The Experts below are selected from a list of 3618 Experts worldwide ranked by ideXlab platform
Nilsalexander Lakomek - One of the best experts on this subject based on the ideXlab platform.
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structural dynamics and transient lipid binding of Synaptobrevin 2 tune snare assembly and membrane fusion
2019Co-Authors: Reinhard Jahn, Nilsalexander Lakomek, Halenur Yavuz, Angel PerezlaraAbstract:Intrinsically disordered proteins (IDPs) and their conformational transitions play an important role in neurotransmitter release at the neuronal synapse. Here, the SNARE proteins are essential by forming the SNARE complex that drives vesicular membrane fusion. While it is widely accepted that the SNARE proteins are intrinsically disordered in their monomeric prefusion form, important mechanistic aspects of this prefusion conformation and its lipid interactions, before forming the SNARE complex, are not fully understood at the molecular level and remain controversial. Here, by a combination of NMR and fluorescence spectroscopy methods, we find that vesicular Synaptobrevin-2 (syb-2) in its monomeric prefusion conformation shows high flexibility, characteristic for an IDP, but also a high dynamic range and increasing rigidity from the N to C terminus. The gradual increase in rigidity correlates with an increase in lipid binding affinity from the N to C terminus. It could also explain the increased rate for C-terminal SNARE zippering, known to be faster than N-terminal SNARE zippering. Also, the syb-2 SNARE motif and, in particular, the linker domain show transient and weak membrane binding, characterized by a high off-rate and low (millimolar) affinity. The transient membrane binding of syb-2 may compensate for the repulsive forces between the two membranes and/or the SNARE motifs and the membranes, helping to destabilize the hydrophilic-hydrophobic boundary in the bilayer. Therefore, we propose that optimum flexibility and membrane binding of syb-2 regulate SNARE assembly and minimize repulsive forces during membrane fusion.
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structural dynamics and transient lipid binding of Synaptobrevin 2 tune snare assembly and membrane fusion
2019Co-Authors: Reinhard Jahn, Nilsalexander Lakomek, Halenur Yavuz, Angel PerezlaraAbstract:Intrinsically disordered proteins (IDPs) and their conformational transitions play an important role in neurotransmitter release at the neuronal synapse. Here, the SNARE proteins are essential by forming the SNARE complex that drives vesicular membrane fusion. While it is widely accepted that the SNARE proteins are intrinsically disordered in their monomeric prefusion form, important mechanistic aspects of this prefusion conformation and its lipid interactions, before forming the SNARE complex, are not fully understood at the molecular level and remain controversial. Here, by a combination of NMR and fluorescence spectroscopy methods, we find that vesicular Synaptobrevin-2 (syb-2) in its monomeric prefusion conformation shows high flexibility, characteristic for an IDP, but also a high dynamic range and increasing rigidity from the N to C terminus. The gradual increase in rigidity correlates with an increase in lipid binding affinity from the N to C terminus. It could also explain the increased rate for C-terminal SNARE zippering, known to be faster than N-terminal SNARE zippering. Also, the syb-2 SNARE motif and, in particular, the linker domain show transient and weak membrane binding, characterized by a high off-rate and low (millimolar) affinity. The transient membrane binding of syb-2 may compensate for the repulsive forces between the two membranes and/or the SNARE motifs and the membranes, helping to destabilize the hydrophilic-hydrophobic boundary in the bilayer. Therefore, we propose that optimum flexibility and membrane binding of syb-2 regulate SNARE assembly and minimize repulsive forces during membrane fusion.
Simon J H Brookes - One of the best experts on this subject based on the ideXlab platform.
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selective coexpression of synaptic proteins α synuclein cysteine string protein α synaptophysin synaptotagmin 1 and Synaptobrevin 2 in vesicular acetylcholine transporter immunoreactive axons in the guinea pig ileum
2013Co-Authors: D F Sharrad, Wei Ping Gai, Simon J H BrookesAbstract:Parkinson's disease is a neurodegenerative disorder characterized by Lewy bodies and neurites composed mainly of the presynaptic protein α-synuclein. Frequently, Lewy bodies and neurites are identified in the gut of Parkinson's disease patients and may underlie associated gastrointestinal dysfunctions. We recently reported selective expression of α-synuclein in the axons of cholinergic neurons in the guinea pig and human distal gut; however, it is not clear whether α-synuclein expression varies along the gut, nor how closely expression is associated with other synaptic proteins. We used multiple-labeling immunohistochemistry to quantify which neurons in the guinea pig ileum expressed α-synuclein, cysteine string protein-α (CSPα), synaptophysin, synaptotagmin-1, or Synaptobrevin-2 in their axons. Among the 10 neurochemically defined axonal populations, a significantly greater proportion of vesicular acetylcholine transporter-immunoreactive (VAChT-IR) varicosities (80% ± 1.7%, n = 4, P < 0.001) contained α-synuclein immunoreactivity, and a significantly greater proportion of α-synuclein-IR axons also contained VAChT immunoreactivity (78% ± 1.3%, n = 4) compared with any of the other nine populations (P < 0.001). Among synaptophysin-, synaptotagmin-1-, Synaptobrevin-2-, and CSPα-IR varicosities, 98% ± 0.7%, 96% ± 0.7%, 88% ± 1.6%, and 85% ± 2.9% (n = 4) contained α-synuclein immunoreactivity, respectively. Among α-synuclein-IR varicosities, 96% ± 0.9%, 99% ± 0.6%, 83% ± 1.9%, and 87% ± 2.3% (n = 4) contained synaptophysin-, synaptotagmin-1-, Synaptobrevin-2-, and CSPα immunoreactivity, respectively. We report a close association between the expression of α-synuclein and the expression of other synaptic proteins in cholinergic axons in the guinea pig ileum. Selective expression of α-synuclein may relate to the neurotransmitter system utilized and predispose cholinergic enteric neurons to degeneration in Parkinson's disease.
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selective coexpression of synaptic proteins α synuclein cysteine string protein α synaptophysin synaptotagmin 1 and Synaptobrevin 2 in vesicular acetylcholine transporter immunoreactive axons in the guinea pig ileum
2013Co-Authors: D F Sharrad, Simon J H BrookesAbstract:Parkinson's disease is a neurodegenerative disorder characterized by Lewy bodies and neurites composed mainly of the presynaptic protein α-synuclein. Frequently, Lewy bodies and neurites are identified in the gut of Parkinson's disease patients and may underlie associated gastrointestinal dysfunctions. We recently reported selective expression of α-synuclein in the axons of cholinergic neurons in the guinea pig and human distal gut; however, it is not clear whether α-synuclein expression varies along the gut, nor how closely expression is associated with other synaptic proteins. We used multiple-labeling immunohistochemistry to quantify which neurons in the guinea pig ileum expressed α-synuclein, cysteine string protein-α (CSPα), synaptophysin, synaptotagmin-1, or Synaptobrevin-2 in their axons. Among the 10 neurochemically defined axonal populations, a significantly greater proportion of vesicular acetylcholine transporter-immunoreactive (VAChT-IR) varicosities (80% ± 1.7%, n = 4, P < 0.001) contained α-synuclein immunoreactivity, and a significantly greater proportion of α-synuclein-IR axons also contained VAChT immunoreactivity (78% ± 1.3%, n = 4) compared with any of the other nine populations (P < 0.001). Among synaptophysin-, synaptotagmin-1-, Synaptobrevin-2-, and CSPα-IR varicosities, 98% ± 0.7%, 96% ± 0.7%, 88% ± 1.6%, and 85% ± 2.9% (n = 4) contained α-synuclein immunoreactivity, respectively. Among α-synuclein-IR varicosities, 96% ± 0.9%, 99% ± 0.6%, 83% ± 1.9%, and 87% ± 2.3% (n = 4) contained synaptophysin-, synaptotagmin-1-, Synaptobrevin-2-, and CSPα immunoreactivity, respectively. We report a close association between the expression of α-synuclein and the expression of other synaptic proteins in cholinergic axons in the guinea pig ileum. Selective expression of α-synuclein may relate to the neurotransmitter system utilized and predispose cholinergic enteric neurons to degeneration in Parkinson's disease. J. Comp. Neurol. 521:2523–2537, 2013. © 2013 Wiley Periodicals, Inc.
Reinhard Jahn - One of the best experts on this subject based on the ideXlab platform.
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structural dynamics and transient lipid binding of Synaptobrevin 2 tune snare assembly and membrane fusion
2019Co-Authors: Reinhard Jahn, Nilsalexander Lakomek, Halenur Yavuz, Angel PerezlaraAbstract:Intrinsically disordered proteins (IDPs) and their conformational transitions play an important role in neurotransmitter release at the neuronal synapse. Here, the SNARE proteins are essential by forming the SNARE complex that drives vesicular membrane fusion. While it is widely accepted that the SNARE proteins are intrinsically disordered in their monomeric prefusion form, important mechanistic aspects of this prefusion conformation and its lipid interactions, before forming the SNARE complex, are not fully understood at the molecular level and remain controversial. Here, by a combination of NMR and fluorescence spectroscopy methods, we find that vesicular Synaptobrevin-2 (syb-2) in its monomeric prefusion conformation shows high flexibility, characteristic for an IDP, but also a high dynamic range and increasing rigidity from the N to C terminus. The gradual increase in rigidity correlates with an increase in lipid binding affinity from the N to C terminus. It could also explain the increased rate for C-terminal SNARE zippering, known to be faster than N-terminal SNARE zippering. Also, the syb-2 SNARE motif and, in particular, the linker domain show transient and weak membrane binding, characterized by a high off-rate and low (millimolar) affinity. The transient membrane binding of syb-2 may compensate for the repulsive forces between the two membranes and/or the SNARE motifs and the membranes, helping to destabilize the hydrophilic-hydrophobic boundary in the bilayer. Therefore, we propose that optimum flexibility and membrane binding of syb-2 regulate SNARE assembly and minimize repulsive forces during membrane fusion.
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structural dynamics and transient lipid binding of Synaptobrevin 2 tune snare assembly and membrane fusion
2019Co-Authors: Reinhard Jahn, Nilsalexander Lakomek, Halenur Yavuz, Angel PerezlaraAbstract:Intrinsically disordered proteins (IDPs) and their conformational transitions play an important role in neurotransmitter release at the neuronal synapse. Here, the SNARE proteins are essential by forming the SNARE complex that drives vesicular membrane fusion. While it is widely accepted that the SNARE proteins are intrinsically disordered in their monomeric prefusion form, important mechanistic aspects of this prefusion conformation and its lipid interactions, before forming the SNARE complex, are not fully understood at the molecular level and remain controversial. Here, by a combination of NMR and fluorescence spectroscopy methods, we find that vesicular Synaptobrevin-2 (syb-2) in its monomeric prefusion conformation shows high flexibility, characteristic for an IDP, but also a high dynamic range and increasing rigidity from the N to C terminus. The gradual increase in rigidity correlates with an increase in lipid binding affinity from the N to C terminus. It could also explain the increased rate for C-terminal SNARE zippering, known to be faster than N-terminal SNARE zippering. Also, the syb-2 SNARE motif and, in particular, the linker domain show transient and weak membrane binding, characterized by a high off-rate and low (millimolar) affinity. The transient membrane binding of syb-2 may compensate for the repulsive forces between the two membranes and/or the SNARE motifs and the membranes, helping to destabilize the hydrophilic-hydrophobic boundary in the bilayer. Therefore, we propose that optimum flexibility and membrane binding of syb-2 regulate SNARE assembly and minimize repulsive forces during membrane fusion.
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two Synaptobrevin molecules are sufficient for vesicle fusion in central nervous system synapses
2011Co-Authors: Raunak Sinha, Reinhard Jahn, Saheeb Ahmed, Jurgen KlingaufAbstract:Exocytosis of synaptic vesicles (SVs) during fast synaptic transmission is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly formed by the coil-coiling of three members of this protein family: vesicle SNARE protein, Synaptobrevin 2 (syb2), and the presynaptic membrane SNAREs syntaxin-1A and SNAP-25. However, it is controversially debated how many SNARE complexes are minimally needed for SV priming and fusion. To quantify this effective number, we measured the fluorescence responses from single fusing vesicles expressing pHluorin (pHl), a pH-sensitive variant of GFP, fused to the luminal domain of the vesicular SNARE syb2 (spH) in cultured hippocampal neurons lacking endogenous syb2. Fluorescence responses were quantal, with the unitary signals precisely corresponding to single pHluorin molecules. Using this approach we found that two copies of spH per SV fully rescued evoked fusion whereas SVs expressing only one spH were unable to rapidly fuse upon stimulation. Thus, two syb2 molecules and likely two SNARE complexes are necessary and sufficient for SV fusion during fast synaptic transmission.
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a novel site of action for α snap in the snare conformational cycle controlling membrane fusion
2007Co-Authors: Marcin Barszczewski, Dirk Fasshauer, John Jia En Chua, Alexander Stein, Ulrike Winter, Rainer Heintzmann, Felipe E Zilly, Thorsten Lang, Reinhard JahnAbstract:Regulated exocytosis in neurons and neuroendocrine cells requires the formation of a stable soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of Synaptobrevin-2/vesicle-associated membrane protein 2, synaptosome-associated protein of 25 kDa (SNAP-25), and syntaxin 1. This complex is subsequently disassembled by the concerted action of α-SNAP and the ATPases associated with different cellular activities-ATPase N-ethylmaleimide-sensitive factor (NSF). We report that NSF inhibition causes accumulation of α-SNAP in clusters on plasma membranes. Clustering is mediated by the binding of α-SNAP to uncomplexed syntaxin, because cleavage of syntaxin with botulinum neurotoxin C1 or competition by using antibodies against syntaxin SNARE motif abolishes clustering. Binding of α-SNAP potently inhibits Ca2+-dependent exocytosis of secretory granules and SNARE-mediated liposome fusion. Membrane clustering and inhibition of both exocytosis and liposome fusion are counteracted by NSF but not when an α-SNAP mutant defective in NSF activation is used. We conclude that α-SNAP inhibits exocytosis by binding to the syntaxin SNARE motif and in turn prevents SNARE assembly, revealing an unexpected site of action for α-SNAP in the SNARE cycle that drives exocytotic membrane fusion.
Vladimir Parpura - One of the best experts on this subject based on the ideXlab platform.
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Astrocytic vesicles and gliotransmitters: Slowness of vesicular release and Synaptobrevin2-laden vesicle nanoarchitecture.
2015Co-Authors: Robert Zorec, Alexei Verkhratsky, José J. Rodríguez, Vladimir ParpuraAbstract:Abstract Neurotransmitters released at synapses activate neighboring astrocytes, which in turn, modulate neuronal activity by the release of diverse neuroactive substances that include classical neurotransmitters such as glutamate, GABA or ATP. Neuroactive substances are released from astrocytes through several distinct molecular mechanisms, for example, by diffusion through membrane channels, by translocation via plasmalemmal transporters or by vesicular exocytosis. Vesicular release regulated by a stimulus-mediated increase in cytosolic calcium involves soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptor (SNARE)-dependent merger of the vesicle membrane with the plasmalemma. Up to 25 molecules of Synaptobrevin 2 (Sb2), a SNARE complex protein, reside at a single astroglial vesicle; an individual neuronal, i.e. synaptic, vesicle contains ∼70 Sb2 molecules. It is proposed that this paucity of Sb2 molecules in astrocytic vesicles may determine the slow secretion. In the present essay we shall overview multiple aspects of vesicular architecture and types of vesicles based on their cargo and dynamics in astroglial cells.
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nanopore sensing of botulinum toxin type b by discriminating an enzymatically cleaved peptide from a synaptic protein Synaptobrevin 2 derivative
2015Co-Authors: Yong Wang, Vedrana Montana, Vladimir Parpura, Vladimir Grubisic, Randy F Stout, Liqun GuAbstract:Botulinum neurotoxins (BoNTs) are the most lethal toxin known to human. Biodefense requires early and rapid detection of BoNTs. Traditionally, BoNTs can be detected by looking for signs of botulism in mice that receive an injection of human material, serum or stool. While the living animal assay remains the most sensitive approach, it is costly, slow and associated with legal and ethical constrains. Various biochemical, optical and mechanical methods have been developed for BoNTs detection with improved speed, but with lesser sensitivity. Here, we report a novel nanopore-based BoNT type B (BoNT-B) sensor that monitors the toxin’s enzymatic activity on its substrate, a recombinant synaptic protein Synaptobrevin 2 derivative. By analyzing the modulation of the pore current caused by the specific BoNT-B-digested peptide as a marker, the presence of BoNT-B at a subnanomolar concentration was identified within minutes. The nanopore detector would fill the niche for a much needed rapid and highly sensitive dete...
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Temporal characteristics of vesicular fusion in astrocytes: examination of Synaptobrevin 2-laden vesicles at single vesicle resolution
2011Co-Authors: Erik B Malarkey, Vladimir ParpuraAbstract:Non-technical summary Astrocytes have been shown to release transmitters by vesicle fusion, in a manner similar to that of neuronal exocytosis. The details of this process in astrocytes are not well understood, so we used a fluorescently labelled vesicle protein, synapto-pHluorin (spH), to track how these fusions occurred. When astrocytes were mechanically stimulated we saw a slow burst of fusions, while other stimuli caused a relatively even sustained rate of fusion. We observed two distinct types of events, transient and full fusions, the proportion of which was stimulus dependent. Similarly, stability of the vesicle fusion pore with the plasma membrane varied with the stimulus. We describe the effects on fusion events resulting from expressing variants of exocytotic proteins, synaptotagmin 1 and SNAP25B. Studying the characteristics of astrocytic exocytosis will aid in the general understanding of this process and also events at the tripartite synapse, both in health and disease. Abstract Astrocytes can release various gliotransmitters in response to stimuli that cause increases in intracellular Ca2+ levels; this secretion occurs via a regulated exocytosis pathway. Indeed, astrocytes express protein components of the vesicular secretory apparatus. However, the detailed temporal characteristics of vesicular fusions in astrocytes are not well understood. In order to start addressing this issue, we used total internal reflection fluorescence microscopy (TIRFM) to visualize vesicular fusion events in astrocytes expressing the fluorescent Synaptobrevin 2 derivative, synapto-pHluorin. Although our cultured astrocytes from visual cortex express synaptosome-associated protein of 23 kDa (SNAP23), but not of 25 kDa (SNAP25), these glial cells exhibited a slow burst of exocytosis under mechanical stimulation; the expression of SNAP25B did not affect bursting behaviour. The relative amount of two distinct types of events observed, transient and full fusions, depended on the applied stimulus. Expression of exogenous synaptotagmin 1 (Syt1) in astrocytes endogenously expressing Syt4, led to a greater proportion of transient fusions when astrocytes were stimulated with bradykinin, a stimulus otherwise resulting in more full fusions. Additionally, we studied the stability of the transient fusion pore by measuring its dwell time, relation to vesicular size, flickering and decay slope; all of these characteristics were secretagogue dependent. The expression of SNAP25B or Syt1 had complex effects on transient fusion pore stability in a stimulus-specific manner. SNAP25B obliterated the appearance of flickers and reduced the dwell time when astrocytes were mechanically stimulated, while astrocytes expressing SNAP25B and stimulated with bradykinin had a reduction in decay slope. Syt1 reduced the dwell time when astrocytes were stimulated either mechanically or with bradykinin. Our detailed study of temporal characteristics of astrocytic exocytosis will not only aid the general understanding of this process, but also the interpretation of the events at the tripartite synapse, both in health and disease.
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age dependent spatial segregation of Synaptobrevin 2 containing vesicles in astrocytes
2011Co-Authors: Oliver C Loson, Vladimir ParpuraAbstract:Astrocytes possess much of the same exocytotic protein machinery as neurons do and can release various gliotransmitters stored in their secretory vesicles. An essential component of this exocytotic machinery is the vesicle-associated membrane protein Synaptobrevin 2 (Sb2). In order to assess whether vesicular age plays a role in determining the intracellular location of vesicles in astrocytes, we generated a fluorescent chimeric form of Sb2. We appended the Sb2 cytosolic N-terminus with the fluorescent ‘timer’ protein DsRedE5, which changes its fluorescence emission from green to red as it ages. We found that Sb2-containing vesicles in astrocytes segregate and localize intracellularly in an age dependent manner. Younger vesicles predominately localize at the periphery of cell somata and processes, while older vesicles predominately locate at the central portion of the cell body. These findings raise the notion that there might be differential astrocyte-neuron signaling at sites away or at the tripartite synapse that could be modulated by the age of vesicles and/or their cargo.
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vesicular transmitter release from astrocytes
2006Co-Authors: Vedrana Montana, Erik B Malarkey, Claudia Verderio, Michela Matteoli, Vladimir ParpuraAbstract:Astrocytes can release a variety of transmitters, including glutamate and ATP, in response to stimuli that induce increases in intracellular Ca2+ levels. This release occurs via a regulated, exocytotic pathway. As evidence of this, astrocytes express protein components of the vesicular secretory apparatus, including Synaptobrevin 2, syntaxin, and SNAP-23. Additionally, astrocytes possess vesicular organelles, the essential morphological elements required for regulated Ca2+-dependent transmitter release. The location of specific exocytotic sites on these cells, however, remains to be unequivocally determined. © 2006 Wiley-Liss, Inc.
Angel Perezlara - One of the best experts on this subject based on the ideXlab platform.
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structural dynamics and transient lipid binding of Synaptobrevin 2 tune snare assembly and membrane fusion
2019Co-Authors: Reinhard Jahn, Nilsalexander Lakomek, Halenur Yavuz, Angel PerezlaraAbstract:Intrinsically disordered proteins (IDPs) and their conformational transitions play an important role in neurotransmitter release at the neuronal synapse. Here, the SNARE proteins are essential by forming the SNARE complex that drives vesicular membrane fusion. While it is widely accepted that the SNARE proteins are intrinsically disordered in their monomeric prefusion form, important mechanistic aspects of this prefusion conformation and its lipid interactions, before forming the SNARE complex, are not fully understood at the molecular level and remain controversial. Here, by a combination of NMR and fluorescence spectroscopy methods, we find that vesicular Synaptobrevin-2 (syb-2) in its monomeric prefusion conformation shows high flexibility, characteristic for an IDP, but also a high dynamic range and increasing rigidity from the N to C terminus. The gradual increase in rigidity correlates with an increase in lipid binding affinity from the N to C terminus. It could also explain the increased rate for C-terminal SNARE zippering, known to be faster than N-terminal SNARE zippering. Also, the syb-2 SNARE motif and, in particular, the linker domain show transient and weak membrane binding, characterized by a high off-rate and low (millimolar) affinity. The transient membrane binding of syb-2 may compensate for the repulsive forces between the two membranes and/or the SNARE motifs and the membranes, helping to destabilize the hydrophilic-hydrophobic boundary in the bilayer. Therefore, we propose that optimum flexibility and membrane binding of syb-2 regulate SNARE assembly and minimize repulsive forces during membrane fusion.
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structural dynamics and transient lipid binding of Synaptobrevin 2 tune snare assembly and membrane fusion
2019Co-Authors: Reinhard Jahn, Nilsalexander Lakomek, Halenur Yavuz, Angel PerezlaraAbstract:Intrinsically disordered proteins (IDPs) and their conformational transitions play an important role in neurotransmitter release at the neuronal synapse. Here, the SNARE proteins are essential by forming the SNARE complex that drives vesicular membrane fusion. While it is widely accepted that the SNARE proteins are intrinsically disordered in their monomeric prefusion form, important mechanistic aspects of this prefusion conformation and its lipid interactions, before forming the SNARE complex, are not fully understood at the molecular level and remain controversial. Here, by a combination of NMR and fluorescence spectroscopy methods, we find that vesicular Synaptobrevin-2 (syb-2) in its monomeric prefusion conformation shows high flexibility, characteristic for an IDP, but also a high dynamic range and increasing rigidity from the N to C terminus. The gradual increase in rigidity correlates with an increase in lipid binding affinity from the N to C terminus. It could also explain the increased rate for C-terminal SNARE zippering, known to be faster than N-terminal SNARE zippering. Also, the syb-2 SNARE motif and, in particular, the linker domain show transient and weak membrane binding, characterized by a high off-rate and low (millimolar) affinity. The transient membrane binding of syb-2 may compensate for the repulsive forces between the two membranes and/or the SNARE motifs and the membranes, helping to destabilize the hydrophilic-hydrophobic boundary in the bilayer. Therefore, we propose that optimum flexibility and membrane binding of syb-2 regulate SNARE assembly and minimize repulsive forces during membrane fusion.
