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Mark L Harlow - One of the best experts on this subject based on the ideXlab platform.
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Synaptic Vesicles contain small ribonucleic acids srnas including transfer rna fragments trfrna and micrornas mirna
Scientific Reports, 2015Co-Authors: Rodolfo Aramayo, Matthew S Sachs, Mark L HarlowAbstract: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.
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Individual Synaptic Vesicles from the electroplaque of Torpedo californica, a classic cholinergic synapse, also contain transporters for glutamate and ATP.
Physiological reports, 2014Co-Authors: Mark L HarlowAbstract: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.
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Synaptic Vesicles Isolated from the Electric Organ of Torpedo Californica Posses Multiple Classes of Neurotransmitter Transporters
Biophysical Journal, 2013Co-Authors: Mark L HarlowAbstract:The fusion of Synaptic Vesicles with the preSynaptic membrane, and the subsequent release of small chemical neurotransmitters is the fundamental process by which neurons communicate at chemical synapses, and it has long been proposed that most neurons release a single type of small molecule neurotransmitter. The simplicity of one neuron, one neurotransmitter has come under intense scrutiny as examples of neurons that appear to co-release two or more neurotransmitters at single synapses (neurotransmitter synergy) have been identified. One such synapse is that of the electric organ of Torpedo californica; Synaptic Vesicles isolated from this single class of neurons appear to contain the neurotransmitter acetylcholine as well as the neurotransmitter ATP. We used immunofluorescence labeling in conjunction with single-molecule TIRF microscopy to observe whether one or more neurotransmitter transporters could be detected on single Synaptic Vesicles. We found that the vesicular acetylcholine transporter (VAChT) and several solute carrier proteins (SLC17A) co-localized to the same Vesicles. The presence of multiple types of neurotransmitter transporters - and potentially neurotransmitters - in individual Synaptic Vesicles raises fundamental questions about chemical Synaptic transmission at the electric organ of T. Californica. In addition, this approach can be applied to other synapses in order to address the prevalence of neurotransmitter synergy and co-release at chemical synapses.
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macromolecular connections of active zone material to docked Synaptic Vesicles and preSynaptic membrane at neuromuscular junctions of mouse
The Journal of Comparative Neurology, 2009Co-Authors: Sharuna Nagwaney, Mark L Harlow, David Ress, Robert M Marshall, Jae Hoon Jung, Joseph A Szule, U J McmahanAbstract:Electron tomography was used to view macromolecules composing active zone material (AZM) in axon terminals at mouse neuromuscular junctions. Connections of the macromolecules to each other, to calcium channels in the preSynaptic membrane, and to Synaptic Vesicles docked on the membrane prior to fusing with it during Synaptic transmission were similar to those of AZM macromolecules at frog neuromuscular junctions previously examined by electron tomography and support the hypothesis that AZM regulates vesicle docking and fusion. A species difference in the arrangement of AZM relative to docked Vesicles may help account for a greater vesicle-preSynaptic membrane contact area during docking and a greater probability of fusion during Synaptic transmission in mouse. Certain AZM macromolecules in mouse were connected to Synaptic Vesicles contacting the preSynaptic membrane at sites where fusion does not occur. These secondary docked Vesicles had a different relationship to the membrane and AZM macromolecules than primary docked Vesicles, consistent with their having a different AZM-regulated behavior. J. Comp. Neurol.
Reinhard Jahn - One of the best experts on this subject based on the ideXlab platform.
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Small-scale isolation of Synaptic Vesicles from mammalian brain
Nature Protocols, 2013Co-Authors: Saheeb Ahmed, Matthew Holt, Dietmar Riedel, Reinhard JahnAbstract:Synaptic Vesicles (SVs) are essential organelles that participate in the release of neurotransmitters from a neuron. Biochemical analysis of purified SVs was instrumental in the identification of proteins involved in exocytotic membrane fusion and neurotransmitter uptake. Although numerous protocols have been published detailing the isolation of SVs from the brain, those that give the highest-purity Vesicles often have low yields. Here we describe a protocol for the small-scale isolation of SVs from mouse and rat brain. The procedure relies on standard fractionation techniques, including differential centrifugation, rate-zonal centrifugation and size-exclusion chromatography, but it has been optimized for minimal vesicle loss while maintaining a high degree of purity. The protocol can be completed in less than 1 d and allows the recovery of ∼150 μg of vesicle protein from a single mouse brain, thus allowing vesicle isolation from transgenic mice.
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molecular machines governing exocytosis of Synaptic Vesicles
Nature, 2012Co-Authors: Reinhard Jahn, Dirk FasshauerAbstract:Calcium-dependent exocytosis of Synaptic Vesicles mediates the release of neurotransmitters. Important proteins in this process have been identified such as the SNAREs, synaptotagmins, complexins, Munc18 and Munc13. Structural and functional studies have yielded a wealth of information about the physiological role of these proteins. However, it has been surprisingly difficult to arrive at a unified picture of the molecular sequence of events from vesicle docking to calcium-triggered membrane fusion. Using mainly a biochemical and biophysical perspective, we briefly survey the molecular mechanisms in an attempt to functionally integrate the key proteins into the emerging picture of the neuronal fusion machine.
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assembly and disassembly of a ternary complex of synaptobrevin syntaxin and snap 25 in the membrane of Synaptic Vesicles
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Henning Otto, Phyllis I Hanson, Reinhard JahnAbstract:The Synaptic membrane proteins synaptobrevin, syntaxin, and SNAP-25 form a ternary complex that can be disassembled by the ATPase N-ethylmaleimide-sensitive factor (NSF) in the presence of soluble cofactors (SNAP proteins). These steps are thought to represent molecular events involved in docking and subsequent exocytosis of Synaptic Vesicles. Using two independent and complementary approaches, we now report that such ternary complexes form in the membrane of highly purified and monodisperse Synaptic Vesicles in the absence of the plasma membrane. Furthermore, the complexes are reversibly dissociated by NSF and SNAP proteins. Thus, ternary complexes can be assembled and disassembled while all three proteins are anchored as neighbors in the same membrane, suggesting that NSF is involved in priming Synaptic Vesicles for exocytosis.
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real time measurement of transmitter release from single Synaptic Vesicles
Nature, 1995Co-Authors: Dieter Bruns, Reinhard JahnAbstract:Neurotransmitter release is mediated by Ca2+ dependent exocytosis of Synaptic Vesicles. Neither the amount of transmitter released from individual Synaptic Vesicles nor the kinetics of this process have yet been directly determined. Using carbon fibres as electrochemical detectors, we have measured release of the neurotransmitter serotonin from cultured neurons of the leech. This technique allowed us to monitor transmitter discharge from single Synaptic Vesicles as spike-like oxidation currents at high time resolution, providing new insight into the mechanism of neuronal exocytosis. Two types of signals were characterized, corresponding to exocytosis of small clear and large dense core Vesicles present in these cells. A small vesicle discharges about 4,700 transmitter molecules with a time constant in the region of 260 microseconds, whereas large Vesicles release their content of approximately 80,000 molecules with a time constant of about 1.3 ms. Release from both vesicle types is initiated rapidly, with a rise time of less than 60 microseconds, suggesting an abrupt opening of a preassembled fusion pore.
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real time measurement of transmitter release from single Synaptic Vesicles
Nature, 1995Co-Authors: Dieter Bruns, Reinhard JahnAbstract:NEUROTRANSMITTER release is mediated by Ca2+ dependent exocytosis of Synaptic Vesicles1. Neither the amount of transmitter released from individual Synaptic Vesicles nor the kinetics of this process have yet been directly determined. Using carbon fibres as electrochemical detectors2,3, we have measured release of the neurotransmitter serotonin from cultured neurons of the leech4. This technique allowed us to monitor transmitter discharge from single Synaptic Vesicles as spike-like oxidation currents at high time resolution, providing new insight into the mechanism of neuronal exocytosis. Two types of signals were characterized, corresponding to exocytosis of small clear and large dense core Vesicles present in these cells. A small vesicle discharges about 4,700 transmitter molecules with a time constant in the region of 260 μs, whereas large Vesicles release their content of approximately 80,000 molecules with a time constant of about 1.3 ms. Release from both vesicle types is initiated rapidly, with a rise time of less than 60 μs, suggesting an abrupt opening of a preassembled fusion pore.
Jae Hoon Jung - One of the best experts on this subject based on the ideXlab platform.
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Synaptic Vesicles having large contact areas with the preSynaptic membrane are preferentially hemifused at active zones of frog neuromuscular junctions fixed during Synaptic activity
International Journal of Molecular Sciences, 2019Co-Authors: Jae Hoon JungAbstract:Synaptic Vesicles dock on the preSynaptic plasma membrane of axon terminals and become ready to fuse with the preSynaptic membrane or primed. Fusion of the vesicle membrane and preSynaptic membrane results in the formation of a pore between the membranes, through which the vesicle’s neurotransmitter is released into the Synaptic cleft. A recent electron tomography study on frog neuromuscular junctions fixed at rest showed that there is no discernible gap between or merging of the membrane of docked Synaptic Vesicles with the preSynaptic membrane, however, the extent of the contact area between the membrane of docked Synaptic Vesicles and the preSynaptic membrane varies 10-fold with a normal distribution. The study also showed that when the neuromuscular junctions are fixed during repetitive electrical nerve stimulation, the portion of large contact areas in the distribution is reduced compared to the portion of small contact areas, suggesting that docked Synaptic Vesicles with the largest contact areas are greatly primed to fuse with the membrane. Furthermore, the finding of several hemifused Synaptic Vesicles among the docked Vesicles was briefly reported. Here, the spatial relationship of 81 Synaptic Vesicles with the preSynaptic membrane at active zones of the neuromuscular junctions fixed during stimulation is described in detail. For the most of the Vesicles, the combined thickness of each of their contact sites was not different from the sum of the membrane thicknesses of the vesicle membrane and preSynaptic membrane, similar to the docked Vesicles at active zones of the resting neuromuscular junctions. However, the combined membrane thickness of a small portion of the Vesicles was considerably less than the sum of the membrane thicknesses, indicating that the membranes at their contact sites were fixed in a state of hemifusion. Moreover, the hemifused Vesicles were found to have large contact areas with the preSynaptic membrane. These findings support the recently proposed hypothesis that, at frog neuromuscular junctions, docked Synaptic Vesicles with the largest contact areas are most primed for fusion with the preSynaptic membrane, and that hemifusion is a fusion intermediate step of the vesicle membrane with the preSynaptic membrane for Synaptic transmission.
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macromolecular connections of active zone material to docked Synaptic Vesicles and preSynaptic membrane at neuromuscular junctions of mouse
The Journal of Comparative Neurology, 2009Co-Authors: Sharuna Nagwaney, Mark L Harlow, David Ress, Robert M Marshall, Jae Hoon Jung, Joseph A Szule, U J McmahanAbstract:Electron tomography was used to view macromolecules composing active zone material (AZM) in axon terminals at mouse neuromuscular junctions. Connections of the macromolecules to each other, to calcium channels in the preSynaptic membrane, and to Synaptic Vesicles docked on the membrane prior to fusing with it during Synaptic transmission were similar to those of AZM macromolecules at frog neuromuscular junctions previously examined by electron tomography and support the hypothesis that AZM regulates vesicle docking and fusion. A species difference in the arrangement of AZM relative to docked Vesicles may help account for a greater vesicle-preSynaptic membrane contact area during docking and a greater probability of fusion during Synaptic transmission in mouse. Certain AZM macromolecules in mouse were connected to Synaptic Vesicles contacting the preSynaptic membrane at sites where fusion does not occur. These secondary docked Vesicles had a different relationship to the membrane and AZM macromolecules than primary docked Vesicles, consistent with their having a different AZM-regulated behavior. J. Comp. Neurol.
Flavia Valtorta - One of the best experts on this subject based on the ideXlab platform.
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synapsin is a novel rab3 effector protein on small Synaptic Vesicles ii functional effects of the rab3a synapsin i interaction
Journal of Biological Chemistry, 2004Co-Authors: Silvia Giovedi, François Darchen, Flavia ValtortaAbstract:Synapsins, a family of neuron-specific phosphoproteins that play an important role in the regulation of Synaptic vesicle trafficking and neurotransmitter release, were recently demonstrated to interact with the Synaptic vesicle-associated small G protein Rab3A within nerve terminals (Giovedi, S., Vaccaro, P., Valtorta, F., Darchen, F., Greengard, P., Cesareni, G., and Benfenati, F. (2004) J. Biol. Chem. 279, 43760–43768). We have analyzed the functional consequences of this interaction on the biological activities of both proteins and on their subcellular distribution within nerve terminals. The presence of synapsin I stimulated GTP binding and GTPase activity of both purified and endogenous Synaptic vesicle-associated Rab3A. Conversely, Rab3A inhibited synapsin I binding to F-actin, as well as synapsin-induced actin bundling and vesicle clustering. Moreover, the amount of Rab3A associated with Synaptic Vesicles was decreased in synapsin knockout mice, and the presence of synapsin I prevented RabGDI-induced Rab3A dissociation from Synaptic Vesicles. The results indicate that an interaction between synapsin I and Rab3A exists on Synaptic Vesicles that modulates the functional properties of both proteins. Given the well recognized importance of both synapsins and Rab3A in Synaptic Vesicles exocytosis, this interaction is likely to play a major role in the modulation of neurotransmitter release.
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Synapsin Is a Novel Rab3 Effector Protein on Small Synaptic Vesicles I. IDENTIFICATION AND CHARACTERIZATION OF THE SYNAPSIN I-Rab3 INTERACTIONS IN VITRO AND IN INTACT NERVE TERMINALS
The Journal of biological chemistry, 2004Co-Authors: Silvia Giovedi, Flavia Valtorta, Paola Vaccaro, François Darchen, Gianni CesareniAbstract:Synapsins, a family of neuron-specific phosphoproteins, have been demonstrated to regulate the availability of Synaptic Vesicles for exocytosis by binding to both Synaptic Vesicles and the actin cytoskeleton in a phosphorylation-dependent manner. Although the above-mentioned observations strongly support a pre-docking role of the synapsins in the assembly and maintenance of a reserve pool of Synaptic Vesicles, recent results suggest that the synapsins may also be involved in some later step of exocytosis. In order to investigate additional interactions of the synapsins with nerve terminal proteins, we have employed phage display library technology to select peptide sequences binding with high affinity to synapsin I. Antibodies raised against the peptide YQYIETSMQ (syn21) specifically recognized Rab3A, a Synaptic vesicle-specific small G protein implicated in multiple steps of exocytosis. The interaction between synapsin I and Rab3A was confirmed by photoaffinity labeling experiments on purified Synaptic Vesicles and by the formation of a chemically cross-linked complex between synapsin I and Rab3A in intact nerve terminals. Synapsin I could be effectively co-precipitated from synaptosomal extracts by immobilized recombinant Rab3A in a GTP-dependent fashion. In vitro binding assays using purified proteins confirmed the binding preference of synapsin I for Rab3A-GTP and revealed that the COOH-terminal regions of synapsin I and the Rab3A effector domain are required for the interaction with Rab3A to occur. The data indicate that synapsin I is a novel Rab3 interactor on Synaptic Vesicles and suggest that the synapsin-Rab3 interaction may participate in the regulation of Synaptic vesicle trafficking within the nerve terminals.
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phosphorylation of vamp synaptobrevin in Synaptic Vesicles by endogenous protein kinases
Journal of Neurochemistry, 2002Co-Authors: H B Nielander, Cesare Montecucco, Flavia Valtorta, Franco Onofri, Giampietro Schiavo, Paul Greengard, Fabio BenfenatiAbstract:VAMP/synaptobrevin (SYB), an integral membrane protein of small Synaptic Vesicles, is specifically cleaved by tetanus neurotoxin and botulinum neurotoxins B, D, F, and G and is thought to play an important role in the docking and/or fusion of Synaptic Vesicles with the preSynaptic membrane. Potential phosphorylation sites for various kinases are present in SYB sequence. We have studied whether SYB is a substrate for protein kinases that are present in nerve terminals and known to modulate neurotransmitter release. SYB can be phosphorylated within the same vesicle by endogenous Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) associated with Synaptic Vesicles. This phosphorylation reaction occurs rapidly and involves serine and threonine residues in the cytoplasmic region of SYB. Similarly to CaMKII, a casein kinase II (CasKII) activity copurifying with Synaptic Vesicles is able to phosphorylate SYB selectively on serine residues of the cytoplasmic region. This phosphorylation reaction is markedly stimulated by sphingosine, a sphingolipid known to activate CasKII and to inhibit CaM-KII and protein kinase C. The results show that SYB is a potential substrate for protein kinases involved in the regulation of neurotransmitter release and open the possibility that phosphorylation of SYB plays a role in modulating the molecular interactions between Synaptic Vesicles and the preSynaptic membrane.
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synapsin i partially dissociates from Synaptic Vesicles during exocytosis induced by electrical stimulation
Neuron, 1992Co-Authors: Torri F Tarelli, Riccardo Fesce, Mario Bossi, Flavia ValtortaAbstract:The distribution of the Synaptic vesicle-associated phosphoprotein synapsin I after electrical stimulation of the frog neuromuscular junction was investigated by immunogold labeling and compared with the distribution of the integral Synaptic vesicle protein synaptophysin. In resting terminals both proteins were localized exclusively on Synaptic Vesicles. In stimulated terminals they appeared also in the axolemma and its infoldings, which however exhibited a lower synapsin I/synaptophysin ratio with respect to Synaptic Vesicles at rest. The value of this ratio was intermediate in Synaptic Vesicles of stimulated terminals, and an increased synapsin I labeling of the cytomatrix was observed. These results indicate that synapsin I undergoes partial dissociation from and reassociation with Synaptic Vesicles, following physiological stimulation, and are consistent with the proposed modulatory role of the protein in neurotransmitter release.
Erik M Jorgensen - One of the best experts on this subject based on the ideXlab platform.
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Synaptic Vesicles transiently dock to refill release sites
Nature Neuroscience, 2020Co-Authors: Grant F. Kusick, Erik M Jorgensen, Morven Chin, Sumana Raychaudhuri, Kristina Lippmann, Kadidia P. Adula, Edward J. Hujber, Thien Vu, M. Wayne Davis, Shigeki WatanabeAbstract:Kusick et al. capture snapshots of Synaptic vesicle docking and fusion using a new time-resolved electron microscopy technique. They find that Vesicles are replaced milliseconds after they fuse, which may contribute to short-term Synaptic plasticity. Synaptic Vesicles fuse with the plasma membrane to release neurotransmitter following an action potential, after which new Vesicles must ‘dock’ to refill vacated release sites. To capture Synaptic vesicle exocytosis at cultured mouse hippocampal synapses, we induced single action potentials by electrical field stimulation, then subjected neurons to high-pressure freezing to examine their morphology by electron microscopy. During synchronous release, multiple Vesicles can fuse at a single active zone. Fusions during synchronous release are distributed throughout the active zone, whereas fusions during asynchronous release are biased toward the center of the active zone. After stimulation, the total number of docked Vesicles across all synapses decreases by ~40%. Within 14 ms, new Vesicles are recruited and fully replenish the docked pool, but this docking is transient and they either undock or fuse within 100 ms. These results demonstrate that the recruitment of Synaptic Vesicles to release sites is rapid and reversible.
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Synaptic Vesicles undock and then transiently dock after an action potential
bioRxiv, 2018Co-Authors: Grant F. Kusick, Erik M Jorgensen, Shigeki Watanabe, Wayne M Davis, Morven Chin, Kristina Lippmann, Kadidia P. AdulaAbstract:Abstract Synaptic Vesicles fuse with the plasma membrane to release neurotransmitter following an action potential, after which new Vesicles must refill vacated release sites. How many Vesicles can fuse at a single active zone, where they fuse within the active zone, and how quickly they are replaced with new Vesicles is not well-established. To capture Synaptic vesicle exocytosis at cultured mouse hippocampal synapses, we induced single action potentials by electrical field stimulation then subjected neurons to high-pressure freezing to examine their morphology by electron microscopy. During synchronous release, multiple Vesicles can fuse at a single active zone; this multivesicular release is augmented by increasing the extracellular calcium concentration. Synchronous fusions are distributed throughout the active zone, whereas asynchronous fusions are biased toward the center of the active zone. Immediately after stimulation a large fraction of Vesicles become undocked. Between 8 and 14 ms, new Vesicles are recruited to the plasma membrane and fully replenish the docked pool, but docking of these Vesicles is transient and they either undock or fuse within 100 ms. These results demonstrate that recruitment of Synaptic Vesicles to release sites is rapid and reversible.
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clathrin regenerates Synaptic Vesicles from endosomes
Nature, 2014Co-Authors: Shigeki Watanabe, Thorsten Trimbuch, Marcial Camachoperez, Benjamin R Rost, Bettina Brokowski, Berit Sohlkielczynski, Annegret Felies, Wayne M Davis, Christian Rosenmund, Erik M JorgensenAbstract:Ultrafast endocytosis can retrieve a single, large endocytic vesicle as fast as 50–100 ms after Synaptic vesicle fusion. However, the fate of the large endocytic Vesicles is not known. Here we demonstrate that these Vesicles transition to a Synaptic endosome about one second after stimulation. The endosome is resolved into coated Vesicles after 3 s, which in turn become small-diameter Synaptic Vesicles 5–6 s after stimulation. We disrupted clathrin function using RNA interference (RNAi) and found that clathrin is not required for ultrafast endocytosis but is required to generate Synaptic Vesicles from the endosome. Ultrafast endocytosis fails when actin polymerization is disrupted, or when neurons are stimulated at room temperature instead of physiological temperature. In the absence of ultrafast endocytosis, Synaptic Vesicles are retrieved directly from the plasma membrane by clathrin-mediated endocytosis. These results may explain discrepancies among published experiments concerning the role of clathrin in Synaptic vesicle endocytosis. Ultrastructural analysis of Synaptic vesicle recycling reveals that clathrin is not required for the initial rapid step of vesicle recycling by ultrafast endocytosis at the plasma membrane and instead clathrin acts later at an endosome to regenerate Synaptic Vesicles; however, when ultrafast endocytosis does not occur (for example, in experiments at room temperature rather than physiological temperature), clathrin-mediated endocytosis does happen at the plasma membrane. Since the discovery of Synaptic vesicle recycling in the 1970s, clathrin has been thought to act at the plasma membrane to reconstitute Synaptic Vesicles about 20 seconds after stimulation of the axon terminal. Erik Jorgensen and colleagues have revisited these original experiments, but combining optogenetic stimulation with high-pressure freezing to rapidly fix the samples after stimulation. This changes the picture: the previously hypothesized mechanism operates at a non-physiological, 'room temperature' of 22°C, when ultra-fast endocytosis is disrupted. At a physiological temperature (34°C), large Vesicles are instead retrieved from the plasma membrane within about 50 milliseconds, through clathrin-independent, ultra-fast endocytosis; these then fuse to form endosomes from which small Synaptic Vesicles are regenerated by clathrin scaffolds, about 5 seconds after stimulation.
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UNC-11, a Caenorhabditis elegans AP180 Homologue, Regulates the Size and Protein Composition of Synaptic Vesicles
Molecular biology of the cell, 1999Co-Authors: Michael L Nonet, Erik M Jorgensen, Erika Hartwieg, Andrea M. Holgado, Faraha Brewer, Craig J. Serpe, Betty A. Norbeck, Julianne Holleran, Liping Wei, Aixa AlfonsoAbstract:The unc-11 gene of Caenorhabditis elegans encodes multiple isoforms of a protein homologous to the mammalian brain-specific clathrin-adaptor protein AP180. The UNC-11 protein is expressed at high levels in the nervous system and at lower levels in other tissues. In neurons, UNC-11 is enriched at preSynaptic terminals but is also present in cell bodies. unc-11 mutants are defective in two aspects of Synaptic vesicle biogenesis. First, the SNARE protein synaptobrevin is mislocalized, no longer being exclusively localized to Synaptic Vesicles. The reduction of synaptobrevin at Synaptic Vesicles is the probable cause of the reduced neurotransmitter release observed in these mutants. Second, unc-11 mutants accumulate large Vesicles at synapses. We propose that the UNC-11 protein mediates two functions during Synaptic vesicle biogenesis: it recruits synaptobrevin to Synaptic vesicle membranes and it regulates the size of the budded vesicle during clathrin coat assembly.
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defective recycling of Synaptic Vesicles in synaptotagmin mutants of caenorhabditis elegans
Nature, 1995Co-Authors: Michael L Nonet, Yishi Jin, Erik M Jorgensen, Erika Hartwieg, Kim Schuske, Robert H HorvitzAbstract:SYNAPTOTAGMIN, an integral membrane protein of the Synaptic vesicle1,2, binds calcium and interacts with proteins of the plasma membrane4–6. These observations suggest several possible functions for synaptotagmin in Synaptic vesicle dynamics: it could facilitate exocytosis by promoting calcium-dependent fusion3, inhibit exocytosis by preventing fusion7, or facilitate endocytosis of Synaptic Vesicles from the plasma membrane by acting as a receptor for the endocytotic proteins of the clathrin AP2 complex8. Here we show that Synaptic Vesicles are depleted at Synaptic terminals in synaptotagmin mutants of the nematode Caenorhabditis elegans. This depletion is not caused by a defect in transport or by increased Synaptic vesicle release, but rather by a defect in retrieval of Synaptic Vesicles from the plasma membrane. Thus we propose that, as well as being involved in exocytosis, synaptotagmin functions in vesicular recycling.
