Edwin Chapman | University of Wisconsin-Madison (original) (raw)

Papers by Edwin Chapman

Neurotransmitter-filled synaptic vesicles (SV) mediate synaptic transmission and are a hallmark s... more Neurotransmitter-filled synaptic vesicles (SV) mediate synaptic transmission and are a hallmark specialization in neuronal axons. Yet, how SV proteins are sorted to presynaptic nerve terminals remains the subject of debate. The leading models posit that these proteins are randomly trafficked throughout neurons and are selectively retained in presynaptic boutons. Here, we used the RUSH system, in conjunction with HaloTag labeling approaches, to study the egress of two distinct SV proteins from the soma of cultured neurons. In sharp contrast to the selective retention model, both proteins selectively and specifically entered axons and did not traffic through dendrites; only upon overexpression do SV proteins spillover into other compartments. Moreover, we observed that SV constituents were first delivered to the presynaptic plasma membrane before incorporation into SVs. These experiments reveal a new-found membrane trafficking pathway in classically polarized mammalian neurons and pro...

Synaptotagmin (SYT) 7 has emerged as key regulator of presynaptic function, but its localization ... more Synaptotagmin (SYT) 7 has emerged as key regulator of presynaptic function, but its localization and precise function in the synaptic vesicle cycle remain unclear. Here, we used iGluSnFR to optically and directly interrogate glutamate release, at the single bouton level, in SYT7 KO dissociated mouse hippocampal neurons. We analyzed asynchronous release, paired pulse facilitation, and synaptic vesicle replenishment, and found that SYT7 contributes to each of these processes to different degrees. ‘Zap-and-freeze’ electron microscopy revealed that loss of SYT7 impairs the docking of synaptic vesicles after a stimulus and the recovery of depleted synaptic vesicles after a stimulus train. To execute these functions, SYT7 must be targeted to the plasma membrane via γ-secretase-mediated cleavage of the amino terminus, followed by palmitoylation. The complex sorting itinerary of SYT7 endows this Ca2+-sensor with the ability to control crucial forms of synaptic function and plasticity. SYT7 ...

eLife, 2021

Synaptotagmin 7 (SYT7) has emerged as a key regulator of presynaptic function, but its localizati... more Synaptotagmin 7 (SYT7) has emerged as a key regulator of presynaptic function, but its localization and precise role in the synaptic vesicle cycle remain the subject of debate. Here, we used iGluSnFR to optically interrogate glutamate release, at the single-bouton level, in SYT7KO-dissociated mouse hippocampal neurons. We analyzed asynchronous release, paired-pulse facilitation, and synaptic vesicle replenishment and found that SYT7 contributes to each of these processes to different degrees. ‘Zap-and-freeze’ electron microscopy revealed that a loss of SYT7 diminishes docking of synaptic vesicles after a stimulus and inhibits the recovery of depleted synaptic vesicles after a stimulus train. SYT7 supports these functions from the axonal plasma membrane, where its localization and stability require both γ-secretase-mediated cleavage and palmitoylation. In summary, SYT7 is a peripheral membrane protein that controls multiple modes of synaptic vesicle (SV) exocytosis and plasticity, in...

Molecular biology of the cell, Jan 14, 2018

In chromaffin cells, the kinetics of fusion pore expansion vary depending on which synaptotagmin ... more In chromaffin cells, the kinetics of fusion pore expansion vary depending on which synaptotagmin isoform (Syt-1 or Syt-7) drives release. Our recent studies have shown that fusion pores of granules harboring Syt-1 expand more rapidly than those harboring Syt-7. Here, we sought to define the structural specificity of synaptotagmin action at the fusion pore by manipulating the Ca-binding C2B module. We generated a chimeric Syt-1 in which its C2B Ca-binding loops had been exchanged for those of Syt-7. Fusion pores of granules harboring a Syt-1 C2B chimera with all three Ca-binding loops of Syt-7 (Syt-1:7C2B) exhibited slower rates of fusion pore expansion and neuropeptide cargo release relative to WT Syt-1. After fusion, this chimera also dispersed more slowly from fusion sites than WT protein. We speculate that the Syt-1:7 C2Band WT Syt-1 are likely to differ in their interactions with Caand membranes. Subsequentanddata demonstrated that the chimera exhibits a higher affinity for phos...

Diabetes, Jul 26, 2016

Synaptotagmin(Syt)-7, a major component of the exocytotic machinery in neurons, is also the major... more Synaptotagmin(Syt)-7, a major component of the exocytotic machinery in neurons, is also the major Syt in rodent pancreatic β-cells shown to mediate glucose-stimulated insulin secretion (GSIS). However, Syt-7's precise exocytotic actions in β-cells remain unknown. We show that Syt-7 is abundant in human β-cells. Adenovirus-shRNA knockdown (KD) of Syt-7 in human islets reduced first- and second-phase GSIS attributed to the reduction of exocytosis of pre-docked and newcomer insulin secretory granules (SGs). Glucose stimulation expectedly induced Syt-7 association in Ca(2+)-dependent manner with syntaxin-3 and syntaxin-1A SNARE complexes known to mediate exocytosis of newcomer and pre-docked SGs, respectively. However, Syt-7-KD did not disrupt SNARE complex assembly. Instead, E.M. analysis showed that Syt-7-KD reduced the recruitment of SGs to the plasma membrane after glucose-stimulated depletion; and which could not be rescued by glucagon-like peptide-1 pretreatment. To assess the...

Journal of Biological Chemistry, 2015

SNARE proteins catalyze many forms of biological membrane fusion, including Ca 2؉-triggered exocy... more SNARE proteins catalyze many forms of biological membrane fusion, including Ca 2؉-triggered exocytosis. Although fusion mediated by SNAREs generally involves proteins anchored to each fusing membrane by a transmembrane domain (TMD), the role of TMDs remains unclear, and previous studies diverge on whether SNAREs can drive fusion without a TMD. This issue is important because it relates to the question of the structure and composition of the initial fusion pore, as well as the question of whether SNAREs mediate fusion solely by creating close proximity between two membranes versus a more active role in transmitting force to the membrane to deform and reorganize lipid bilayer structure. To test the role of membrane attachment, we generated four variants of the synaptic v-SNARE synaptobrevin-2 (syb2) anchored to the membrane by lipid instead of protein. These constructs were tested for functional efficacy in three different systems as follows: Ca 2؉-triggered dense core vesicle exocytosis, spontaneous synaptic vesicle exocytosis, and Ca 2؉-synaptotagmin-enhanced SNARE-mediated liposome fusion. Lipid-anchoring motifs harboring one or two lipid acylation sites completely failed to support fusion in any of these assays. Only the lipid-anchoring motif from cysteine string protein-␣, which harbors many lipid acylation sites, provided support for fusion but at levels well below that achieved with wild type syb2. Thus, lipid-anchored syb2 provides little or no support for exocytosis, and anchoring syb2 to a membrane by a TMD greatly improves its function. The low activity seen with syb2-cysteine string protein-␣ may reflect a slower alternative mode of SNARE-mediated membrane fusion.

Journal of Cell Biology, 2007

Botulinum neurotoxins (BoNTs) target presynaptic nerve terminals by recognizing specific neuronal... more Botulinum neurotoxins (BoNTs) target presynaptic nerve terminals by recognizing specific neuronal surface receptors. Two homologous synaptic vesicle membrane proteins, synaptotagmins (Syts) I and II, bind toxins BoNT/B and G. However, a direct demonstration that Syts I/II mediate toxin binding and entry into neurons is lacking. We report that BoNT/B and G fail to bind and enter hippocampal neurons cultured from Syt I knockout mice. Wild-type Syts I and II (but not Syt I loss-of-function toxin-binding domain mutants) restored binding and entry of BoNT/B and G in Syt I–null neurons, thus demonstrating that Syts I/II are protein receptors for BoNT/B and G. Furthermore, mice lacking complex gangliosides exhibit reduced sensitivity to BoNT/G, and binding and entry of BoNT/A, B, and G into hippocampal neurons lacking gangliosides is diminished. These data suggest that gangliosides are the shared coreceptor for BoNT/A, B, and G, supporting a double-receptor model for these three BoNTs for ...

Journal of Cell Biology, 2003

Botulinum neurotoxins (BoNTs) cause botulism by entering neurons and cleaving proteins that media... more Botulinum neurotoxins (BoNTs) cause botulism by entering neurons and cleaving proteins that mediate neurotransmitter release; disruption of exocytosis results in paralysis and death. The receptors for BoNTs are thought to be composed of both proteins and gangliosides; however, protein components that mediate toxin entry have not been identified. Using gain-of-function and loss-of-function approaches, we report here that the secretory vesicle proteins, synaptotagmins (syts) I and II, mediate the entry of BoNT/B (but not BoNT/A or E) into PC12 cells. Further, we demonstrate that BoNT/B entry into PC12 cells and rat diaphragm motor nerve terminals was activity dependent and can be blocked using fragments of syt II that contain the BoNT/B-binding domain. Finally, we show that syt II fragments, in conjunction with gangliosides, neutralized BoNT/B in intact mice. These findings establish that syts I and II can function as protein receptors for BoNT/B.

Science, 2004

We investigated the effect of synaptotagmin I on membrane fusion mediated by neuronal SNARE prote... more We investigated the effect of synaptotagmin I on membrane fusion mediated by neuronal SNARE proteins, SNAP-25, syntaxin, and synaptobrevin, which were reconstituted into vesicles. In the presence of Ca 2+ , the cytoplasmic domain of synaptotagmin I (syt) strongly stimulated membrane fusion when synaptobrevin densities were similar to those found in native synaptic vesicles. The Ca 2+ dependence of syt-stimulated fusion was modulated by changes in lipid composition of the vesicles and by a truncation that mimics cleavage of SNAP-25 by botulinum neurotoxin A. Stimulation of fusion was abolished by disrupting the Ca 2+ -binding activity, or by severing the tandem C2 domains, of syt. Thus, syt and SNAREs are likely to represent the minimal protein complement for Ca 2+ -triggered exocytosis.

Science, 2004

The fusion pore of regulated exocytosis is a channel that connects and spans the vesicle and plas... more The fusion pore of regulated exocytosis is a channel that connects and spans the vesicle and plasma membranes. The molecular composition of this important intermediate structure of exocytosis is unknown. Here, we found that mutations of some residues within the transmembrane segment of syntaxin (Syx), a plasma membrane protein essential for exocytosis, altered neurotransmitter flux through fusion pores and altered pore conductance. The residues that influenced fusion-pore flux lay along one face of an α-helical model. Thus, the fusion pore is formed at least in part by a circular arrangement of 5 to 8 Syx transmembrane segments in the plasma membrane.

Proceedings of the National Academy of Sciences, 2002

Synaptotagmin (syt) I, an integral membrane protein localized to secretory vesicles, is a putativ... more Synaptotagmin (syt) I, an integral membrane protein localized to secretory vesicles, is a putative Ca 2+ sensor for exocytosis. Its N terminus spans the membrane once, and its cytoplasmic domain contains two conserved C2 domains, designated C2A and C2B. The isolated C2A domain penetrates membranes in response to Ca 2+ ; isolated C2B does not. Here, we have addressed the function of each C2 domain, but in the context of the intact cytoplasmic domain (C2A-C2B), by using fluorescent reporters placed in the Ca 2+ -binding loops of either C2A or C2B. Surprisingly, these reporters revealed that, analogous to C2A, a Ca 2+ -binding loop in C2B directly penetrates into lipid bilayers. Penetration of each C2 domain was very rapid ( k on ≈10 10 M −1 ⋅s −1 ) and resulted in high affinity C2A-C2B–liposome complexes ( K d ≈13–14 nM). C2B-bilayer penetration strictly depended on the presence, but not the membrane binding activity, of an adjacent C2A domain, severing C2A from C2B after protein synt...

Proceedings of the National Academy of Sciences, 2003

Neuronal exocytosis is mediated by Ca 2+ -triggered rearrangements between proteins and lipids th... more Neuronal exocytosis is mediated by Ca 2+ -triggered rearrangements between proteins and lipids that result in the opening and dilation of fusion pores. Synaptotagmin I (syt I) is a Ca 2+ -sensing protein proposed to regulate fusion pore dynamics via Ca 2+ -promoted binding of its cytoplasmic domain (C2A-C2B) to effector molecules, including anionic phospholipids and other copies of syt. Functional studies indicate that Ca 2+ -triggered oligomerization of syt is a critical step in excitation–secretion coupling; however, this activity has recently been called into question. Here, we show that Ca 2+ does not drive the oligomerization of C2A-C2B in solution. However, analysis of Ca 2+ ⋅C2A-C2B bound to lipid monolayers, using electron microscopy, revealed the formation of ring-like heptameric oligomers that are ≈11 nm long and ≈11 nm in diameter. In some cases, C2A-C2B also assembled into long filaments. Oligomerization, but not membrane binding, was disrupted by neutralization of two l...

PLoS Pathogens, 2010

Tetanus neurotoxin causes the disease tetanus, which is characterized by rigid paralysis. The tox... more Tetanus neurotoxin causes the disease tetanus, which is characterized by rigid paralysis. The toxin acts by inhibiting the release of neurotransmitters from inhibitory neurons in the spinal cord that innervate motor neurons and is unique among the clostridial neurotoxins due to its ability to shuttle from the periphery to the central nervous system. Tetanus neurotoxin is thought to interact with a high affinity receptor complex that is composed of lipid and protein components; however, the identity of the protein receptor remains elusive. In the current study, we demonstrate that toxin binding, to dissociated hippocampal and spinal cord neurons, is greatly enhanced by driving synaptic vesicle exocytosis. Moreover, tetanus neurotoxin entry and subsequent cleavage of synaptobrevin II, the substrate for this toxin, was also dependent on synaptic vesicle recycling. Next, we identified the potential synaptic vesicle binding protein for the toxin and found that it corresponded to SV2; tetanus neurotoxin was unable to cleave synaptobrevin II in SV2 knockout neurons. Toxin entry into knockout neurons was rescued by infecting with viruses that express SV2A or SV2B. Tetanus toxin elicited the hyper excitability in dissociated spinal cord neurons-due to preferential loss of inhibitory transmission-that is characteristic of the disease. Surprisingly, in dissociated cortical cultures, low concentrations of the toxin preferentially acted on excitatory neurons. Further examination of the distribution of SV2A and SV2B in both spinal cord and cortical neurons revealed that SV2B is to a large extent localized to excitatory terminals, while SV2A is localized to inhibitory terminals. Therefore, the distinct effects of tetanus toxin on cortical and spinal cord neurons are not due to differential expression of SV2 isoforms. In summary, the findings reported here indicate that SV2A and SV2B mediate binding and entry of tetanus neurotoxin into central neurons.

Neuroscience, 2010

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

Nature Neuroscience, 2011

Synaptotagmin I (syt1) is required for normal rates of synaptic vesicle endo-and exocytosis. Howe... more Synaptotagmin I (syt1) is required for normal rates of synaptic vesicle endo-and exocytosis. However, whether the kinetic defects observed during endocytosis in syt1 knockout neurons are secondary to defective exocytosis, or whether syt1 directly regulates the rate of vesicle retrieval, remains unresolved. In order to address this question, it is necessary to dissociate these two activities. Here, we have uncoupled the function of syt1 in exo-and endocytosis by re-targeting of the protein, or via mutagenesis of its tandem C2-domains; the impact of these manipulations on exo-and endocytosis were analyzed via electrophysiology, in conjunction with optical imaging of the vesicle cycle. These experiments uncovered a direct role for syt1 in endocytosis. Surprisingly, either C2-domain of syt1-C2A or C2B-was able to function as Ca 2+-sensor for endocytosis. Hence, syt1 functions as a dual Ca 2+ sensor for both endo-and exocytosis, potentially coupling these two limbs of the vesicle cycle. Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Nature Neuroscience, 2009

Synaptotagmin-IV (syt-IV) is a membrane trafficking protein that influences learning and memory, ... more Synaptotagmin-IV (syt-IV) is a membrane trafficking protein that influences learning and memory, but its localization and role in synaptic function remain unclear. Here we discovered that syt-IV localizes to BDNF-containing vesicles in hippocampal neurons. Syt-IV/BDNF-harboring vesicles undergo exocytosis in both axons and dendrites, and syt-IV inhibits BDNF release at both sites. Knockout of syt-IV increases, and over-expression decreases, the rate of FM dye destaining from presynaptic terminals indirectly via changes in post-synaptic release of BDNF. Hence, postsynaptic syt-IV regulates the trans-synaptic action of BDNF to control presynaptic vesicle dynamics. Furthermore, selective loss of presynaptic syt-IV increased spontaneous quantal release, while loss of post-synaptic syt-IV increased quantal amplitude. Finally, syt-IV knockout mice exhibit enhanced LTP, which depends entirely on disinhibition of BDNF release. Thus, regulation of BDNF secretion by syt-IV emerges as a mechanism to maintain synaptic strength within a useful range during long-term potentiation.

Journal of Neurophysiology, 2004

We show that activation of postsynaptic inositol 1,4,5-tris-phosphate receptors (IP3Rs) with the ... more We show that activation of postsynaptic inositol 1,4,5-tris-phosphate receptors (IP3Rs) with the IP3R agonist adenophostin A (AdA) produces large increases in AMPA receptor (AMPAR) excitatory postsynaptic current (EPSC) amplitudes at hippocampal CA1 synapses. Co-perfusion of the Ca2+chelator bis-( o-aminophenoxy)- N,N,N′,N′-tetraacetic acid strongly inhibited AdA-enhanced increases in EPSC amplitudes. We examined the role of AMPAR insertion/anchoring in basal synaptic transmission. Perfusion of an inhibitor of synaptotagmin-soluble n-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor SNARE-mediated exocytosis depressed basal EPSC amplitudes, whereas a peptide that inhibits GluR2/3 interactions with postsynaptic density-95 (PDZ) domain proteins glutamate receptor interacting protein (GRIP)/protein interacting with C-kinase-1 (PICK1) enhanced basal synaptic transmission. These results suggest that constitutive trafficking and anchoring of AMPARs help maintain basal syn...

The Journal of General Physiology, 2010

The Journal of General Physiology, 2012

Neurotransmitter-filled synaptic vesicles (SV) mediate synaptic transmission and are a hallmark s... more Neurotransmitter-filled synaptic vesicles (SV) mediate synaptic transmission and are a hallmark specialization in neuronal axons. Yet, how SV proteins are sorted to presynaptic nerve terminals remains the subject of debate. The leading models posit that these proteins are randomly trafficked throughout neurons and are selectively retained in presynaptic boutons. Here, we used the RUSH system, in conjunction with HaloTag labeling approaches, to study the egress of two distinct SV proteins from the soma of cultured neurons. In sharp contrast to the selective retention model, both proteins selectively and specifically entered axons and did not traffic through dendrites; only upon overexpression do SV proteins spillover into other compartments. Moreover, we observed that SV constituents were first delivered to the presynaptic plasma membrane before incorporation into SVs. These experiments reveal a new-found membrane trafficking pathway in classically polarized mammalian neurons and pro...

Synaptotagmin (SYT) 7 has emerged as key regulator of presynaptic function, but its localization ... more Synaptotagmin (SYT) 7 has emerged as key regulator of presynaptic function, but its localization and precise function in the synaptic vesicle cycle remain unclear. Here, we used iGluSnFR to optically and directly interrogate glutamate release, at the single bouton level, in SYT7 KO dissociated mouse hippocampal neurons. We analyzed asynchronous release, paired pulse facilitation, and synaptic vesicle replenishment, and found that SYT7 contributes to each of these processes to different degrees. ‘Zap-and-freeze’ electron microscopy revealed that loss of SYT7 impairs the docking of synaptic vesicles after a stimulus and the recovery of depleted synaptic vesicles after a stimulus train. To execute these functions, SYT7 must be targeted to the plasma membrane via γ-secretase-mediated cleavage of the amino terminus, followed by palmitoylation. The complex sorting itinerary of SYT7 endows this Ca2+-sensor with the ability to control crucial forms of synaptic function and plasticity. SYT7 ...

eLife, 2021

Synaptotagmin 7 (SYT7) has emerged as a key regulator of presynaptic function, but its localizati... more Synaptotagmin 7 (SYT7) has emerged as a key regulator of presynaptic function, but its localization and precise role in the synaptic vesicle cycle remain the subject of debate. Here, we used iGluSnFR to optically interrogate glutamate release, at the single-bouton level, in SYT7KO-dissociated mouse hippocampal neurons. We analyzed asynchronous release, paired-pulse facilitation, and synaptic vesicle replenishment and found that SYT7 contributes to each of these processes to different degrees. ‘Zap-and-freeze’ electron microscopy revealed that a loss of SYT7 diminishes docking of synaptic vesicles after a stimulus and inhibits the recovery of depleted synaptic vesicles after a stimulus train. SYT7 supports these functions from the axonal plasma membrane, where its localization and stability require both γ-secretase-mediated cleavage and palmitoylation. In summary, SYT7 is a peripheral membrane protein that controls multiple modes of synaptic vesicle (SV) exocytosis and plasticity, in...

Molecular biology of the cell, Jan 14, 2018

In chromaffin cells, the kinetics of fusion pore expansion vary depending on which synaptotagmin ... more In chromaffin cells, the kinetics of fusion pore expansion vary depending on which synaptotagmin isoform (Syt-1 or Syt-7) drives release. Our recent studies have shown that fusion pores of granules harboring Syt-1 expand more rapidly than those harboring Syt-7. Here, we sought to define the structural specificity of synaptotagmin action at the fusion pore by manipulating the Ca-binding C2B module. We generated a chimeric Syt-1 in which its C2B Ca-binding loops had been exchanged for those of Syt-7. Fusion pores of granules harboring a Syt-1 C2B chimera with all three Ca-binding loops of Syt-7 (Syt-1:7C2B) exhibited slower rates of fusion pore expansion and neuropeptide cargo release relative to WT Syt-1. After fusion, this chimera also dispersed more slowly from fusion sites than WT protein. We speculate that the Syt-1:7 C2Band WT Syt-1 are likely to differ in their interactions with Caand membranes. Subsequentanddata demonstrated that the chimera exhibits a higher affinity for phos...

Diabetes, Jul 26, 2016

Synaptotagmin(Syt)-7, a major component of the exocytotic machinery in neurons, is also the major... more Synaptotagmin(Syt)-7, a major component of the exocytotic machinery in neurons, is also the major Syt in rodent pancreatic β-cells shown to mediate glucose-stimulated insulin secretion (GSIS). However, Syt-7's precise exocytotic actions in β-cells remain unknown. We show that Syt-7 is abundant in human β-cells. Adenovirus-shRNA knockdown (KD) of Syt-7 in human islets reduced first- and second-phase GSIS attributed to the reduction of exocytosis of pre-docked and newcomer insulin secretory granules (SGs). Glucose stimulation expectedly induced Syt-7 association in Ca(2+)-dependent manner with syntaxin-3 and syntaxin-1A SNARE complexes known to mediate exocytosis of newcomer and pre-docked SGs, respectively. However, Syt-7-KD did not disrupt SNARE complex assembly. Instead, E.M. analysis showed that Syt-7-KD reduced the recruitment of SGs to the plasma membrane after glucose-stimulated depletion; and which could not be rescued by glucagon-like peptide-1 pretreatment. To assess the...

Journal of Biological Chemistry, 2015

SNARE proteins catalyze many forms of biological membrane fusion, including Ca 2؉-triggered exocy... more SNARE proteins catalyze many forms of biological membrane fusion, including Ca 2؉-triggered exocytosis. Although fusion mediated by SNAREs generally involves proteins anchored to each fusing membrane by a transmembrane domain (TMD), the role of TMDs remains unclear, and previous studies diverge on whether SNAREs can drive fusion without a TMD. This issue is important because it relates to the question of the structure and composition of the initial fusion pore, as well as the question of whether SNAREs mediate fusion solely by creating close proximity between two membranes versus a more active role in transmitting force to the membrane to deform and reorganize lipid bilayer structure. To test the role of membrane attachment, we generated four variants of the synaptic v-SNARE synaptobrevin-2 (syb2) anchored to the membrane by lipid instead of protein. These constructs were tested for functional efficacy in three different systems as follows: Ca 2؉-triggered dense core vesicle exocytosis, spontaneous synaptic vesicle exocytosis, and Ca 2؉-synaptotagmin-enhanced SNARE-mediated liposome fusion. Lipid-anchoring motifs harboring one or two lipid acylation sites completely failed to support fusion in any of these assays. Only the lipid-anchoring motif from cysteine string protein-␣, which harbors many lipid acylation sites, provided support for fusion but at levels well below that achieved with wild type syb2. Thus, lipid-anchored syb2 provides little or no support for exocytosis, and anchoring syb2 to a membrane by a TMD greatly improves its function. The low activity seen with syb2-cysteine string protein-␣ may reflect a slower alternative mode of SNARE-mediated membrane fusion.

Journal of Cell Biology, 2007

Botulinum neurotoxins (BoNTs) target presynaptic nerve terminals by recognizing specific neuronal... more Botulinum neurotoxins (BoNTs) target presynaptic nerve terminals by recognizing specific neuronal surface receptors. Two homologous synaptic vesicle membrane proteins, synaptotagmins (Syts) I and II, bind toxins BoNT/B and G. However, a direct demonstration that Syts I/II mediate toxin binding and entry into neurons is lacking. We report that BoNT/B and G fail to bind and enter hippocampal neurons cultured from Syt I knockout mice. Wild-type Syts I and II (but not Syt I loss-of-function toxin-binding domain mutants) restored binding and entry of BoNT/B and G in Syt I–null neurons, thus demonstrating that Syts I/II are protein receptors for BoNT/B and G. Furthermore, mice lacking complex gangliosides exhibit reduced sensitivity to BoNT/G, and binding and entry of BoNT/A, B, and G into hippocampal neurons lacking gangliosides is diminished. These data suggest that gangliosides are the shared coreceptor for BoNT/A, B, and G, supporting a double-receptor model for these three BoNTs for ...

Journal of Cell Biology, 2003

Botulinum neurotoxins (BoNTs) cause botulism by entering neurons and cleaving proteins that media... more Botulinum neurotoxins (BoNTs) cause botulism by entering neurons and cleaving proteins that mediate neurotransmitter release; disruption of exocytosis results in paralysis and death. The receptors for BoNTs are thought to be composed of both proteins and gangliosides; however, protein components that mediate toxin entry have not been identified. Using gain-of-function and loss-of-function approaches, we report here that the secretory vesicle proteins, synaptotagmins (syts) I and II, mediate the entry of BoNT/B (but not BoNT/A or E) into PC12 cells. Further, we demonstrate that BoNT/B entry into PC12 cells and rat diaphragm motor nerve terminals was activity dependent and can be blocked using fragments of syt II that contain the BoNT/B-binding domain. Finally, we show that syt II fragments, in conjunction with gangliosides, neutralized BoNT/B in intact mice. These findings establish that syts I and II can function as protein receptors for BoNT/B.

Science, 2004

We investigated the effect of synaptotagmin I on membrane fusion mediated by neuronal SNARE prote... more We investigated the effect of synaptotagmin I on membrane fusion mediated by neuronal SNARE proteins, SNAP-25, syntaxin, and synaptobrevin, which were reconstituted into vesicles. In the presence of Ca 2+ , the cytoplasmic domain of synaptotagmin I (syt) strongly stimulated membrane fusion when synaptobrevin densities were similar to those found in native synaptic vesicles. The Ca 2+ dependence of syt-stimulated fusion was modulated by changes in lipid composition of the vesicles and by a truncation that mimics cleavage of SNAP-25 by botulinum neurotoxin A. Stimulation of fusion was abolished by disrupting the Ca 2+ -binding activity, or by severing the tandem C2 domains, of syt. Thus, syt and SNAREs are likely to represent the minimal protein complement for Ca 2+ -triggered exocytosis.

Science, 2004

The fusion pore of regulated exocytosis is a channel that connects and spans the vesicle and plas... more The fusion pore of regulated exocytosis is a channel that connects and spans the vesicle and plasma membranes. The molecular composition of this important intermediate structure of exocytosis is unknown. Here, we found that mutations of some residues within the transmembrane segment of syntaxin (Syx), a plasma membrane protein essential for exocytosis, altered neurotransmitter flux through fusion pores and altered pore conductance. The residues that influenced fusion-pore flux lay along one face of an α-helical model. Thus, the fusion pore is formed at least in part by a circular arrangement of 5 to 8 Syx transmembrane segments in the plasma membrane.

Proceedings of the National Academy of Sciences, 2002

Synaptotagmin (syt) I, an integral membrane protein localized to secretory vesicles, is a putativ... more Synaptotagmin (syt) I, an integral membrane protein localized to secretory vesicles, is a putative Ca 2+ sensor for exocytosis. Its N terminus spans the membrane once, and its cytoplasmic domain contains two conserved C2 domains, designated C2A and C2B. The isolated C2A domain penetrates membranes in response to Ca 2+ ; isolated C2B does not. Here, we have addressed the function of each C2 domain, but in the context of the intact cytoplasmic domain (C2A-C2B), by using fluorescent reporters placed in the Ca 2+ -binding loops of either C2A or C2B. Surprisingly, these reporters revealed that, analogous to C2A, a Ca 2+ -binding loop in C2B directly penetrates into lipid bilayers. Penetration of each C2 domain was very rapid ( k on ≈10 10 M −1 ⋅s −1 ) and resulted in high affinity C2A-C2B–liposome complexes ( K d ≈13–14 nM). C2B-bilayer penetration strictly depended on the presence, but not the membrane binding activity, of an adjacent C2A domain, severing C2A from C2B after protein synt...

Proceedings of the National Academy of Sciences, 2003

Neuronal exocytosis is mediated by Ca 2+ -triggered rearrangements between proteins and lipids th... more Neuronal exocytosis is mediated by Ca 2+ -triggered rearrangements between proteins and lipids that result in the opening and dilation of fusion pores. Synaptotagmin I (syt I) is a Ca 2+ -sensing protein proposed to regulate fusion pore dynamics via Ca 2+ -promoted binding of its cytoplasmic domain (C2A-C2B) to effector molecules, including anionic phospholipids and other copies of syt. Functional studies indicate that Ca 2+ -triggered oligomerization of syt is a critical step in excitation–secretion coupling; however, this activity has recently been called into question. Here, we show that Ca 2+ does not drive the oligomerization of C2A-C2B in solution. However, analysis of Ca 2+ ⋅C2A-C2B bound to lipid monolayers, using electron microscopy, revealed the formation of ring-like heptameric oligomers that are ≈11 nm long and ≈11 nm in diameter. In some cases, C2A-C2B also assembled into long filaments. Oligomerization, but not membrane binding, was disrupted by neutralization of two l...

PLoS Pathogens, 2010

Tetanus neurotoxin causes the disease tetanus, which is characterized by rigid paralysis. The tox... more Tetanus neurotoxin causes the disease tetanus, which is characterized by rigid paralysis. The toxin acts by inhibiting the release of neurotransmitters from inhibitory neurons in the spinal cord that innervate motor neurons and is unique among the clostridial neurotoxins due to its ability to shuttle from the periphery to the central nervous system. Tetanus neurotoxin is thought to interact with a high affinity receptor complex that is composed of lipid and protein components; however, the identity of the protein receptor remains elusive. In the current study, we demonstrate that toxin binding, to dissociated hippocampal and spinal cord neurons, is greatly enhanced by driving synaptic vesicle exocytosis. Moreover, tetanus neurotoxin entry and subsequent cleavage of synaptobrevin II, the substrate for this toxin, was also dependent on synaptic vesicle recycling. Next, we identified the potential synaptic vesicle binding protein for the toxin and found that it corresponded to SV2; tetanus neurotoxin was unable to cleave synaptobrevin II in SV2 knockout neurons. Toxin entry into knockout neurons was rescued by infecting with viruses that express SV2A or SV2B. Tetanus toxin elicited the hyper excitability in dissociated spinal cord neurons-due to preferential loss of inhibitory transmission-that is characteristic of the disease. Surprisingly, in dissociated cortical cultures, low concentrations of the toxin preferentially acted on excitatory neurons. Further examination of the distribution of SV2A and SV2B in both spinal cord and cortical neurons revealed that SV2B is to a large extent localized to excitatory terminals, while SV2A is localized to inhibitory terminals. Therefore, the distinct effects of tetanus toxin on cortical and spinal cord neurons are not due to differential expression of SV2 isoforms. In summary, the findings reported here indicate that SV2A and SV2B mediate binding and entry of tetanus neurotoxin into central neurons.

Neuroscience, 2010

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

Nature Neuroscience, 2011

Synaptotagmin I (syt1) is required for normal rates of synaptic vesicle endo-and exocytosis. Howe... more Synaptotagmin I (syt1) is required for normal rates of synaptic vesicle endo-and exocytosis. However, whether the kinetic defects observed during endocytosis in syt1 knockout neurons are secondary to defective exocytosis, or whether syt1 directly regulates the rate of vesicle retrieval, remains unresolved. In order to address this question, it is necessary to dissociate these two activities. Here, we have uncoupled the function of syt1 in exo-and endocytosis by re-targeting of the protein, or via mutagenesis of its tandem C2-domains; the impact of these manipulations on exo-and endocytosis were analyzed via electrophysiology, in conjunction with optical imaging of the vesicle cycle. These experiments uncovered a direct role for syt1 in endocytosis. Surprisingly, either C2-domain of syt1-C2A or C2B-was able to function as Ca 2+-sensor for endocytosis. Hence, syt1 functions as a dual Ca 2+ sensor for both endo-and exocytosis, potentially coupling these two limbs of the vesicle cycle. Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Nature Neuroscience, 2009

Synaptotagmin-IV (syt-IV) is a membrane trafficking protein that influences learning and memory, ... more Synaptotagmin-IV (syt-IV) is a membrane trafficking protein that influences learning and memory, but its localization and role in synaptic function remain unclear. Here we discovered that syt-IV localizes to BDNF-containing vesicles in hippocampal neurons. Syt-IV/BDNF-harboring vesicles undergo exocytosis in both axons and dendrites, and syt-IV inhibits BDNF release at both sites. Knockout of syt-IV increases, and over-expression decreases, the rate of FM dye destaining from presynaptic terminals indirectly via changes in post-synaptic release of BDNF. Hence, postsynaptic syt-IV regulates the trans-synaptic action of BDNF to control presynaptic vesicle dynamics. Furthermore, selective loss of presynaptic syt-IV increased spontaneous quantal release, while loss of post-synaptic syt-IV increased quantal amplitude. Finally, syt-IV knockout mice exhibit enhanced LTP, which depends entirely on disinhibition of BDNF release. Thus, regulation of BDNF secretion by syt-IV emerges as a mechanism to maintain synaptic strength within a useful range during long-term potentiation.

Journal of Neurophysiology, 2004

We show that activation of postsynaptic inositol 1,4,5-tris-phosphate receptors (IP3Rs) with the ... more We show that activation of postsynaptic inositol 1,4,5-tris-phosphate receptors (IP3Rs) with the IP3R agonist adenophostin A (AdA) produces large increases in AMPA receptor (AMPAR) excitatory postsynaptic current (EPSC) amplitudes at hippocampal CA1 synapses. Co-perfusion of the Ca2+chelator bis-( o-aminophenoxy)- N,N,N′,N′-tetraacetic acid strongly inhibited AdA-enhanced increases in EPSC amplitudes. We examined the role of AMPAR insertion/anchoring in basal synaptic transmission. Perfusion of an inhibitor of synaptotagmin-soluble n-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor SNARE-mediated exocytosis depressed basal EPSC amplitudes, whereas a peptide that inhibits GluR2/3 interactions with postsynaptic density-95 (PDZ) domain proteins glutamate receptor interacting protein (GRIP)/protein interacting with C-kinase-1 (PICK1) enhanced basal synaptic transmission. These results suggest that constitutive trafficking and anchoring of AMPARs help maintain basal syn...

The Journal of General Physiology, 2010

The Journal of General Physiology, 2012