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Patrick Brennwald - One of the best experts on this subject based on the ideXlab platform.
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the Tomosyn homologue sro7 is a direct effector of the rab gtpase sec4 in post golgi vesicle tethering
Molecular Biology of the Cell, 2018Co-Authors: Guendalina Rossi, Kelly Watson, Wade Kennedy, Patrick BrennwaldAbstract:The Tomosyn/Sro7 family is thought to play an important role in cell surface trafficking both as an effector of Rab family GTPases and as a regulator of plasma-membrane SNARE function. Recent work has determined the binding site of GTP-bound Sec4 on Sro7. Here we examine the effect of mutations in Sro7 that block Sec4 binding in determining the role of this interaction in Sro7 function. Using an in vitro vesicle:vesicle tethering assay, we find that most of Sro7’s ability to tether vesicles is blocked by mutations that disrupt binding to Sec4-GTP. Similarly, genetic analysis demonstrates that the interaction with Sec4 is important for most of Sro7’s functions in vivo. The interaction of Sro7 with Sec4 appears to be particularly important when exocyst function is compromised. This provides strong evidence that Sro7 and the exocyst act as dual effector pathways downstream of Sec4. We also demonstrate that Sro7 tethering requires the presence of Sec4 on both opposing membranes and that homo-oligomerization of Sro7 occurs during vesicle tethering. This suggests a simple model for Sro7 function as a Rab effector in tethering post-Golgi vesicles to the plasma membrane in a pathway parallel to that of the exocyst complex.
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structural basis for recognition of the sec4 rab gtpase by its effector the lgl Tomosyn homologue sro7
Molecular Biology of the Cell, 2015Co-Authors: Kelly Watson, Guendalina Rossi, Brenda Temple, Patrick BrennwaldAbstract:Members of the Tomosyn/Lgl/Sro7 family play important roles in vesicle trafficking and cell polarity in eukaryotic cells. The yeast homologue, Sro7, is believed to act as a downstream effector of the Sec4 Rab GTPase to promote soluble N-ethylmaleimide–sensitive factor adaptor protein receptor (SNARE) assembly during Golgi-to–cell surface vesicle transport. Here we describe the identification of a Sec4 binding site on the surface of Sro7 that is contained within a cleft created by the junction of two adjacent β-propellers that form the core structure of Sro7. Computational docking experiments suggested four models for interaction of GTP-Sec4 with the Sro7 binding cleft. Further mutational and biochemical analyses confirmed that only one of the four docking arrangements is perfectly consistent with our genetic and biochemical interaction data. Close examination of this docking model suggests a structural basis for the high substrate and nucleotide selectivity in effector binding by Sro7. Finally, analysis of the surface variation within the homologous interaction site on Tomosyn-1 and Lgl-1 structural models suggests a possible conserved Rab GTPase effector function in Tomosyn vertebrate homologues.
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Structural basis for recognition of the Sec4 Rab GTPase by its effector, the Lgl/Tomosyn homologue, Sro7
Molecular Biology of the Cell, 2015Co-Authors: Kelly Watson, Guendalina Rossi, Brenda Temple, Patrick BrennwaldAbstract:Members of the Tomosyn/Lgl/Sro7 family play important roles in vesicle trafficking and cell polarity in eukaryotic cells. The yeast homologue, Sro7, is believed to act as a downstream effector of the Sec4 Rab GTPase to promote soluble N-ethylmaleimide–sensitive factor adaptor protein receptor (SNARE) assembly during Golgi-to–cell surface vesicle transport. Here we describe the identification of a Sec4 binding site on the surface of Sro7 that is contained within a cleft created by the junction of two adjacent β-propellers that form the core structure of Sro7. Computational docking experiments suggested four models for interaction of GTP-Sec4 with the Sro7 binding cleft. Further mutational and biochemical analyses confirmed that only one of the four docking arrangements is perfectly consistent with our genetic and biochemical interaction data. Close examination of this docking model suggests a structural basis for the high substrate and nucleotide selectivity in effector binding by Sro7. Finally, analysis of the surface variation within the homologous interaction site on Tomosyn-1 and Lgl-1 structural models suggests a possible conserved Rab GTPase effector function in Tomosyn vertebrate homologues.
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in vitro reconstitution of rab gtpase dependent vesicle clustering by the yeast lethal giant larvae Tomosyn homolog sro7
Journal of Biological Chemistry, 2015Co-Authors: Guendalina Rossi, Kelly Watson, Brenda Temple, Mallory Demonch, Patrick BrennwaldAbstract:Abstract Intracellular traffic in yeast between the Golgi and the cell surface is mediated by vesicular carriers which tether and fuse in a fashion that depends on the function of the Rab GTPase, Sec4. Overexpression of either of two Sec4 effectors, Sro7 or Sec15, results in the formation of a cluster of post-Golgi vesicles within the cell. Here we describe a novel assay which recapitulates post-Golgi vesicle clustering in vitro utilizing purified Sro7 and vesicles isolated from late secretory mutants. We show clustering in vitro closely replicates the in vivo clustering process as it is highly dependent on both Sro7 and GTP-Sec4. We also make use of this assay to characterize a novel mutant form of Sro7 which results in a protein which is specifically defective in vesicle clustering both in vivo and in vitro. We show that this mutation acts by effecting a conformational change in Sro7 from the closed to a more open structure. Our analysis demonstrates that the N-terminal propeller needs to be able to engage the C-terminal tail for vesicle clustering to occur. Consistent with this we show that occupancy of the N terminus of Sro7 by the t-SNARE Sec9 which results in the open conformation of Sro7, also acts to inhibit vesicle cluster formation by Sro7. This suggests a model by which a conformational switch in Sro7 acts to coordinate Rab-mediated vesicle tethering with SNARE assembly by requiring a single conformational state for both of these processes to occur.
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In Vitro Reconstitution of Rab GTPase-dependent Vesicle Clustering by the Yeast Lethal Giant Larvae/Tomosyn Homolog, Sro7
Journal of Biological Chemistry, 2014Co-Authors: Guendalina Rossi, Kelly Watson, Brenda Temple, Mallory Demonch, Patrick BrennwaldAbstract:Abstract Intracellular traffic in yeast between the Golgi and the cell surface is mediated by vesicular carriers which tether and fuse in a fashion that depends on the function of the Rab GTPase, Sec4. Overexpression of either of two Sec4 effectors, Sro7 or Sec15, results in the formation of a cluster of post-Golgi vesicles within the cell. Here we describe a novel assay which recapitulates post-Golgi vesicle clustering in vitro utilizing purified Sro7 and vesicles isolated from late secretory mutants. We show clustering in vitro closely replicates the in vivo clustering process as it is highly dependent on both Sro7 and GTP-Sec4. We also make use of this assay to characterize a novel mutant form of Sro7 which results in a protein which is specifically defective in vesicle clustering both in vivo and in vitro. We show that this mutation acts by effecting a conformational change in Sro7 from the closed to a more open structure. Our analysis demonstrates that the N-terminal propeller needs to be able to engage the C-terminal tail for vesicle clustering to occur. Consistent with this we show that occupancy of the N terminus of Sro7 by the t-SNARE Sec9 which results in the open conformation of Sro7, also acts to inhibit vesicle cluster formation by Sro7. This suggests a model by which a conformational switch in Sro7 acts to coordinate Rab-mediated vesicle tethering with SNARE assembly by requiring a single conformational state for both of these processes to occur.
Guendalina Rossi - One of the best experts on this subject based on the ideXlab platform.
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the Tomosyn homologue sro7 is a direct effector of the rab gtpase sec4 in post golgi vesicle tethering
Molecular Biology of the Cell, 2018Co-Authors: Guendalina Rossi, Kelly Watson, Wade Kennedy, Patrick BrennwaldAbstract:The Tomosyn/Sro7 family is thought to play an important role in cell surface trafficking both as an effector of Rab family GTPases and as a regulator of plasma-membrane SNARE function. Recent work has determined the binding site of GTP-bound Sec4 on Sro7. Here we examine the effect of mutations in Sro7 that block Sec4 binding in determining the role of this interaction in Sro7 function. Using an in vitro vesicle:vesicle tethering assay, we find that most of Sro7’s ability to tether vesicles is blocked by mutations that disrupt binding to Sec4-GTP. Similarly, genetic analysis demonstrates that the interaction with Sec4 is important for most of Sro7’s functions in vivo. The interaction of Sro7 with Sec4 appears to be particularly important when exocyst function is compromised. This provides strong evidence that Sro7 and the exocyst act as dual effector pathways downstream of Sec4. We also demonstrate that Sro7 tethering requires the presence of Sec4 on both opposing membranes and that homo-oligomerization of Sro7 occurs during vesicle tethering. This suggests a simple model for Sro7 function as a Rab effector in tethering post-Golgi vesicles to the plasma membrane in a pathway parallel to that of the exocyst complex.
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structural basis for recognition of the sec4 rab gtpase by its effector the lgl Tomosyn homologue sro7
Molecular Biology of the Cell, 2015Co-Authors: Kelly Watson, Guendalina Rossi, Brenda Temple, Patrick BrennwaldAbstract:Members of the Tomosyn/Lgl/Sro7 family play important roles in vesicle trafficking and cell polarity in eukaryotic cells. The yeast homologue, Sro7, is believed to act as a downstream effector of the Sec4 Rab GTPase to promote soluble N-ethylmaleimide–sensitive factor adaptor protein receptor (SNARE) assembly during Golgi-to–cell surface vesicle transport. Here we describe the identification of a Sec4 binding site on the surface of Sro7 that is contained within a cleft created by the junction of two adjacent β-propellers that form the core structure of Sro7. Computational docking experiments suggested four models for interaction of GTP-Sec4 with the Sro7 binding cleft. Further mutational and biochemical analyses confirmed that only one of the four docking arrangements is perfectly consistent with our genetic and biochemical interaction data. Close examination of this docking model suggests a structural basis for the high substrate and nucleotide selectivity in effector binding by Sro7. Finally, analysis of the surface variation within the homologous interaction site on Tomosyn-1 and Lgl-1 structural models suggests a possible conserved Rab GTPase effector function in Tomosyn vertebrate homologues.
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Structural basis for recognition of the Sec4 Rab GTPase by its effector, the Lgl/Tomosyn homologue, Sro7
Molecular Biology of the Cell, 2015Co-Authors: Kelly Watson, Guendalina Rossi, Brenda Temple, Patrick BrennwaldAbstract:Members of the Tomosyn/Lgl/Sro7 family play important roles in vesicle trafficking and cell polarity in eukaryotic cells. The yeast homologue, Sro7, is believed to act as a downstream effector of the Sec4 Rab GTPase to promote soluble N-ethylmaleimide–sensitive factor adaptor protein receptor (SNARE) assembly during Golgi-to–cell surface vesicle transport. Here we describe the identification of a Sec4 binding site on the surface of Sro7 that is contained within a cleft created by the junction of two adjacent β-propellers that form the core structure of Sro7. Computational docking experiments suggested four models for interaction of GTP-Sec4 with the Sro7 binding cleft. Further mutational and biochemical analyses confirmed that only one of the four docking arrangements is perfectly consistent with our genetic and biochemical interaction data. Close examination of this docking model suggests a structural basis for the high substrate and nucleotide selectivity in effector binding by Sro7. Finally, analysis of the surface variation within the homologous interaction site on Tomosyn-1 and Lgl-1 structural models suggests a possible conserved Rab GTPase effector function in Tomosyn vertebrate homologues.
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in vitro reconstitution of rab gtpase dependent vesicle clustering by the yeast lethal giant larvae Tomosyn homolog sro7
Journal of Biological Chemistry, 2015Co-Authors: Guendalina Rossi, Kelly Watson, Brenda Temple, Mallory Demonch, Patrick BrennwaldAbstract:Abstract Intracellular traffic in yeast between the Golgi and the cell surface is mediated by vesicular carriers which tether and fuse in a fashion that depends on the function of the Rab GTPase, Sec4. Overexpression of either of two Sec4 effectors, Sro7 or Sec15, results in the formation of a cluster of post-Golgi vesicles within the cell. Here we describe a novel assay which recapitulates post-Golgi vesicle clustering in vitro utilizing purified Sro7 and vesicles isolated from late secretory mutants. We show clustering in vitro closely replicates the in vivo clustering process as it is highly dependent on both Sro7 and GTP-Sec4. We also make use of this assay to characterize a novel mutant form of Sro7 which results in a protein which is specifically defective in vesicle clustering both in vivo and in vitro. We show that this mutation acts by effecting a conformational change in Sro7 from the closed to a more open structure. Our analysis demonstrates that the N-terminal propeller needs to be able to engage the C-terminal tail for vesicle clustering to occur. Consistent with this we show that occupancy of the N terminus of Sro7 by the t-SNARE Sec9 which results in the open conformation of Sro7, also acts to inhibit vesicle cluster formation by Sro7. This suggests a model by which a conformational switch in Sro7 acts to coordinate Rab-mediated vesicle tethering with SNARE assembly by requiring a single conformational state for both of these processes to occur.
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In Vitro Reconstitution of Rab GTPase-dependent Vesicle Clustering by the Yeast Lethal Giant Larvae/Tomosyn Homolog, Sro7
Journal of Biological Chemistry, 2014Co-Authors: Guendalina Rossi, Kelly Watson, Brenda Temple, Mallory Demonch, Patrick BrennwaldAbstract:Abstract Intracellular traffic in yeast between the Golgi and the cell surface is mediated by vesicular carriers which tether and fuse in a fashion that depends on the function of the Rab GTPase, Sec4. Overexpression of either of two Sec4 effectors, Sro7 or Sec15, results in the formation of a cluster of post-Golgi vesicles within the cell. Here we describe a novel assay which recapitulates post-Golgi vesicle clustering in vitro utilizing purified Sro7 and vesicles isolated from late secretory mutants. We show clustering in vitro closely replicates the in vivo clustering process as it is highly dependent on both Sro7 and GTP-Sec4. We also make use of this assay to characterize a novel mutant form of Sro7 which results in a protein which is specifically defective in vesicle clustering both in vivo and in vitro. We show that this mutation acts by effecting a conformational change in Sro7 from the closed to a more open structure. Our analysis demonstrates that the N-terminal propeller needs to be able to engage the C-terminal tail for vesicle clustering to occur. Consistent with this we show that occupancy of the N terminus of Sro7 by the t-SNARE Sec9 which results in the open conformation of Sro7, also acts to inhibit vesicle cluster formation by Sro7. This suggests a model by which a conformational switch in Sro7 acts to coordinate Rab-mediated vesicle tethering with SNARE assembly by requiring a single conformational state for both of these processes to occur.
Uri Ashery - One of the best experts on this subject based on the ideXlab platform.
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dynamic partitioning of synaptic vesicle pools by the snare binding protein Tomosyn
The Journal of Neuroscience, 2016Co-Authors: Victor A. Cazares, Uri Ashery, Johnny J. Saldate, Arasakumar Subramani, Yoav Bensimon, Meredith M Njus, Amanda Manly, Michael A Sutton, Edward L StuenkelAbstract:Neural networks engaged in high-frequency activity rely on sustained synaptic vesicle recycling and coordinated recruitment from functionally distinct synaptic vesicle (SV) pools. However, the molecular pathways matching neural activity to SV dynamics and release requirements remain unclear. Here we identify unique roles of SNARE-binding Tomosyn1 (Tomo1) proteins as activity-dependent substrates that regulate dynamics of SV pool partitioning at rat hippocampal synapses. Our analysis is based on monitoring changes in distinct functionally defined SV pools via V-Glut1-pHluorin fluorescence in cultured hippocampal neurons in response to alterations in presynaptic protein expression. Specifically, we find knockdown of Tomo1 facilitates release efficacy from the Readily Releasable Pool (RRP), and regulates SV distribution to the Total Recycling Pool (TRP), which is matched by a decrease in the SV Resting Pool. Notably, these effects were reversed by Tomo1 rescue and overexpression. Further, we identify that these actions of Tomo1 are regulated via activity-dependent phosphorylation by cyclin-dependent kinase 5 (Cdk5). Assessment of molecular interactions that may contribute to these actions identified Tomo1 interaction with the GTP-bound state of Rab3A, an SV GTPase involved in SV targeting and presynaptic membrane tethering. In addition, Tomo1 via Rab3A-GTP was also observed to interact with Synapsin 1a/b cytoskeletal interacting proteins. Finally, our data indicate that Tomo1 regulation of SV pool sizes serves to adapt presynaptic neurotransmitter release to chronic silencing of network activity. Overall, the results establish Tomo1 proteins as central mediators in neural activity-dependent changes in SV distribution among SV pools. SIGNIFICANCE STATEMENT Although information transfer at central synapses via sustained high-frequency neural activity requires coordinated synaptic vesicle (SV) recycling, the mechanism(s) by which synapses sense and dynamically modify SV pools to match network demands remains poorly defined. To advance understanding, we quantified SV pool sizes and their sensitivity to neural activity while altering Tomo1 expression, a putative regulator of the presynaptic Readily Releasable Pool. Remarkably, we find Tomo1 actions to extend beyond the Readily Releasable Pool to mediate the Total Recycling Pool and SV Resting Pool distribution, and this action is sensitive to neural activity through Cdk5 phosphorylation of Tomo1. Moreover, Tomo1 appears to exert these actions through interaction with Rab3A-GTP and synapsin proteins. Together, our results argue that Tomo1 is a central mediator of SV availability for neurotransmission.
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a combined optogenetic knockdown strategy reveals a major role of Tomosyn in mossy fiber synaptic plasticity
Cell Reports, 2015Co-Authors: Yoav Bensimon, Edward L Stuenkel, Alma Rodenasruano, Karina Alvina, Pablo E Castillo, Uri AsheryAbstract:Summary Neurotransmitter release probability (P r ) largely determines the dynamic properties of synapses. While much is known about the role of presynaptic proteins in transmitter release, their specific contribution to synaptic plasticity is unclear. One such protein, Tomosyn, is believed to reduce P r by interfering with the SNARE complex formation. Tomosyn is enriched at hippocampal mossy fiber-to-CA3 pyramidal cell synapses (MF-CA3), which characteristically exhibit low P r , strong synaptic facilitation, and pre-synaptic protein kinase A (PKA)-dependent long-term potentiation (LTP). To evaluate Tomosyn's role in MF-CA3 function, we used a combined knockdown (KD)-optogenetic strategy whereby presynaptic neurons with reduced Tomosyn levels were selectively activated by light. Using this approach in mouse hippocampal slices, we found that facilitation, LTP, and PKA-induced potentiation were significantly impaired at Tomosyn-deficient synapses. These findings not only indicate that Tomosyn is a key regulator of MF-CA3 plasticity but also highlight the power of a combined KD-optogenetic approach to determine the role of presynaptic proteins.
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differential interaction of Tomosyn with syntaxin and snap25 depends on domains in the wd40 β propeller core and determines its inhibitory activity
Journal of Biological Chemistry, 2014Co-Authors: Noa Bielopolski, Edward L Stuenkel, Dana Baron, Markus Sauer, Uri AsheryAbstract:Neuronal exocytosis depends on efficient formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and is regulated by Tomosyn, a SNARE-binding protein. To gain new information about Tomosyn's activity, we characterized its mobility and organization on the plasma membrane (PM) in relation to other SNARE proteins and inhibition of exocytosis. By using direct stochastic optical reconstruction microscopy (dSTORM), we found Tomosyn to be organized in small clusters adjacent to syntaxin clusters. In addition, we show that Tomosyn is present in both syntaxin-Tomosyn complexes and syntaxin-SNAP25-Tomosyn complexes. Tomosyn mutants that lack residues 537–578 or 897–917 from its β-propeller core diffused faster on the PM and exhibited reduced binding to SNAP25, suggesting that these mutants shift the equilibrium between Tomosyn-syntaxin-SNAP25 complexes on the PM to Tomosyn-syntaxin complexes. As these deletion mutants impose less inhibition on exocytosis, we suggest that Tomosyn inhibition is mediated via Tomosyn-syntaxin-SNAP25 complexes and not Tomosyn-syntaxin complexes. These findings characterize, for the first time, Tomosyn's dynamics at the PM and its relation to its inhibition of exocytosis.
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structural and functional analysis of Tomosyn identifies domains important in exocytotic regulation
Journal of Biological Chemistry, 2011Co-Authors: Antionette L Williams, Noa Bielopolski, Uri Ashery, Daphna Meroz, Daniel R Passmore, Nir Bental, Stephen A Ernst, Edward L StuenkelAbstract:Tomosyn is a 130-kDa cytosolic R-SNARE protein that associates with Q-SNAREs and reduces exocytotic activity. Two paralogous genes, Tomosyn-1 and -2, occur in mammals and produce seven different isoforms via alternative splicing. Here, we map the structural differences between the yeast homologue of m-Tomosyn-1, Sro7, and Tomosyn genes/isoforms to identify domains critical to the regulation of exocytotic activity to Tomosyn that are outside the soluble N-ethylmaleimide-sensitive attachment receptor motif. Homology modeling of m-Tomosyn-1 based on the known structure of yeast Sro7 revealed a highly conserved functional conformation but with Tomosyn containing three additional loop domains that emanate from a β-propeller core. Notably, deletion of loops 1 and 3 eliminates Tomosyn inhibitory activity on secretion without altering its soluble N-ethylmaleimide-sensitive attachment receptor pairing with syntaxin1A. By comparison, deletion of loop 2, which contains the hypervariable splice region, did not reduce the ability of Tomosyn to inhibit regulated secretion. However, exon variation within the hypervariable splice region resulted in significant differences in protein accumulation of Tomosyn-2 isoforms. Functional analysis of s-Tomosyn-1, m-Tomosyn-1, m-Tomosyn-2, and xb-Tomosyn-2 demonstrated that they exert similar inhibitory effects on elevated K+-induced secretion in PC12 cells, although m-Tomosyn-2 was novel in strongly augmenting basal secretion. Finally, we report that m-Tomosyn-1 is a target substrate for SUMO 2/3 conjugation and that mutation of this small ubiquitin-related modifier target site (Lys-730) enhances m-Tomosyn-1 inhibition of secretion without altering interaction with syntaxin1A. Together these results suggest that multiple domains outside the R-SNARE of Tomosyn are critical to the efficacy of inhibition by Tomosyn on exocytotic secretion.
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Tomosyn expression pattern in the mouse hippocampus suggests both presynaptic and postsynaptic functions
Frontiers in Neuroanatomy, 2010Co-Authors: Boaz Barak, Antionette L Williams, Noa Bielopolski, Irit Gottfried, Eitan Okun, Meghan A Brown, Ulf Matti, Jens Rettig, Edward L Stuenkel, Uri AsheryAbstract:The protein Tomosyn decreases synaptic transmission and release probability of vesicles, and is essential for modulating synaptic transmission in neurons. In this study, we provide a detailed description of the expression and localization patterns of Tomosyn1 and Tomosyn2 in the subareas of the mouse hippocampus. Using confocal and two-photon high-resolution microscopy we demonstrate that Tomosyn colocalizes with several pre- and postsynaptic markers and is found mainly in glutamatergic synapses. Specifically, we show that Tomosyn1 is differentially distributed in the mouse hippocampus and concentrated mainly in the hilus and mossy fibers. Surprisingly, we found that Tomosyn2 is expressed in the subiculum, CA1 and CA2 pyramidal cell bodies, dendrites and spines, and colocalizes with PSD95, suggesting a postsynaptic role. These results suggest that in addition to the well-characterized presynaptic function of Tomosyn in neurotransmitter release, Tomosyn2 might have a postsynaptic function, and place Tomosyn as a more general regulator of synaptic transmission and plasticity.
Toshiaki Sakisaka - One of the best experts on this subject based on the ideXlab platform.
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roles of Tomosyn in neurotransmitter release
2015Co-Authors: Yasunori Yamamoto, Toshiaki SakisakaAbstract:SNARE complexes and synaptotagmin mediate synaptic vesicle fusion with the plasma membrane of the active zone for the neurotransmitter release from presynaptic nerve terminals responding to neuronal signals. Many regulatory proteins for the SNARE complex formation have been identified. Among them, our originally identified protein, Tomosyn, is likely to be a key molecule for the regulation of the SNARE complex-involved pre-fusion step and the Ca2+-triggered synaptic vesicle fusion step. Tomosyn inhibits SNARE complex formation and thereby inhibits synaptic vesicle fusion by sequestering target SNAREs through its C-terminal VAMP-like domain in a Ca2+-independent manner. The N-terminal WD40 repeats are the site for its binding to synaptotagmin-1, a Ca2+-sensor protein, in a Ca2+-dependent manner. The interaction negatively regulates the Ca2+-dependent synaptic vesicle fusion mediated by synaptotagmin-1. Thus, Tomosyn is a potent inhibitor, temporally and stepwisely regulating the synaptic vesicle fusion at the active zone, for the synchronized and fast neurotransmitter release.
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Tomosyn inhibits synaptotagmin 1 mediated step of ca2 dependent neurotransmitter release through its n terminal wd40 repeats
Journal of Biological Chemistry, 2010Co-Authors: Yasunori Yamamoto, Kohei Fujikura, Sumiko Mochida, Takao Kurooka, Naoyuki Miyazaki, Katsuhisa Kawai, Kenji Iwasaki, Toshiaki SakisakaAbstract:Abstract Neurotransmitter release is triggered by Ca2+ binding to a low affinity Ca2+ sensor, mostly synaptotagmin-1, which catalyzes SNARE-mediated synaptic vesicle fusion. Tomosyn negatively regulates Ca2+-dependent neurotransmitter release by sequestering target SNAREs (t-SNAREs) through the C-terminal VAMP-like domain. In addition to the C-terminus, the N-terminal WD40 repeats of Tomosyn also have potent inhibitory activity toward Ca2+-dependent neurotransmitter release, although the molecular mechanism underlying this effect remains elusive. Here, we show that through its N-terminal WD40 repeats, Tomosyn directly binds to synaptotagmin-1 in a Ca2+-dependent manner. The N-terminal WD40 repeats impaired the activities of synaptotagmin-1 to promote SNARE complex-mediated membrane fusion and to bend the lipid bilayers. Decreased acetylcholine release from N-terminal WD40 repeats-microinjected superior cervical ganglion neurons was relieved by microinjection of the cytoplasmic domain of synaptotagmin-1. These results indicate that, upon direct binding, the N-terminal WD40 repeats negatively regulate the synaptotagmin-1-mediated step of Ca2+-dependent neurotransmitter release. Furthermore, we show that synaptotagmin-1 binding enhances the t-SNARE-sequestering activity of Tomosyn. These results suggest that the interplay between Tomosyn and synaptotagmin-1 underlies inhibitory control of Ca2+-dependent neurotransmitter release.
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the tail domain of Tomosyn controls membrane fusion through Tomosyn displacement by vamp2
Biochemical and Biophysical Research Communications, 2010Co-Authors: Yasunori Yamamoto, Kohei Fujikura, Mio Sakaue, Kenjiro Okimura, Yuta Kobayashi, Toshihiro Nakamura, Toshiaki SakisakaAbstract:Abstract Neurotransmitter release is regulated by SNARE complex-mediated synaptic vesicle fusion. Tomosyn sequesters target SNAREs (t-SNAREs) through its C-terminal VAMP-like domain (VLD). Cumulative biochemical results suggest that the Tomosyn–SNARE complex is so tight that VAMP2 cannot displace Tomosyn. Based on these results, the Tomosyn–SNARE complex has been believed to be a dead-end complex to inhibit neurotransmitter release. On the other hand, some studies using siRNA depletion of Tomosyn suggest that Tomosyn positively regulates exocytosis. Therefore, it is still controversial whether Tomosyn is a simple inhibitor for neurotransmitter release. We recently reported that the inhibitory activity of Tomosyn is regulated by the tail domain binding to the VLD. In this study, we employed the liposome fusion assay in order to further understand modes of action of Tomosyn in detail. The tail domain unexpectedly had no effect on binding of the VLD to t-SNARE-bearing liposomes. Nonetheless, the tail domain decreased the inhibitory activity of the VLD on the SNARE complex-mediated liposome fusion. These results indicate that the tail domain controls membrane fusion through Tomosyn displacement by VAMP2. Deletion of the tail domain-binding region in the VLD retained the binding to t-SNAREs and promoted the liposome fusion. Together, we propose here a novel mechanism of Tomosyn that controls synaptic vesicle fusion positively by serving as a placeholder for VAMP2.
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reciprocal intramolecular interactions of Tomosyn control its inhibitory activity on snare complex formation
Journal of Biological Chemistry, 2009Co-Authors: Yasunori Yamamoto, Sumiko Mochida, Takao Kurooka, Toshiaki SakisakaAbstract:Neurotransmitter release from presynaptic nerve terminals is regulated by SNARE complex-mediated synaptic vesicle fusion. Tomosyn, a negative regulator of neurotransmitter release, which is composed of N-terminal WD40 repeats, a tail domain, and a C-terminal VAMP-like domain, is known to inhibit SNARE complex formation by sequestering target SNAREs (t-SNAREs) upon interaction of its C-terminal VAMP-like domain with t-SNAREs. However, it remains unclear how the inhibitory activity of Tomosyn is regulated. Here we show that the tail domain functions as a regulator of the inhibitory activity of Tomosyn through intramolecular interactions. The binding of the tail domain to the C-terminal VAMP-like domain interfered with the interaction of the C-terminal VAMP-like domain with t-SNAREs, and thereby repressed the inhibitory activity of Tomosyn on the SNARE complex formation. The repressed inhibitory activity of Tomosyn was restored by the binding of the tail domain to the N-terminal WD40 repeats. These results indicate that the probable conformational change of Tomosyn mediated by the intramolecular interactions of the tail domain controls its inhibitory activity on the SNARE complex formation, leading to a regulated inhibition of neurotransmitter release.
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dual inhibition of snare complex formation by Tomosyn ensures controlled neurotransmitter release
Journal of Cell Biology, 2008Co-Authors: Toshiaki Sakisaka, Yasunori Yamamoto, Sumiko Mochida, Michiko Nakamura, Kouki Nishikawa, Hiroyoshi Ishizaki, Miki Okamototanaka, Jun Miyoshi, Yoshinori Fujiyoshi, Toshiya ManabeAbstract:Neurotransmitter release from presynaptic nerve terminals is regulated by soluble NSF attachment protein receptor (SNARE) complex–mediated synaptic vesicle fusion. Tomosyn inhibits SNARE complex formation and neurotransmitter release by sequestering syntaxin-1 through its C-terminal vesicle-associated membrane protein (VAMP)–like domain (VLD). However, in Tomosyn-deficient mice, the SNARE complex formation is unexpectedly decreased. In this study, we demonstrate that the N-terminal WD-40 repeat domain of Tomosyn catalyzes the oligomerization of the SNARE complex. Microinjection of the Tomosyn N-terminal WD-40 repeat domain into neurons prevented stimulated acetylcholine release. Thus, Tomosyn inhibits neurotransmitter release by catalyzing oligomerization of the SNARE complex through the N-terminal WD-40 repeat domain in addition to the inhibitory activity of the C-terminal VLD.
Janet E Richmond - One of the best experts on this subject based on the ideXlab platform.
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Tomosyn dependent regulation of synaptic transmission is required for a late phase of associative odor memory
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Kaiyun Chen, Antje Richlitzki, David E Featherstone, Martin Schwarzel, Janet E RichmondAbstract:Synaptic vesicle secretion requires the assembly of fusogenic SNARE complexes. Consequently proteins that regulate SNARE complex formation can significantly impact synaptic strength. The SNARE binding protein Tomosyn has been shown to potently inhibit exocytosis by sequestering SNARE proteins in nonfusogenic complexes. The Tomosyn–SNARE interaction is regulated by protein kinase A (PKA), an enzyme implicated in learning and memory, suggesting Tomosyn could be an important effector in PKA-dependent synaptic plasticity. We tested this hypothesis in Drosophila, in which the role of the PKA pathway in associative learning has been well established. We first determined that panneuronal Tomosyn knockdown by RNAi enhanced synaptic strength at the Drosophila larval neuromuscular junction, by increasing the evoked response duration. We next assayed memory performance 3 min (early memory) and 3 h (late memory) after aversive olfactory learning. Whereas early memory was unaffected by Tomosyn knockdown, late memory was reduced by 50%. Late memory is a composite of stable and labile components. Further analysis determined that Tomosyn was specifically required for the anesthesia-sensitive, labile component, previously shown to require cAMP signaling via PKA in mushroom bodies. Together these data indicate that Tomosyn has a conserved role in the regulation of synaptic transmission and provide behavioral evidence that Tomosyn is involved in a specific component of late associative memory.
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in vivo analysis of conserved c elegans Tomosyn domains
PLOS ONE, 2011Co-Authors: Anna O Burdina, Susan M. Klosterman, Ludmila Shtessel, Shawn Ahmed, Janet E RichmondAbstract:Neurosecretion is critically dependent on the assembly of a macromolecular complex between the SNARE proteins syntaxin, SNAP-25 and synaptobrevin. Evidence indicates that the binding of Tomosyn to syntaxin and SNAP-25 interferes with this assembly, thereby negatively regulating both synaptic transmission and peptide release. Tomosyn has two conserved domains: an N-terminal encompassing multiple WD40 repeats predicted to form two β-propeller structures and a C-terminal SNARE-binding motif. To assess the function of each domain, we performed an in vivo analysis of the N- and C- terminal domains of C. elegans Tomosyn (TOM-1) in a tom-1 mutant background. We verified that both truncated TOM-1 constructs were transcribed at levels comparable to rescuing full-length TOM-1, were of the predicted size, and localized to synapses. Unlike full-length TOM-1, expression of the N- or C-terminal domains alone was unable to restore inhibitory control of synaptic transmission in tom-1 mutants. Similarly, co-expression of both domains failed to restore TOM-1 function. In addition, neither the N- nor C-terminal domain inhibited release when expressed in a wild-type background. Based on these results, we conclude that the ability of Tomosyn to regulate neurotransmitter release in vivo depends on the physical integrity of the protein, indicating that both N- and C-terminal domains are necessary but not sufficient for effective inhibition of release in vivo.
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Differential Regulation of Synaptic Vesicle Tethering and Docking by UNC-18 and TOM-1
Frontiers in Synaptic Neuroscience, 2010Co-Authors: Elena O Gracheva, Ed B. Maryon, Martine Berthelot-grosjean, Janet E RichmondAbstract:The assembly of SNARE complexes between syntaxin, SNAP-25 and synaptobrevin is required to prime synaptic vesicles for fusion. Since Munc18 and Tomosyn compete for syntaxin interactions, the interplay between these proteins is predicted to be important in regulating synaptic transmission. We explored this possibility, by examining genetic interactions between C. elegans unc-18(Munc18), unc-64(syntaxin) and tom-1(Tomosyn). We have previously demonstrated that unc-18 mutants have reduced synaptic transmission, whereas tom-1 mutants exhibit enhanced release. Here we show that the unc-18 mutant release defect is associated with loss of two morphologically distinct vesicle pools; those tethered within 25 nm of the plasma membrane and those docked with the plasma membrane. In contrast, priming defective unc-13 mutants accumulate tethered vesicles, while docked vesicles are greatly reduced, indicating tethering is UNC-18-dependent and occurs in the absence of priming. C. elegans unc-64 mutants phenocopy unc-18 mutants, losing both tethered and docked vesicles, whereas overexpression of open syntaxin preferentially increases vesicle docking, suggesting UNC-18/closed syntaxin interactions are responsible for vesicle tethering. Given the competition between vertebrate Tomosyn and Munc18, for syntaxin binding, we hypothesized that C. elegans TOM-1 may inhibit both UNC-18-dependent vesicle targeting steps. Consistent with this hypothesis, tom-1 mutants exhibit enhanced UNC-18 plasma membrane localization and a concomitant increase in both tethered and docked synaptic vesicles. Furthermore, in tom-1;unc-18 double mutants the docked, primed vesicle pool is preferentially rescued relative to unc-18 single mutants. Together these data provide evidence for the differential regulation of two vesicle targeting steps by UNC-18 and TOM-1 through competitive interactions with syntaxin.
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functional interactions among the snare regulators unc 13 Tomosyn and unc 18
2008Co-Authors: Robby M Weimer, Janet E RichmondAbstract:Neurotransmitters are released from secretory vesicles following calcium-triggered fusion with the plasma membrane. These exocytotic events are driven by assembly of tertiary soluble N-ethylmaleimide–sensitive factor attachment receptor (SNARE) complexes among the vesicle SNARE, synaptobrevin, the plasma membrane-associated SNAREs, syntaxin, and synaptosome-associated protein of 25 kDa (SNAP-25). Proteins that effect SNARE complex assembly are thus important regulators of synaptic strength. This chapter reviews our current understanding of the roles played by three SNARE interacting proteins: UNC-13(Munc13), TOM-1(Tomosyn) and UNC-18(Munc18). We discuss studies from both invertebrate and vertebrate model systems, highlighting recent advances, the current consensus on molecular mechanisms of action, and unresolved aspects of their function.
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Tomosyn negatively regulates both synaptic transmitter and neuropeptide release at the c elegans neuromuscular junction
The Journal of Physiology, 2007Co-Authors: Elena O Gracheva, Anna O Burdina, Denis Touroutine, Martine Berthelotgrosjean, Hetal Parekh, Janet E RichmondAbstract:The SNARE proteins, syntaxin, SNAP-25 and synaptobrevin form a tertiary complex essential for vesicle fusion. Proteins that influence SNARE complex assembly are therefore likely to be important regulators of fusion events. In this study we have focused on Tomosyn, a highly conserved, neuronally enriched, syntaxin-binding protein that has been implicated in the regulation of vesicle exocytosis. To directly test the role of Tomosyn in neurosecretion we analysed loss-of-function mutants in the single Caenorhabditis elegans Tomosyn gene, tom-1. These mutants exhibit enhanced synaptic transmission based on electrophysiological analysis of neuromuscular junction activity. This phenotype is the result of increased synaptic vesicle priming. In addition, we present evidence that tom-1 mutants also exhibit enhanced peptide release from dense core vesicles. These results indicate that Tomosyn negatively regulates secretion for both vesicle types, possibly through a common mechanism, interfering with SNARE complex formation, thereby inhibiting vesicle fusion.