Browsing by Subject "Synaptic Vesicles"
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Item Functional characterization of synaptic proteins in calcium triggered exocytosis(2008-09-12) Chang, Wen-Pin; Sudhof, 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 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 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 Role of SNARE Interactions in Neurotransmitter Release(2005-05-04) Chen, Xiaocheng; Kavalali, EgeThe 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.