Browsing by Subject "Synaptic Vesicles"
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Item Endolysosomal Function in Neuronal Maintenance(2018-06-14) Jin, Eugene Jennifer; Bezprozvanny, Ilya; Herz, Joachim; Terman, Jonathan R.; Huber, Kimberly M.; Hiesinger, Peter RobinEndolysosomal degradation of membrane proteins is crucial for the maintenance of synaptic function and neuronal health. Neurons can live for the lifetime of an organism and therefore rely on robust membrane turnover mechanisms to clear old, dysfunctional, excess or possibly also functional membrane proteins. A key regulator of canonical endolysosomal degradation is Rab7, a ubiquitous small GTPase required for endosomal maturation. Based on the observation that Rab7 expression is strongly neuron-enriched during Drosophila development, I first tested specific requirement of Rab7 in neurons. I found that loss of rab7 does not affect development, but causes activity-dependent degeneration that starts at synapses in Drosophila photoreceptors. Four point mutations in Rab7 are associated with the peripheral neuropathy Charcot-Marie-Tooth Type 2B (CMT2B) disease and my data suggest a partial loss of function mechanism. Together, these findings highlight that neurons are particularly sensitive to the dosage of Rab7-dependent endolysosomal degradation. Several other membrane turnover mechanisms, including autophagy and a neuron-specific branch of the endolysosomal system, called 'neuronal-sort-and-degrade' (NSD), are also required for neuronal maintenance. However, it remained unclear what cargoes these different membrane turnover mechanisms degrade, and where cargoes are degraded. Given that NSD is a neuron-specific mechanism whereas Rab7-dependent endolysosomal degradation and autophagy are ubiquitous mechanisms, I hypothesized that NSD may specifically sort and degrade synaptic membrane proteins, whereas the Rab7-dependent canonical endolysosomal degradation and autophagy unbiasedly degrade all membrane proteins. I tested this hypothesis by live imaging of an acidification-sensing degradation probe for a synaptic vesicle (SV)-specific cargo and a general membrane cargo to directly and quantitatively measure the sorting and degradation of these cargoes at Drosophila photoreceptor axon terminals. I found that both cargoes are sorted and degraded locally at axon terminals. Interestingly, the two cargoes are sorted into two distinct 'hub compartments' for degradation. Rab7 and NSD are required for the sorting and degradation of the two cargoes separately: sorting and degradation of general cargo is Rab7-dependent, whereas that of SV cargo is NSD-dependent. In sum, this work highlights neuron-specific mechanisms for cargo-specific membrane protein degradation that keep synapses healthy and functional.Item Functional Characterization of Synaptic Proteins in Calcium Triggered Exocytosis(2008-09-12) Chang, Wen-Pin; Südhof, Thomas C.Release of neurotransmitter involves fusion of the membrane of synaptic vesicle with the presynaptic plasma membrane, a process that is tightly regulated by calcium. One of the central goals is to understand the molecular machinery underline the fundamental fusion mechanism used at all synapses, thus it is important to characterize the physiological function of unknown synaptic proteins and to identify new members that might have functions in synaptic vesicle fusion. In this thesis, I first characterize the function of synaptic vesicle protein 2 (SV2), which is one of the first synaptic vesicle proteins identified. SV2 is essential for survival in mice; its deletion impairs neurotransmitter release, although the exact point at which step of release is affected remains unclear. Using electrophysiological approaches, our data demonstrate that SV2 acts downstream of the priming, but upstream of the Ca2+-triggering of vesicle fusion. By using rescue experiments, we also demonstrate that mutations of charged residues within the transmembrane regions or of the intravesicular glycosylation sequences of SV2 block its function, probably by impairing the folding and trafficking of SV2. In contrast, deletion of the conserved N-terminal putative synaptotagmin-binding sequence of SV2 did not abolish SV2 function, nor did mutation of another conserved cytoplasmic sequence. These observations suggest that SV2 functions in a maturation step of primed vesicles that converts the vesicles into a Ca2+- and synaptotagmin-responsive state. Second, SNAREs and Sec1/Munc18 (SM) proteins are critical for intracellular membrane fusion. The neuronal SM protein Munc18-1 binds to SNARE complexes and syntaxin-1. The interaction to SNARE complex likely represents the general mode of SMARE/SM protein coupling, but the understanding of its physiological relevance to vesicle fusion and precise point of its function during the process is hindered by the duality of Munc18-1/SNARE binding modes. Here we designed three mutations that preserve Munc18-1/syntaxin-1 binding but differentially impairs the Munc18-1/SNARE complex binding. By utilizing rescue experiments, we showed that the impairment correlates with disruption of vesicle priming and evoked release, and suggest that Munc18-1/SNARE complex assemblies generally govern membrane traffic. Third, we reported the primary structure and biochemical properties of a family of evolutionarily conserved mammalian proteins, E-Syts, which contain multiple C2 domains, a common Ca2+ binding module, and a transmembrane region. Our findings suggest that E-Syts function as Ca2+-regulated intrinsic membrane proteins and expand the repertoire of multiple C2 domains proteins to a fourth class beyond synaptotagmins, ferlins, and MTCPs (multiple C2 domain and transmembrane region proteins).Item Genetic Dissection of Synaptic Vesicle Endocytosis(2019-03-28) Afuwape, Olusoji Adeyemi; Terman, Jonathan R.; Kavalali, Ege T.; Schmid, Sandra; Monteggia, Lisa; Krämer, HelmutSynaptic transmission is mediated by the quantal release of neurotransmitters through the fusion of discrete synaptic vesicles with the presynaptic membrane. To maintain reliable transmission, synaptic vesicles and proteins must be recycled after release of neurotransmitters. A key protein in this recycling process is dynamin. Dynamin is a GTPase that catalyzes the scission of a budding endosome off its parent membrane. The mammalian brain expresses three isoforms of dynamin. Using genetically modified mouse hippocampal neurons, I analyzed the functional significance of dynamin in synaptic vesicle endocytosis. Specifically, I assessed dynamin 2 function in synaptic vesicle recycling and neurotransmission and investigated the role of dynamin independent endocytosis at the synapse. My data demonstrates that synaptic transmission after post-natal knockout of dynamin 2 remains intact and synaptic vesicle endocytosis, assessed by the trafficking of vesicular glutamate transporter fused to pHluorin, is unperturbed. Synaptic vesicle endocytosis in the absence of dynamin 2 was assessed at both room temperature and 32 oC. At both temperatures, my results reveal that synaptic vesicle recycling functions independent of dynamin 2 but also, the kinetics of single vesicle recycling is unaffected by changes in temperature suggesting that a single, temperature insensitive (within the limits of testing) form of endocytosis mediates single synaptic vesicle endocytosis. Further experiments reveal that the retrieval of single synaptic vesicles persists after the knockout of all dynamin isoforms. However, after multivesicular release, my results show an overall decrease in synaptic vesicle pool size and a retardation of subsequent vesicle endocytosis in neurons lacking all dynamin isoforms suggesting dynamin function at the synapse is activity dependent. This finding is consistent with prior reports showing dynamin function at the synapse is dependent on its dephosphorylation by the Ca2= dependent phosphatase, calcineurin. My results also demonstrate a dichotomy in the dependence of dynamin for synaptic neurotransmission. Whereas I observe a decrease in evoked amplitude, release probability and frequency of spontaneously released events in glutamatergic synapses, I observe no discernable defects in GABAergic neurotransmission. This result suggests inhibitory synapses are better equipped with compensatory mechanisms to deal with the loss of dynamins 1,2 and 3. Overall, my data demonstrate that dynamin is crucial but not essential for synaptic vesicle endocytosis. Dynamin is an activity dependent GTPase that is required for synaptic vesicle recycling after exocytosis of multiple vesicles. However, the underlying mechanism of single synaptic vesicle endocytosis is both dynamin and temperature independent.Item Insights into the Functions of Munc18-1 in Neurotransmitter Release(2013-03-07) Su, Lijing; Rizo-Rey, José; Luo, Xuelian; Kavalali, Ege T.; Liu, YiNeurotransmitter release is an exquisitely regulated process that transmits signals between neurons. The release process includes: docking of synaptic vesicles at the active zone of the pre-synaptic plasma membrane, priming to a release ready state, and then membrane fusion and release of neurotransmitters triggered by Ca2+. Several conserved proteins are involved in regulating the entire process. The central membrane fusion machinery in neurons includes Munc18-1 and the SNARE proteins syntaxin-1, SNAP-25 and synaptobrevin. The SNAREs form tight SNARE complexes that bring the vesicle membrane and plasma membrane into close proximity and provide forces to induce membrane fusion. Munc18-1 is essential because deletion of Munc18-1 in mice leads to a complete loss of neurotransmitter secretion. However, its molecular mechanism of action is still unclear. This work is aimed to unravel the critical roles of Munc18-1 in regulating neurotransmitter release. The functions of Munc18-1 discovered so far are related to the SNAREs. Recently we found that Munc18-1 interacts with synaptobrevin and the SNARE four-helix bundle with week affinity, which have been shown to stimulate in vitro SNARE-dependent liposome fusion. Biophysical characterization of these two interactions could provide important information to uncover the roles of Munc18-1 in membrane fusion. Cross-linking and NMR spectroscopy experiments showed that Munc18-1 interacts with the C-terminus of the synaptobrevin SNARE motif through some positively charged residues located in its domain 3a. NMR spectroscopy and ITC experiments revealed that the Munc18-1 N-terminal domain interacts with the C-terminal part of synaptobrevin and syntaxin-1 on the SNARE four-helix bundle, and that the affinity is higher than full length Munc18-1. In our in vitro reconstitution experiments that try to establish the vital functions of Munc18-1 and Munc13 in neurotransmitter release, I found that Munc18-1 displaces SNAP-25 from syntaxin-1/SNAP-25 complex to form Munc18-1/syntaxin-1 complex on liposomes in the presence of NSF/α-SNAP/ATP. When NSF/α-SNAP were incorporated in the lipid mixing assays between synaptobrevin-liposmes and syntaxin-1/SNAP-25-liposomes, Munc18-1 together with Munc13 activate lipid mixing that is inhibited by NSF/α-SNAP. These results suggest that Munc18-1 functions with Munc13 to promote SNARE complex formation in an NSF/α-SNAP resistant manner and to guide the synaptic vesicle exocytosis through a tightly regulated pathway.Item Mechanistic Insights into the Role of Munc13 in Synaptic Vesicle Docking, Priming, and Fusion(2019-03-06) Quade, Bradley Jackson; Luo, Xuelian; Rizo-Rey, José; Rice, Luke M.; Zhang, XuewuNeurotransmitter release is a fundamental aspect of neuronal communication that relies on the fusion of synaptic vesicles with the presynaptic membrane. These fusion events are tightly regulated by the influx of Ca2+, which is sensed by the complex protein machinery at the axon terminal. In order for these Ca2+-mediated fusion events to occur in the correct time and place, protein machines interact with neurotransmitter filled vesicles to dock and prime them for release. Munc13 is one of the essential components of the docking, priming, and fusion machinery. To understand the role of Munc13 in docking and priming I attempted to structurally characterize the MUN domain and SNARE protein interactions using nuclear magnetic resonance spectroscopy. Paramagnetic relaxation enhancement and pseudocontact shift experiments were performed to identify the binding site of SNAREs or the SNARE complex on the MUN domain and in both cases the data suggested that there may be binding in multiple locations or that the interactions are promiscuous. I also attempted to crystallize the C2C domain of Munc13 alone and in the context of larger fragments. I was able to grow crystals of various fragments of Munc13 containing C2C and adjacent domains, but these crystals were fragile and diffracted poorly. In lieu of a crystal structure, I modeled the C2C domain based on homologous C2 domains and performed sequence conservation analysis to identify functionally important regions of C2C that may bind membranes. Using structural information coupled with reconstitutions, dynamic light scattering, and cryo-electron tomography I explored the functional relevance of membrane binding within the C1, C2B, and C2C domains of Munc13. The C2C domain was identified as a critical component of Munc13 that enables bridging between liposomes with synaptic vesicle and plasma membrane composition in vitro and this bridging ability is integral for synaptic vesicle docking in vivo. The C1C2B area has a large membrane binding interface that changes depending on the ligands present in the system and this enables Munc13 to modulate the distance between membranes. The ability of Munc13 to regulate the distance between membranes in response to ligands may underlie its role in synaptic plasticity.Item Molecular and Functional Determinants of Synaptic Vesicle Recycling In CNS Synapses(2007-05-23) Virmani, Tuhin; Kavalali, Ege T.Chemical neurotransmission is the basis for information processing in the brain, and presynaptic terminals respond to a large range of stimulation patterns including baseline rhythms of activity that coordinate neuronal ensembles, to short bursts of activity that encode information. They also release neurotransmitter spontaneously in the absence of any activity. The question then is how can a single subcellular compartment with approximately 100 synaptic vesicles coordinate these complex functions? We used a multifaceted approach to address this question. We first studied the role of synaptotagmin 7 (syt7), a highly alternatively spliced synaptic plasma membrane protein, whose short splice forms inhibit clathrin-mediated endocytosis. We found that in hippocampal synapses, the splice variants formed a bi-directional molecular switch targeting vesicles to kinetically distinct recycling pathways. Additionally, syt7 knockout synapses had less fast endocytosis, while calcium binding site mutant synapses showed increased vesicle endocytosis. We further investigated the slower recycling pathways by exploring rab5 function. A dominant negative rab5 mutation did not alter synaptic function, but constitutively active rab5 or the inhibition of vesicle budding from endosomes by the PI3-kinase inhibitor wortmannin, decreased vesicle pool size and release kinetics. This suggests that central synapses are tuned towards faster modes of recycling. The model of spontaneous neurotransmitter release from this same evoked recycling pool places additional constraints on this system. We explored this hypothesis by directly visualizing presynaptic recycling of spontaneous vesicles using FM dyes, syt1 antibodies and HRP uptake. We found that there are actually two sets of vesicle pools, one for evoked release, and one for spontaneous release that have minimal interaction with one another. Can presynaptic function be a substrate for diseases of the CNS? To test this important question, we studied a mouse model for infantile Batten disease. We found that underlying synaptic deficits in vesicle pool size and mini frequency could produce the neurological phenotypes exhibited by patients well before the onset of neurodegeneration. Taken together, these results show that synaptic vesicle recycling is a very plastic entity and that synapses have the intrinsic ability to modulate their vesicle trafficking pathways in response to the varying demands placed on them.Item Molecular Determinants of Synaptic Vesicle Exocytosis and Endocytosis Coupling(2016-07-12) Li, Ying Chuan; Rizo-Rey, José; Krämer, Helmut; Xu, WeiSynaptic vesicle recycling is essential for maintaining normal synaptic function. The reuse of vesicles maintains the size of the presynaptic terminal and ensures the availability of synaptic vesicles for subsequent exocytosis. The coupling of exocytosis and endocytosis allows for continued rapid synaptic transmission; however, the molecular mechanisms of this process are not well understood. This coupling is assumed to be Ca2+-dependent but the exact role of Ca2+ and its key effectors in the regulation of endocytosis are not clear. Using a genetically encoded pH-sensitive GFP tag expressed in cultured hippocampal neurons, I analyzed synaptic vesicle trafficking in high resolution optical experiments. By manipulating the expression of various effectors of vesicle fusion I was able to dissect out the relationship between exocytic pathway and subsequent endocytic kinetics. My results showed that the slowed endocytosis phenotype previously reported after synaptotagmin 1 loss-of-function can also be triggered by other manipulations that promote asynchronous release such as Sr2+ substitution and complexin loss-of-function. The link between asynchronous release and slowed endocytosis was due to selective targeting of fused synaptic vesicles towards slow retrieval by the asynchronous release Ca2+ sensor synaptotagmin7. This divergence in Ca2+ sensor function supports findings that VAMP4 selectively drive asynchronous release through a population of vesicles that do not interact with synaptotagmin 1 or complexins. At the single synaptic vesicle level, synaptotagmin 1 acted as an essential determinant of synaptic vesicle endocytosis time course by increasing the kinetics of vesicle retrieval in response to increasing Ca2+ levels. In contrast, synaptotagmin 1 did not affect the rapid retrieval of spontaneously fused vesicles. Taken together, these results suggest that exocytic pathways dictate endocytic kinetics as asynchronously fused vesicles are retrieved slowly while spontaneously fused vesicles are rapidly retrieved. These mechanisms may diversify the molecular compositions of synaptic vesicles regenerated after fusion to provide presynaptic terminals with a wide range of synaptic vesicle populations with distinct biogenesis properties and exo-endocytosis kinetics.Item Neuronal Maintenance via a Neuron-Specific Degradation Pathway(2015-01-26) Schmidt, Taylor; Jin, Eugene Jennifer; Ozel, Mehmet Neset; Epstein, Daniel; Marchant, Corey; Hiesinger, RobinBACKGROUND: Neurons can survive for decades via cell maintenance and protein degradation. This process includes the general protein endolysosomal degradation pathway, an integral part of which is the Rab GTPase proteins. Recently, components of a neuron-specific protein degradation pathway were discovered, which include the neuronal vesicle ATPase component V100 and the synaptic vesicle protein neuronal Synaptobrevin (n-Syb). While this neuron-specific degradation pathway has been shown as necessary for neuronal maintenance in adult Drosophila melanogaster fruit flies, it is not known what this neuron-specific degradation pathway does, nor how it interacts with the general protein degradation pathway. Our research aimed to fill this gap in knowledge. Such research may be salient because the misregulation of protein degradation in neurons leads to neurodegenerative diseases like dementia. OBJECTIVE: We hypothesized that neurons either have an increased or a specialized need for protein degradation in comparison to other cells. METHODS: 1. The lab chose a myristoylated protein (myr) to represent general proteins found in every cell, and Synaptotagmin1 (Syt1) to represent neuron-specific proteins. The acidification-sensitive tag mCherry-pHluorin, which changes color with a decrease in pH, was placed on Syt1 and myr to visualize acidification and degradation of the two proteins. 2. The lab generated Drosophila lines to compare acidification and degradation of Syt1 and myr in wild-type versus the following three mutants: rab7 mutants to disrupt general protein degradation, v100 to disrupt the neuron-specific protein degradation, and synaptobrevin also to disrupt neuron-specific degradation. 3. We performed live imaging to visualize acidification and protein degradation at synaptic terminals. Brains of Drosophila pupae from each cross were dissected, mounted onto Petri dishes, and surrounded with a culture medium to be kept alive. A resonant confocal microscope was used to observe the brain's lamina, a layer of neurons between the eye and the brain. At the lamina, we recorded 30-minute videos showing changes in fluorescence representing protein degradation. RESULTS AND CONCLUSION: Preliminary data show that nsyb and v100 mutations may cause defects in the degradation of neuron-specific cargo. Such evidence suggests that the neuron-specific endolysosomal degradation pathway specifically degrades the synaptic vesicle protein Synaptotagmin1. Also, the experiments indicate that disruption of either the neuron-specific or the general endolysosomal degradation pathway has no effect on the acidification of the myristoylated protein. Such evidence implies that the general pathway of protein degradation occurs at synapses, but has no specificity for protein cargo. A greater sample size is needed for future experiments, as well as quantitative analysis.Item Optical Quantal Analysis of Evoked and Spontaneous Single-Vesicle Fusion(2014-07-14) Leitz, Jeremy Thomas Sheng; Hilgemann, Donald W.; Hiesinger, Peter Robin; Albanesi, Joseph P.; Kavalali, Ege T.Synaptic vesicle recycling is critical for the maintenance and proper function of neurotransmission. Neurotransmission can proceed through action-potential evoked vesicle fusion where, upon depolarization, Ca2+ enters the nerve terminal though voltage-gated channels, interacts with vesicle-associated proteins to promote fusion with the terminal membrane, and causes release of vesicle contents. Neurotransmission can also occur spontaneously in the absence of stimulation, although this process is still Ca2+-dependent. Regardless of the mode of vesicle fusion, the vesicle lipids and protein components must be removed from the terminal membrane; the vesicle must be reconstituted and re-filled with neurotransmitter, so that it may ultimately be reused. Uncoupling the roles of Ca2+ in synaptic vesicle fusion and retrieval has been difficult to date as studies have relied on measurements of bulk synaptic vesicle retrieval. Here, to dissect the role of Ca2+ in these processes, we utilized low signal-to-noise pHluorin-tagged vesicular probes to monitor single synaptic vesicle recycling of both action-potential evoked and spontaneous fusion vesicles in rat hippocampal neurons. We show that during stimulation, increasing extracellular Ca2+ increases synaptic vesicle fusion probability, but decreases the rate of synaptic vesicle retrieval. This negative regulation of synaptic vesicle retrieval is blocked by the Ca2+ chelation as well as inhibition of calcineurin, a Ca2+-calmodulin-dependent phosphatase. Indeed, the slow time course of aggregate synaptic vesicle retrieval detected during repetitive activity can be explained by a progressive decrease in the rate of synaptic vesicle retrieval during the stimulation train. These results indicate Ca2+ entry during single action potentials slows the pace of subsequent synaptic vesicle recycling. Conversely, we found that synaptic vesicles that undergo spontaneous fusion are retrieved very rapidly and this retrieval time is Ca2+-independent. Interestingly, we found that within a single synaptic bouton, the rate of spontaneous neurotransmission is independent of evoked fusion probability, suggesting there are fundamental regulatory differences between these forms of neurotransmission. Moreover, we found that the glycoprotein Reelin can act presynaptically to enhance spontaneous neurotransmission without affecting evoked neurotransmission by mobilizing a molecularly specific subset of synaptic vesicles. These data illustrate fundamental differences in vesicle recycling between modes of neurotransmission at the single-vesicle level.Item Regulation of Synaptic Vesicle Trafficking at Central Synapses(2009-09-04) Chung, Chihye; Kavalali, Ege T.Synapses are where electrical information is converted to chemical signaling, allowing for careful regulation of inter-neuronal communication in the brain. At presynaptic terminals, synaptic vesicles fuse with plasma membrane in response to electrical stimulation, followed by rapid retrieval to the terminal and re-organization for reuse. Thus, synaptic vesicle trafficking is of interest as to where presynaptic regulations of synaptic transmission begins to occur. The first two chapters explored a novel secretagogue, lanthanum (La³⁺), and its potential usage as a probe to study vesicle recycling at central synapses. Chapter two describes the characteristics of La³⁺-evoked transmission at hippocampal synapses. La³⁺ has two separate actions on transmission, with a different time course and underlying mechanism of action. This newly characterized rapid action of La³⁺ is intracellular Ca²⁺-independent, in contrast to its delayed action, yet requires functional SNARE complex formation. Therefore, chapter three took advantage of La³⁺-evoked transmission as a tool to investigate the coupling between exo- and endocytosis in SNARE-dependent fusion. Using multifaceted approaches, I propose that La³⁺ induces transmitter release via narrow fusion pore opening and closure, or a ‘kiss-and-run’ mode of exo- and endocytosis. Chapter four investigates the molecular requirement for the synaptic vesicle recycling pathway. I analyzed the impact of one of main players in endocytosis, dynamin in different forms of release. Acute inhibition of dynamin in central synapses impairs activity-dependent synaptic vesicle recycling while leaves spontaneous recycling intact, suggesting the operation of two parallel recycling pathways in central synapses as well as proposing the molecular signature between spontaneously and activity-dependently recycling pathways. In chapter five, I further investigated the origins of spontaneously recycling synaptic vesicles by simultaneous monitoring of spectrally separable FM dyes, as chapter suggested four that they are originated from an isolated pool. This chapter includes comprehensive analysis of the endocytic pathway operating at rest and its molecular participants –specifically dynamin, which was implicated to play a role in the endocytic pathway from observations I made in chapter four. Chapter six expands the investigation as to how presynaptic signaling regulates synaptic vesicle trafficking in glutamatergic synapses. I focused on the impact of ambient glutamate concentration on vesicle recycling as a feedback signal to rapid synaptic reuse to impact short-term synaptic plasticity. Taken together, these results suggest that synaptic vesicle trafficking is an actively regulated process, impacting various aspects of information cascades between neurons.Item Unraveling the Functions of Synaptotagmin and Munc13 in Neurotransmitter Release(2015-01-30) Seven, Alpay Burak; Rosen, Michael K.; Rizo-Rey, José; Albanesi, Joseph P.; Jiang, Qiu-XingNeurotransmitter release is a central event in interneuronal communication. The release machinery includes three SNAREs (soluble N-ethylmaleimide sensitive factor adaptor protein receptor) and Munc18-1 as core components. Munc13, synaptotagmin and complexin underlie the exquisitely tight regulation of synaptic exocytosis. The SNAREs form the SNARE complex that brings synaptic vesicles and plasma membrane together. This complex is disassembled by NSF/α-SNAP (soluble N-ethylmaleimide sensitive fusion protein/NSF attachment protein). Munc18 binds to syntaxin, which keeps syntaxin in its closed confirmation and prevents to form the SNARE complex. Interactions between Munc13, Munc18, and syntaxin perform a vital role in regulation of the SNARE complex formation. Tight regulation of the release machinery requires other factors such as Ca2+ sensor Synaptotagmin-1 and negatively charged lipids. Synaptotagmin interacts with the SNARE complex and the negatively charged lipids, and initiates the synchronous fast release by sensing the Ca2+ influx. It is crucial to investigate functions of individual proteins to understand the mechanism of membrane fusion and neurotransmitter release. Therefore, we investigated the mechanism of membrane bridging by synaptotagmin. Our cryo-electron microscopy (cryo-EM) images showed that a majority of synaptotagmin fragment containing both C¬2A and C2B domains (C2AB) molecules bridge membranes directly. Fluorescence spectroscopy demonstrates that the bottom of the C2B domain contacts the membrane in a substantial population of membrane-bound synaptotagmin fragments. NMR analysis of C2AB-nanodiscs shows that a fraction of C2AB molecules binds to membranes with antiparallel orientations of the C2 domains. Together with previous studies, these results show that direct bridging constitutes the prevalent mechanism of membrane bridging by synaptotagmin, suggesting that this mechanism underlies the function of synaptotagmin-1 in neurotransmitter release. We have also discovered that Munc13-1 can bridge membranes in a Ca2+-independent manner, which shed light to the docking activity of Munc13-1. We also showed that Munc13-1 can cause efficient lipid mixing and slow content mixing together with Munc18-1 in the absence of synaptotagmin-1. Addition of synaptotagmin facilitates the fusion pore formation in content mixing assays. Recently, we were able to reconstitute key components of the release machinery. We are further investigating our observations with cryo-electron microscopy to understand the mechanism of membrane fusion.Item Unraveling the Role of SNARE Interactions in Neurotransmitter Release(2005-05-04) Chen, Xiaocheng; Kavalali, Ege T.The release of neurotransmitters by Ca2+-triggered synaptic vesicle exocytosis is tightly controlled by an intricate protein machinery. Essential components of this machinery are the synaptic vesicle protein synaptobrevin and the plasma membrane proteins syntaxin 1 and SNAP-25, which are collectively known as SNAREs and form a tight complex (the core complex). The assembly of the core complex may mediate membrane fusion. Complexin is a highly conserved cytoplasmic protein that binds tightly to the SNARE complex. Analysis of the interaction between complexin and the SNARE complex showed that complexin binds to the groove between the synaptobrevin and syntaxin helices, and the binding stabilizes the syntaxin/synaptobrevin interface. These results led to a model whereby complexin stabilizes the fully assembled SNARE complex, which is critical for the fast Ca2+-triggered neurotransmitter release. The N-terminal domain of syntaxin 1 folds back and forms a 'closed' conformation, which interacts with munc18-1, an essential protein in the neurotransmitter release. It has been proposed that the binding of munc18-1 might change the closed conformation. To test this model, I solved the solution structure of the N-terminal domain within the closed conformation of syntaxin 1 and structure comparisons showed that the N-terminal domain adopts the same conformation whether it is isolated, bound to Munc18-1, or within the closed conformation. Analysis of the Ca2+-binding properties of the core complex revealed that it contains several low affinity Ca2+ binding sites and most of them are nonspecific for Ca2+. A SNAP-25 mutation that causes a change in the Ca2+-dependence of secretion in chromaffin cells has no effect on the SNARE/synaptotagmin 1 interactions, but has a conspicuous effect on core complex assembly. Thus, the SNAREs are unlikely to directly act as Ca2+ sensors, but SNARE complex assembly is tightly coupled to Ca2+ sensing in neurotransmitter release. To directly test SNARE function, I reconstituted v- and t-SNAREs into separate liposomes and carefully characterized the proteoliposomes containing v- and t-SNAREs. Fusion between the v- and t-SNARE proteoliposomes was then monitored with a lipid mixing assay. Interestingly, little fusion was observed. The results suggest that the SNAREs alone are not sufficient to mediate membrane fusion.