release takes place within seconds of illumination in an explosive burst. We will discuss the destabilization and payload release kinetics, as revealed by high resolution microscopy at the single particle level, as well as potential applications in single cell biomodulation.

  9. Optical manipulation of vesicles for optofluidic applications

    NASA Astrophysics Data System (ADS)

    Vasdekis, A. E.; Scott, E. A.; O'Neil, C. P.; Psaltis, D.; Hubbell, J. A.

    2013-09-01

    In this report, we review our recent results in the optical micromanipulation of vesicles. Traditionally, vesicle manipulation has been possible by employing photon momentum and optical trapping, giving rise to unique observations of vesicle shape changes and soft matter mechanics. Contrary to these attempts, we employ photon energy rather than momentum, by sensitizing vesicles with an oxidizing moiety. The later converts incident photons to reactive oxygen species, which in turn attack and compromise the stability of the vesicle membrane. Both coherent and incoherent radiation was employed. Polymersome re-organization into smaller diameter vesicles was possible by focusing the excitation beam in the vicinity of the polymersomes. Extended vesicle illumination with a collimated beam lead to their complete destabilization and micelle formation. Single particle analysis revealed that payload release takes place within seconds of illumination in an explosive burst. We will discuss the destabilization and payload release kinetics, as revealed by high resolution microscopy at the single particle level, as well as potential applications in single cell biomodulation.

  10. Molecular mechanisms of synaptic remodeling in alcoholism.

    PubMed

    Kyzar, Evan J; Pandey, Subhash C

    2015-08-01

    Alcohol use and alcohol addiction represent dysfunctional brain circuits resulting from neuroadaptive changes during protracted alcohol exposure and its withdrawal. Alcohol exerts a potent effect on synaptic plasticity and dendritic spine formation in specific brain regions, providing a neuroanatomical substrate for the pathophysiology of alcoholism. Epigenetics has recently emerged as a critical regulator of gene expression and synaptic plasticity-related events in the brain. Alcohol exposure and withdrawal induce changes in crucial epigenetic processes in the emotional brain circuitry (amygdala) that may be relevant to the negative affective state defined as the "dark side" of addiction. Here, we review the literature concerning synaptic plasticity and epigenetics, with a particular focus on molecular events related to dendritic remodeling during alcohol abuse and alcoholism. Targeting epigenetic processes that modulate synaptic plasticity may yield novel treatments for alcoholism.

  11. Memory Formation Shaped by Astroglia

    PubMed Central

    Zorec, Robert; Horvat, Anemari; Vardjan, Nina; Verkhratsky, Alexei

    2015-01-01

    Astrocytes, the most heterogeneous glial cells in the central nervous system (CNS), execute a multitude of homeostatic functions and contribute to memory formation. Consolidation of synaptic and systemic memory is a prolonged process and hours are required to form long-term memory. In the past, neurons or their parts have been considered to be the exclusive cellular sites of these processes, however, it has now become evident that astrocytes provide an important and essential contribution to memory formation. Astrocytes participate in the morphological remodeling associated with synaptic plasticity, an energy-demanding process that requires mobilization of glycogen, which, in the CNS, is almost exclusively stored in astrocytes. Synaptic remodeling also involves bidirectional astroglial-neuronal communication supported by astroglial receptors and release of gliosignaling molecules. Astroglia exhibit cytoplasmic excitability that engages second messengers, such as Ca2+, for phasic, and cyclic adenosine monophosphate (cAMP), for tonic signal coordination with neuronal processes. The detection of signals by astrocytes and the release of gliosignaling molecules, in particular by vesicle-based mechanisms, occurs with a significant delay after stimulation, orders of magnitude longer than that present in stimulus–secretion coupling in neurons. These particular arrangements position astrocytes as integrators ideally tuned to support time-dependent memory formation. PMID:26635551

  12. Structural alterations in lecithin-cholesterol vesicles following interactions with monomeric and micellar bile salts: physical-chemical basis for subselection of biliary lecithin species and aggregative states of biliary lipids during bile formation.

    PubMed

    Cohen, D E; Angelico, M; Carey, M C

    1990-01-01

    Using complementary physical-chemical methods including turbidimetry, quasielastic light scattering, gel filtration, and phase analysis, we examined the interactions between dilute concentrations of the common bile salt, taurochenodeoxycholate (TCDC), and uni- and multilamellar vesicles (MLVs) composed of defined molecular species of lecithin (L) and varying contents of cholesterol (Ch). Dissolution rates of MLVs with micellar TCDC, as assessed by turbidimetry, were more rapid with vesicles composed of sn-1 palmitoyl species, typical of biliary L, compared with those composed of the more hydrophobic sn-1 stearoyl species. Incorporation of Ch retarded MLV dissolution rates in proportion to the Ch content, and only at high Ch contents were dissolution rates appreciably influenced by the sn-2 fatty acid composition of L. When MLVs contained Ch in amounts characteristic of intracellular membranes (Ch/L approximately 0.1), the dissolution rates of the individual L species by TCDC accurately predicted the steady state L composition of human bile. TCDC interacted with small unilamellar L/Ch vesicles (SUVs) at concentrations well below, as well as appreciably above, its critical micellar concentration. In accordance with the TCDC-egg yolk L-H2O phase diagram, perimicellar concentrations of TCDC interacted with SUVs to form aggregates that were approximately twice the size of the SUVs. These were consistent with the formation of a dispersed hexagonal (rod-like) phase, which co-existed with aqueous bile salt (BS) monomers and either micellar or unilamellar SUV phases. Micellar TCDC completely solubilized SUVs as mixed micelles, putatively via this transient hexagonal phase. With modest Ch-supersaturation, dissolution was followed by the reemergence of a new vesicle population that coexisted metastably with mixed micelles. With high Ch supersaturation, TCDC extracted L and Ch molecules from SUVs in different proportions to form Ch-supersaturated mixed micelles and Ch

  13. Active endocannabinoids are secreted on extracellular membrane vesicles.

    PubMed

    Gabrielli, Martina; Battista, Natalia; Riganti, Loredana; Prada, Ilaria; Antonucci, Flavia; Cantone, Laura; Matteoli, Michela; Maccarrone, Mauro; Verderio, Claudia

    2015-02-01

    Endocannabinoids primarily influence neuronal synaptic communication within the nervous system. To exert their function, endocannabinoids need to travel across the intercellular space. However, how hydrophobic endocannabinoids cross cell membranes and move extracellularly remains an unresolved problem. Here, we show that endocannabinoids are secreted through extracellular membrane vesicles produced by microglial cells. We demonstrate that microglial extracellular vesicles carry on their surface N-arachidonoylethanolamine (AEA), which is able to stimulate type-1 cannabinoid receptors (CB1), and inhibit presynaptic transmission, in target GABAergic neurons. This is the first demonstration of a functional role of extracellular vesicular transport of endocannabinoids.

  14. Structure suggests function: the case for synaptic ribbons as exocytotic nanomachines.

    PubMed

    Lenzi, D; von Gersdorff, H

    2001-09-01

    Synaptic ribbons, the organelles identified in electron micrographs of the sensory synapses involved in vision, hearing, and balance, have long been hypothesized to play an important role in regulating presynaptic function because they associate with synaptic vesicles at the active zone. Their physiology and molecular composition have, however, remained largely unknown. Recently, a series of elegant studies spurred by technical innovation have finally begun to shed light on the ultrastructure and function of ribbon synapses. Electrical capacitance measurements have provided sub-millisecond resolution of exocytosis, evanescent-wave microscopy has filmed the fusion of single 30 nm synaptic vesicles, electron tomography has revealed the 3D architecture of the synapse, and molecular cloning has begun to identify the proteins that make up ribbons. These results are consistent with the ribbon serving as a vesicle "conveyor belt" to resupply the active zone, and with the suggestion that ribbon and conventional chemical synapses have much in common.

  15. Temporal separation of vesicle release from vesicle fusion during exocytosis.

    PubMed

    Troyer, Kevin P; Wightman, R Mark

    2002-08-01

    During exocytosis, vesicles in secretory cells fuse with the cellular membrane and release their contents in a Ca2+-dependent process. Release occurs initially through a fusion pore, and its rate is limited by the dissociation of the matrix-associated contents. To determine whether this dissociation is promoted by osmotic forces, we have examined the effects of elevated osmotic pressure on release and extrusion from vesicles at mast and chromaffin cells. The identity of the molecules released and the time course of extrusion were measured with fast scan cyclic voltammetry at carbon fiber microelectrodes. In external solutions of high osmolarity, release events following entry of divalent ions (Ba2+ or Ca2+) were less frequent. However, the vesicles appeared to be fused to the membrane without extruding their contents, since the maximal observed concentrations of events were less than 7% of those evoked in isotonic media. Such an isolated, intermediate fusion state, which we term "kiss-and-hold," was confirmed by immunohistochemistry at chromaffin cells. Transient exposure of cells in the kiss and hold state to isotonic solutions evoked massive release. These results demonstrate that an osmotic gradient across the fusion pore is an important driving force for exocytotic extrusion of granule contents from secretory cells following fusion pore formation. PMID:12034731

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

    SciTech Connect

    Stenovec, Matjaz Kreft, Marko Grilc, Sonja Potokar, Maja Kreft, Mateja Erdani Pangrsic, Tina Zorec, Robert

    2007-11-01

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

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

    PubMed Central

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

    2015-01-01

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

  18. Spontaneous formation of bilayers and vesicles in mixtures of single-chain alkyl carboxylates: effect of pH and aging and cytotoxicity studies.

    PubMed

    Vlachy, N; Merle, C; Touraud, D; Schmidt, J; Talmon, Y; Heilmann, J; Kunz, W

    2008-09-16

    We report the observation of bilayer fragments, some of which close to form vesicles, over a large range of pH at room temperature from mixtures of single-chain biocompatible commercially available nontoxic alkyl carboxylic surfactants after neutralization with HCl. The pH at which the morphological transitions occur is varied only by changing the ratio between two surfactants: the alkyloligoethyleneoxide carboxylate and sodium laurate. The effect of aging of the mixed surfactant systems in the pH region desired for dermatologic application (4.5 < pH < 7) is also studied. Finally, we show results of cytotoxicity studies on the surfactant mixtures.

  19. Association of Endophilin B1 with Cytoplasmic Vesicles.

    PubMed

    Li, Jinhui; Barylko, Barbara; Eichorst, John P; Mueller, Joachim D; Albanesi, Joseph P; Chen, Yan

    2016-08-01

    Endophilins are SH3- and BAR domain-containing proteins implicated in membrane remodeling and vesicle formation. Endophilins A1 and A2 promote the budding of endocytic vesicles from the plasma membrane, whereas endophilin B1 has been implicated in vesicle budding from intracellular organelles, including the trans-Golgi network and late endosomes. We previously reported that endophilins A1 and A2 exist almost exclusively as soluble dimers in the cytosol. Here, we present results of fluorescence fluctuation spectroscopy analyses indicating that, in contrast, the majority of endophilin B1 is present in multiple copies on small, highly mobile cytoplasmic vesicles. Formation of these vesicles was enhanced by overexpression of wild-type dynamin 2, but suppressed by expression of a catalytically inactive dynamin 2 mutant. Using dual-color heterospecies partition analysis, we identified the epidermal growth factor receptor on endophilin B1 vesicles. Moreover, a proportion of endophilin B1 vesicles also contained caveolin, whereas clathrin was almost undetectable on those vesicles. These results raise the possibility that endophilin B1 participates in dynamin 2-dependent formation of a population of transport vesicles distinct from those generated by A-type endophilins. PMID:27508440

  20. A synaptic mechanism for temporal filtering of visual signals.

    PubMed

    Baden, Tom; Nikolaev, Anton; Esposti, Federico; Dreosti, Elena; Odermatt, Benjamin; Lagnado, Leon

    2014-10-01

    The visual system transmits information about fast and slow changes in light intensity through separate neural pathways. We used in vivo imaging to investigate how bipolar cells transmit these signals to the inner retina. We found that the volume of the synaptic terminal is an intrinsic property that contributes to different temporal filters. Individual cells transmit through multiple terminals varying in size, but smaller terminals generate faster and larger calcium transients to trigger vesicle release with higher initial gain, followed by more profound adaptation. Smaller terminals transmitted higher stimulus frequencies more effectively. Modeling global calcium dynamics triggering vesicle release indicated that variations in the volume of presynaptic compartments contribute directly to all these differences in response dynamics. These results indicate how one neuron can transmit different temporal components in the visual signal through synaptic terminals of varying geometries with different adaptational properties. PMID:25333637

  1. A Synaptic Mechanism for Temporal Filtering of Visual Signals

    PubMed Central

    Esposti, Federico; Dreosti, Elena; Odermatt, Benjamin; Lagnado, Leon

    2014-01-01

    The visual system transmits information about fast and slow changes in light intensity through separate neural pathways. We used in vivo imaging to investigate how bipolar cells transmit these signals to the inner retina. We found that the volume of the synaptic terminal is an intrinsic property that contributes to different temporal filters. Individual cells transmit through multiple terminals varying in size, but smaller terminals generate faster and larger calcium transients to trigger vesicle release with higher initial gain, followed by more profound adaptation. Smaller terminals transmitted higher stimulus frequencies more effectively. Modeling global calcium dynamics triggering vesicle release indicated that variations in the volume of presynaptic compartments contribute directly to all these differences in response dynamics. These results indicate how one neuron can transmit different temporal components in the visual signal through synaptic terminals of varying geometries with different adaptational properties. PMID:25333637

  2. Analysis of Synaptic Gene Expression in the Neocortex of Primates Reveals Evolutionary Changes in Glutamatergic Neurotransmission

    PubMed Central

    Muntané, Gerard; Horvath, Julie E.; Hof, Patrick R.; Ely, John J.; Hopkins, William D.; Raghanti, Mary Ann; Lewandowski, Albert H.; Wray, Gregory A.; Sherwood, Chet C.

    2015-01-01

    Increased relative brain size characterizes the evolution of primates, suggesting that enhanced cognition plays an important part in the behavioral adaptations of this mammalian order. In addition to changes in brain anatomy, cognition can also be regulated by molecular changes that alter synaptic function, but little is known about modifications of synapses in primate brain evolution. The aim of the current study was to investigate the expression patterns and evolution of 20 synaptic genes from the prefrontal cortex of 12 primate species. The genes investigated included glutamate receptors, scaffolding proteins, synaptic vesicle components, as well as factors involved in synaptic vesicle release and structural components of the nervous system. Our analyses revealed that there have been significant changes during primate brain evolution in the components of the glutamatergic signaling pathway in terms of gene expression, protein expression, and promoter sequence changes. These results could entail functional modifications in the regulation of specific genes related to processes underlying learning and memory. PMID:24408959

  3. Analysis of synaptic gene expression in the neocortex of primates reveals evolutionary changes in glutamatergic neurotransmission.

    PubMed

    Muntané, Gerard; Horvath, Julie E; Hof, Patrick R; Ely, John J; Hopkins, William D; Raghanti, Mary Ann; Lewandowski, Albert H; Wray, Gregory A; Sherwood, Chet C

    2015-06-01

    Increased relative brain size characterizes the evolution of primates, suggesting that enhanced cognition plays an important part in the behavioral adaptations of this mammalian order. In addition to changes in brain anatomy, cognition can also be regulated by molecular changes that alter synaptic function, but little is known about modifications of synapses in primate brain evolution. The aim of the current study was to investigate the expression patterns and evolution of 20 synaptic genes from the prefrontal cortex of 12 primate species. The genes investigated included glutamate receptors, scaffolding proteins, synaptic vesicle components, as well as factors involved in synaptic vesicle release and structural components of the nervous system. Our analyses revealed that there have been significant changes during primate brain evolution in the components of the glutamatergic signaling pathway in terms of gene expression, protein expression, and promoter sequence changes. These results could entail functional modifications in the regulation of specific genes related to processes underlying learning and memory. PMID:24408959

  4. DGKθ Catalytic Activity is Required for Efficient Recycling of Presynaptic Vesicles at Excitatory Synapses

    PubMed Central

    Goldschmidt, Hana L.; Tu-Sekine, Becky; Volk, Lenora; Anggono, Victor; Huganir, Richard L.; Raben, Daniel M.

    2015-01-01

    Summary Synaptic transmission relies on coordinated coupling of synaptic vesicle (SV) exocytosis and endocytosis. While much attention has focused on characterizing proteins involved in SV recycling, the roles of membrane lipids and their metabolism remain poorly understood. Diacylglycerol, a major signaling lipid produced at synapses during synaptic transmission, is regulated by diacylglycerol kinase (DGK). Here we report a role for DGKθ in the mammalian central nervous system in facilitating recycling of presynaptic vesicles at excitatory synapses. Using synaptophysin- and vGlut1-pHluorin optical reporters, we found that acute and chronic deletion of DGKθ attenuated the recovery of SVs following neuronal stimulation. Rescue of recycling kinetics required DGKθ kinase activity. Our data establish a role for DGK catalytic activity and its byproduct, phosphatidic acid, at the presynaptic nerve terminal in SV recycling. Together these data suggest DGKθ supports synaptic transmission during periods of elevated neuronal activity. PMID:26748701

  5. Polypeptide vesicles with densely packed multilayer membranes.

    PubMed

    Song, Ziyuan; Kim, Hojun; Ba, Xiaochu; Baumgartner, Ryan; Lee, Jung Seok; Tang, Haoyu; Leal, Cecilia; Cheng, Jianjun

    2015-05-28

    Multilamellar membranes are important building blocks for constructing self-assembled structures with improved barrier properties, such as multilamellar lipid vesicles. Polymeric vesicles (polymersomes) have attracted growing interest, but multilamellar polymersomes are much less explored. Here, we report the formation of polypeptide vesicles with unprecedented densely packed multilayer membrane structures with poly(ethylene glycol)-block-poly(γ-(4,5-dimethoxy-2-nitrobenzyl)-l-glutamate) (PEG-b-PL), an amphiphilic diblock rod-coil copolymer containing a short PEG block and a short hydrophobic rod-like polypeptide segment. The polypeptide rods undergo smectic ordering with PEG buried between the hydrophobic polypeptide layers. The size of both blocks and the rigidity of the hydrophobic polypeptide block are critical in determining the membrane structures. Increase of the PEG length in PEG-b-PL results in the formation of bilayer sheets, while using random-coil polypeptide block leads to the formation of large compound micelles. UV treatment causes ester bond cleavage of the polypeptide side chain, which induces helix-to-coil transition, change of copolymer amphiphilicity, and eventual disassembly of vesicles. These polypeptide vesicles with unique membrane structures provide a new insight into self-assembly structure control by precisely tuning the composition and conformation of polymeric amphiphiles.

  6. Unitary assembly of presynaptic active zones from Piccolo-Bassoon transport vesicles.

    PubMed

    Shapira, Mika; Zhai, R Grace; Dresbach, Thomas; Bresler, Tal; Torres, Viviana I; Gundelfinger, Eckart D; Ziv, Noam E; Garner, Craig C

    2003-04-24

    Recent studies indicate that active zones (AZs)-sites of neurotransmitter release-may be assembled from preassembled AZ precursor vesicles inserted into the presynaptic plasma membrane. Here we report that one putative AZ precursor vesicle of CNS synapses-the Piccolo-Bassoon transport vesicle (PTV)-carries a comprehensive set of AZ proteins genetically and functionally coupled to synaptic vesicle exocytosis. Time-lapse imaging reveals that PTVs are highly mobile, consistent with a role in intracellular transport. Quantitative analysis reveals that the Bassoon, Piccolo, and RIM content of individual PTVs is, on average, half of that of individual presynaptic boutons and shows that the synaptic content of these molecules can be quantitatively accounted for by incorporation of integer numbers (typically two to three) of PTVs into presynaptic membranes. These findings suggest that AZs are assembled from unitary amounts of AZ material carried on PTVs.

  7. The cell biology of synaptic specificity during development

    PubMed Central

    Christensen, Ryan; Shao, Zhiyong; Colón-Ramos, Daniel A

    2013-01-01

    Proper circuit connectivity is critical for nervous system function. Connectivity derives from the interaction of two interdependent modules: synaptic specificity and synaptic assembly. Specificity involves both targeting of neurons to specific laminar regions and the formation of synapses onto defined subcellular areas. In this review, we focus discussion on recently elucidated molecular mechanisms that control synaptic specificity and link them to synapse assembly. We use these molecular pathways to underscore fundamental cell biological concepts that underpin, and help explain, the rules governing synaptic specificity. PMID:23932598

  8. The Arabidopsis P4-ATPase ALA3 localizes to the golgi and requires a beta-subunit to function in lipid translocation and secretory vesicle formation.

    PubMed

    Poulsen, Lisbeth Rosager; López-Marqués, Rosa Laura; McDowell, Stephen C; Okkeri, Juha; Licht, Dirk; Schulz, Alexander; Pomorski, Thomas; Harper, Jeffrey F; Palmgren, Michael Gjedde

    2008-03-01

    Vesicle budding in eukaryotes depends on the activity of lipid translocases (P(4)-ATPases) that have been implicated in generating lipid asymmetry between the two leaflets of the membrane and in inducing membrane curvature. We show that Aminophospholipid ATPase3 (ALA3), a member of the P(4)-ATPase subfamily in Arabidopsis thaliana, localizes to the Golgi apparatus and that mutations of ALA3 result in impaired growth of roots and shoots. The growth defect is accompanied by failure of the root cap to release border cells involved in the secretion of molecules required for efficient root interaction with the environment, and ala3 mutants are devoid of the characteristic trans-Golgi proliferation of slime vesicles containing polysaccharides and enzymes for secretion. In yeast complementation experiments, ALA3 function requires interaction with members of a novel family of plant membrane-bound proteins, ALIS1 to ALIS5 (for ALA-Interacting Subunit), and in this host ALA3 and ALIS1 show strong affinity for each other. In planta, ALIS1, like ALA3, localizes to Golgi-like structures and is expressed in root peripheral columella cells. We propose that the ALIS1 protein is a beta-subunit of ALA3 and that this protein complex forms an important part of the Golgi machinery required for secretory processes during plant development.

  9. Alkanols and chlorophenols cause different physiological adaptive responses on the level of cell surface properties and membrane vesicle formation in Pseudomonas putida DOT-T1E.

    PubMed

    Baumgarten, Thomas; Vazquez, José; Bastisch, Christian; Veron, Wilfried; Feuilloley, Marc G J; Nietzsche, Sandor; Wick, Lukas Y; Heipieper, Hermann J

    2012-01-01

    In order to cope with the toxicity imposed by the exposure to environmental hydrocarbons, many bacteria have developed specific adaptive responses such as modifications in the cell envelope. Here we compared the influence of n-alkanols and chlorophenols on the surface properties of the solvent-tolerant bacterium Pseudomonas putida DOT-T1E. In the presence of toxic concentrations of n-alkanols, this strain significantly increased its cell surface charge and hydrophobicity with changes depending on the chain length of the added n-alkanols. The adaptive response occurred within 10 min after the addition of the solvent and was demonstrated to be of physiological nature. Contrary to that, chlorophenols of similar hydrophobicity and potential toxicity as the corresponding alkanols caused only minor effects in the surface properties. To our knowledge, this is the first observation of differences in the cellular adaptive response of bacteria to compound classes of quasi equal hydrophobicity and toxicity. The observed adaptation of the physico-chemical surface properties of strain DOT-T1E to the presence of alkanols was reversible and correlated with changes in the composition of the lipopolysaccharide content of the cells. The reaction is explained by previously described reactions allowing the release of membrane vesicles that was demonstrated for cells affected by 1-octanol and heat shock, whereas no membrane vesicles were released after the addition of chlorophenols.

  10. Molecular assemblies of 4-(hexadecyloxy)-n-(pyridinylmethylene)anilines at the air-water interface and Cu(II)-promoted vesicle formation via metal coordination.

    PubMed

    Wang, Haibo; Miao, Wangen; Liu, Huijin; Zhang, Xianfeng; Du, Xuezhong

    2010-09-01

    The molecular assemblies of 4-(hexadecyloxy)-N-(pyridinylmethylene)anilines (HPA) at the air-water interface on pure water and aqueous Cu(II) subphases have been investigated using in situ infrared reflection absorption spectroscopy (IRRAS). The Schiff base units were oriented with their long axes almost perpendicular to the water surface, and both imine and pyridinyl nitrogen atoms of the Schiff base units were coordinated to Cu(II) ions together with their geometrical conversions. The alkyl chains in the monolayers were quantitatively determined on the assumption that the HPA monolayers at the air-water interface were composed of sublayers of alkyl chains and Schiff base units, and the chain orientation angle on pure water was 30 +/- 2 degrees and increased to 37 +/- 2 degrees on the aqueous Cu(II) subphase. The HPA amphiphiles could not be dispersed in pure water but could self-organize into vesicles with metal-coordinated headgroups and interdigitated-packed alkyl chains in the presence of Cu(II) ions in aqueous solution. Transmission electron microscopy (TEM), differential scanning calorimetry (DSC), UV-vis spectroscopy, and small-angle X-ray diffraction (XRD) were used to investigate the aggregate structures and specific properties of the coordinated vesicles. PMID:20698514

  11. Absence of synaptotagmin disrupts excitation-secretion coupling during synaptic transmission.

    PubMed Central

    Broadie, K; Bellen, H J; DiAntonio, A; Littleton, J T; Schwarz, T L

    1994-01-01

    Synaptotagmin is an integral synaptic vesicle protein proposed to be involved in Ca(2+)-dependent exocytosis during synaptic transmission. Null mutations in synaptotagmin have been made in Drosophila, and the protein's in vivo function has been assayed at the neuromuscular synapse. In the absence of synaptotagmin, synaptic transmission is dramatically impaired but is not abolished. In null mutants, evoked vesicle release is decreased by a factor of 10. Moreover, the fidelity of excitation-secretion coupling is impaired so that a given stimulus generates a more variable amount of secretion. However, this residual evoked release shows Ca(2+)-dependence similar to normal release, suggesting either that synaptotagmin is not the Ca2+ sensor or that a second, independent Ca2+ sensor exists. While evoked transmission is suppressed, the rate of spontaneous vesicle fusion is increased by a factor of 5. We conclude that synaptotagmin is not an absolutely essential component of the Ca(2+)-dependent secretion pathway in synaptic transmission but is necessary for normal levels of transmission. Our data support a model in which synaptotagmin functions as a negative regulator of spontaneous vesicle fusion and acts to increase the efficiency of excitation-secretion coupling during synaptic transmission. Images PMID:7938019

  12. Role of Rab27 in synaptic transmission at the squid giant synapse.

    PubMed

    Yu, Eunah; Kanno, Eiko; Choi, Soonwook; Sugimori, Mutsuyuki; Moreira, Jorge E; Llinás, Rodolfo R; Fukuda, Mitsunori

    2008-10-14

    Small GTPase Rab is a member of a large family of Ras-related proteins, highly conserved in eukaryotic cells, and thought to regulate specific type(s) and/or specific step(s) in intracellular membrane trafficking. Given our interest in synaptic transmission, we addressed the possibility that Rab27 (a close isoform of Rab3) could be involved in cytosolic synaptic vesicle mobilization. Indeed, preterminal injection of a specific antibody against squid Rab27 (anti-sqRab27 antibody) combined with confocal microscopy demonstrated that Rab27 is present on squid synaptic vesicles. Electrophysiological study of injected synapses showed that the anti-sqRab27 antibody inhibited synaptic release in a stimulation-dependent manner without affecting presynaptic action potentials or inward Ca(2+) current. This result was confirmed in in vitro synaptosomes by using total internal reflection fluorescence microscopy. Thus, synaptosomal Ca(2+)-stimulated release of FM1-43 dye was greatly impaired by intraterminal anti-sqRab27 antibody. Ultrastructural analysis of the injected giant preterminal further showed a reduced number of docked synaptic vesicles and an increase in nondocked vesicular profiles distant from the active zone. These results, taken together, indicate that Rab27 is primarily involved in the maturation of recycled vesicles and/or their transport to the presynaptic active zone in the squid giant synapse.

  13. Role of Rab27 in synaptic transmission at the squid giant synapse

    PubMed Central

    Yu, Eunah; Kanno, Eiko; Choi, Soonwook; Sugimori, Mutsuyuki; Moreira, Jorge E.; Llinás, Rodolfo R.; Fukuda, Mitsunori

    2008-01-01

    Small GTPase Rab is a member of a large family of Ras-related proteins, highly conserved in eukaryotic cells, and thought to regulate specific type(s) and/or specific step(s) in intracellular membrane trafficking. Given our interest in synaptic transmission, we addressed the possibility that Rab27 (a close isoform of Rab3) could be involved in cytosolic synaptic vesicle mobilization. Indeed, preterminal injection of a specific antibody against squid Rab27 (anti-sqRab27 antibody) combined with confocal microscopy demonstrated that Rab27 is present on squid synaptic vesicles. Electrophysiological study of injected synapses showed that the anti-sqRab27 antibody inhibited synaptic release in a stimulation-dependent manner without affecting presynaptic action potentials or inward Ca2+ current. This result was confirmed in in vitro synaptosomes by using total internal reflection fluorescence microscopy. Thus, synaptosomal Ca2+-stimulated release of FM1-43 dye was greatly impaired by intraterminal anti-sqRab27 antibody. Ultrastructural analysis of the injected giant preterminal further showed a reduced number of docked synaptic vesicles and an increase in nondocked vesicular profiles distant from the active zone. These results, taken together, indicate that Rab27 is primarily involved in the maturation of recycled vesicles and/or their transport to the presynaptic active zone in the squid giant synapse. PMID:18840683

  14. Sucrose induces vesicle accumulation and autophagy.

    PubMed

    Higuchi, Takahiro; Nishikawa, Jun; Inoue, Hiroko

    2015-04-01

    It has been shown that the treatment of mammalian cells with sucrose leads to vacuole accumulation associated with lysosomes and upregulation of lysosomal enzyme expression and activity. Autophagy is an evolutionarily conserved homeostatic process by which cells deliver cytoplasmic material for degradation into lysosomes, thus it is probable that sucrose affects the autophagic activity. The role of sucrose in autophagy is unknown; however, another disaccharide, trehalose has been shown to induce autophagy. In the current study, we used mouse embryonic fibroblasts to investigate whether sucrose induces autophagy and whether vesicle formation is associated with autophagy. The results showed that sucrose induces autophagy while being accumulated within the endosomes/lysosomes. These vesicles were swollen and packed within the cytoplasm. Furthermore, trehalose and the trisaccharide raffinose, which are not hydrolyzed in mammalian cells, increased the rate of vesicles accumulation and LC3-II level (a protein marker of autophagy). However, fructose and maltose did not show the same effects. The correlation between the two processes, vesicle accumulation and autophagy induction, was confirmed by treatment of cells with sucrose plus invertase, or maltose plus acarbose-the α-glucosidase inhibitor-and by sucrose deprivation. Results also showed that vesicle accumulation was not affected by autophagy inhibition. Therefore, the data suggest that sucrose-induced autophagy through accumulation of sucrose-containing vesicles is caused by the absence of hydrolysis enzymes.

  15. Sucrose induces vesicle accumulation and autophagy.

    PubMed

    Higuchi, Takahiro; Nishikawa, Jun; Inoue, Hiroko

    2015-04-01

    It has been shown that the treatment of mammalian cells with sucrose leads to vacuole accumulation associated with lysosomes and upregulation of lysosomal enzyme expression and activity. Autophagy is an evolutionarily conserved homeostatic process by which cells deliver cytoplasmic material for degradation into lysosomes, thus it is probable that sucrose affects the autophagic activity. The role of sucrose in autophagy is unknown; however, another disaccharide, trehalose has been shown to induce autophagy. In the current study, we used mouse embryonic fibroblasts to investigate whether sucrose induces autophagy and whether vesicle formation is associated with autophagy. The results showed that sucrose induces autophagy while being accumulated within the endosomes/lysosomes. These vesicles were swollen and packed within the cytoplasm. Furthermore, trehalose and the trisaccharide raffinose, which are not hydrolyzed in mammalian cells, increased the rate of vesicles accumulation and LC3-II level (a protein marker of autophagy). However, fructose and maltose did not show the same effects. The correlation between the two processes, vesicle accumulation and autophagy induction, was confirmed by treatment of cells with sucrose plus invertase, or maltose plus acarbose-the α-glucosidase inhibitor-and by sucrose deprivation. Results also showed that vesicle accumulation was not affected by autophagy inhibition. Therefore, the data suggest that sucrose-induced autophagy through accumulation of sucrose-containing vesicles is caused by the absence of hydrolysis enzymes. PMID:25389129

  16. Coated vesicles: a diversity of form and function.

    PubMed

    Schmid, S L; Damke, H

    1995-11-01

    In every well-characterized example, the small transport vesicles that mediate membrane trafficking between intracellular organelles are encased in a protein coat. In general, the coat proteins assemble from cytosolic pools onto the membrane and play a critical role in vesicle formation. Recent reviews have emphasized the clear similarities in the mechanisms that drive vesicle budding at distinct cellular locations. Here we focus on the diversity of solutions to an apparently related biological task. These mechanistic differences are likely to be physiologically important determinants of the diversity in form, and function of coated transport vesicles. PMID:7589986

  17. From Vesicles to Protocells: The Roles of Amphiphilic Molecules

    PubMed Central

    Sakuma, Yuka; Imai, Masayuki

    2015-01-01

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

  18. Small molecule-mediated stabilization of vesicle-associated helical α-synuclein inhibits pathogenic misfolding and aggregation.

    PubMed

    Fonseca-Ornelas, Luis; Eisbach, Sybille E; Paulat, Maria; Giller, Karin; Fernández, Claudio O; Outeiro, Tiago F; Becker, Stefan; Zweckstetter, Markus

    2014-01-01

    α-synuclein is an abundant presynaptic protein that is important for regulation of synaptic vesicle trafficking, and whose misfolding plays a key role in Parkinson's disease. While α-synuclein is disordered in solution, it folds into a helical conformation when bound to synaptic vesicles. Stabilization of helical, folded α-synuclein might therefore interfere with α-synuclein-induced neurotoxicity. Here we show that several small molecules, which delay aggregation of α-synuclein in solution, including the Parkinson's disease drug selegiline, fail to interfere with misfolding of vesicle-bound α-synuclein. In contrast, the porphyrin phtalocyanine tetrasulfonate directly binds to vesicle-bound α-synuclein, stabilizes its helical conformation and thereby delays pathogenic misfolding and aggregation. Our study suggests that small-molecule-mediated stabilization of helical vesicle-bound α-synuclein opens new possibilities to target Parkinson's disease and related synucleinopathies. PMID:25524885

  19. Early Exposure to General Anesthesia Disrupts Spatial Organization of Presynaptic Vesicles in Nerve Terminals of the Developing Rat Subiculum.

    PubMed

    Lunardi, N; Oklopcic, A; Prillaman, M; Erisir, A; Jevtovic-Todorovic, V

    2015-10-01

    Exposure to general anesthesia (GA) during critical stages of brain development induces widespread neuronal apoptosis and causes long-lasting behavioral deficits in numerous animal species. Although several studies have focused on the morphological fate of neurons dying acutely by GA-induced developmental neuroapoptosis, the effects of an early exposure to GA on the surviving synapses remain unclear. The aim of this study is to study whether exposure to GA disrupts the fine regulation of the dynamic spatial organization and trafficking of synaptic vesicles in presynaptic terminals. We exposed postnatal day 7 (PND7) rat pups to a clinically relevant anesthetic combination of midazolam, nitrous oxide, and isoflurane and performed a detailed ultrastructural analysis of the synaptic vesicle architecture at presynaptic terminals in the subiculum of rats at PND 12. In addition to a significant decrease in the density of presynaptic vesicles, we observed a reduction of docked vesicles, as well as a reduction of vesicles located within 100 nm from the active zone, in animals 5 days after an initial exposure to GA. We also found that the synaptic vesicles of animals exposed to GA are located more distally with respect to the plasma membrane than those of sham control animals and that the distance between presynaptic vesicles is increased in GA-exposed animals compared to sham controls. We report that exposure of immature rats to GA during critical stages of brain development causes significant disruption of the strategic topography of presynaptic vesicles within the nerve terminals of the subiculum. PMID:26048670

  20. Preferential localization of a vesicular monoamine transporter to dense core vesicles in PC12 cells

    PubMed Central

    1994-01-01

    Neurons and endocrine cells have two types of secretory vesicle that undergo regulated exocytosis. Large dense core vesicles (LDCVs) store neural peptides whereas small clear synaptic vesicles store classical neurotransmitters such as acetylcholine, gamma-aminobutyric acid (GABA), glycine, and glutamate. However, monoamines differ from other classical transmitters and have been reported to appear in both LDCVs and smaller vesicles. To localize the transporter that packages monoamines into secretory vesicles, we have raised antibodies to a COOH- terminal sequence from the vesicular amine transporter expressed in the adrenal gland (VMAT1). Like synaptic vesicle proteins, the transporter occurs in endosomes of transfected CHO cells, accounting for the observed vesicular transport activity. In rat pheochromocytoma PC12 cells, the transporter occurs principally in LDCVs by both immunofluorescence and density gradient centrifugation. Synaptic-like microvesicles in PC12 cells contain relatively little VMAT1. The results appear to account for the storage of monoamines by LDCVs in the adrenal medulla and indicate that VMAT1 provides a novel membrane protein marker unique to LDCVs. PMID:7962100

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

    PubMed Central

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

    2001-01-01

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