Browsing by Subject "Exocytosis"
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Item Characterization of the FXYD Protein Family in the Regulation of Insulin Exocytosis(2004-05-04) Hays, Lori Beth; Rhodes, Christopher J.; Roth, Michael G.; Cobb, Melanie H.; Uyeda, KosakuInsulin exocytosis is a complex, regulated process involving numerous exocytotic proteins to coordinate the release of insulin. Syncollin has been implicated in zymogen granule exocytosis in acinar cells. It was hypothesized that either syncollin or a ‘syncollin-like’ protein may be expressed in β-cells and influence insulin exocytosis. Adenoviral mediated expression of either long or short forms of syncollin in isolated islets and INS-1 cells showed both forms underwent N-terminal signal peptide cleavage to yield the same 14kD mature protein. Immunofluorescence revealed that adenovirally-expressed syncollin was specifically targeted to the ß-granule lumen. In perifused islets, syncollin expression significantly inhibited first-phase glucose-induced insulin secretion compared to AdV-GFP infected islets. GLP-1 and glyburide potentiation of insulin secretion was inhibited; whereas constitutive secretion and insulin content were normal in syncollin-infected islets indicating syncollin-mediated inhibition of insulin secretion was not due to inadequate insulin production or secondary stimulus-coupling signals. Thus, syncollin likely inhibited the distal stages of insulin exocytosis providing the first evidence that an intragranular protein is capable of influencing regulated insulin secretion. Syncollin fluorescent fusion proteins were localized to ß-granules, but did not influence insulin secretion implicating these chimeras as ß-granule specific markers for emerging imaging technology. Real-time confocal microscopy demonstrated syncollin-GFP could be used to examine spatiotemporal dynamics of exocytosis. Furthermore, consecutive infection of syncollin-GFP and syncollin-dsRFP labeled distinct pools of β-granules. Expression of syncollin was not identified in β-cells; however, a 10Kd ‘syncollin-like’ protein was expressed, which when sequenced corresponded to FXYD6. Comparison of syncollin and FXYD6 protein structure revealed several conserved domains, indicating syncollin is likely a pseudo-FXYD family member. FXYD6 was the only FXYD protein endogenously expressed in β-cells, which localized to distinct regions of the plasma membrane. Overexpression of FXYD6-Myc enhanced β-granule transport to distinct regions of the plasma membrane that also expressed FXYD6; however, there was no significant effect on glucose-stimulated insulin secretion in isolated islets. SiRNA-mediated reduction of FXYD6 resulted in no obvious changes in β-granule distribution; however, β-granule movement during glucose stimulation was erratic and misdirected. These data implicate FXYD6 as a molecular beacon on the plasma membrane guiding β-granules to the active site of exocytosis.Item Competition Between Synaptotagmin 1 and Complexin for SNARE Complex Binding, Controls Fast Synaptic Vesicle Exocytosis(2007-05-23) Tang, Jiong; Südhof, Thomas C.Calcium binding to synaptotagmin 1 triggers fast exocytosis of synaptic vesicles that were primed for release by SNARE complex assembly. Besides synaptotagmin 1, fast Ca2+- triggered exocytosis requires complexins. Synaptotagmin 1 and complexins both bind to assembled SNARE complexes, but it is unclear how their functions are coupled. To clarify previous debates on calcium dependent and independent binding between synaptotagmin 1 and SNARE proteins, I systematically examined the interactions between synaptotagmin 1 and purified SNARE monomer, heterodimer and core complex separately. This would avoid the problem of doing binding assays in an undefined protein mixture. We found the calcium dependency of synaptotagmin 1 and SNARE interactions relied on the accurate binding conditions that include protein concentration and ionic strength. In addition, at physiological conditions, calcium dependent binding is favored. Based on this system, I discovered the competition between complexin and synaptotagmin 1 for SNARE complex binding. Although in hydrophilic environment, complexin shows much higher affinity for SNARE complex than synaptotagmin 1, synaptotagmin 1 can more efficiently replace complexin from membrane embedded SNARE complex in a strictly calcium dependent manner. Expression of synaptic vesicle targeted complexin (by fusion to synaptobrevin 2) in cultured cortical neurons severely blocks fast synchronous release, but not asynchronous release, which is very similar to that of synaptotagmin 1 knockout mice. Based on electrophysiological data and biochemical confirmation of competition, we suggest that the phenotype could result from the replacement of synaptotagmin 1 from SNARE complex by local high concentration of fused complexin. We propose our model as: complexin binding promotes the assembly of SNARE complex and further stabilizes it. As a result, vesicles are activated into a "superprimed" metastable state, and are clamped at the same time waiting for triggering signals. Synaptotagmin 1 replaces complexin and releases this clamp through SNARE complex binding upon calcium entry. The simultaneous binding of synaptotagmin 1 with SNARE complex and phospholipids finally triggers membrane fusion and vesicle release.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 Structural and Functional Studies of the Munc13 MUN Domain and the RIM C2B Domain(2007-12-17) Guan, Rong; Rizo-Rey, JoséNeurotransmitter release is essential for normal brain function and is achieved through exocytosis of synaptic vesicles. Many proteins are involved the regulation of neurotransmission. The central fusion machinery includes the SNARE proteins and Munc18- 1. Besides these universal components, many other neuronal specific proteins are also involved in regulating Ca2+-triggered neurotransmitter release, such as the key priming factors RIMs and Munc13s. Munc13s are essential for vesicle priming. RIMs form a protein scaffold in the presynaptic nerve terminal. My studies have focused on the structures and functions of the Munc13 MUN domain and the RIM C2B domain. I have studied the structure and function of the Munc13 MUN domain. On one hand, I have tried to determine the three dimensional structure of the Munc13 MUN domain by Xray crystallography. I have successfully obtained crystals of the Munc13-1 MUN domain, Munc13-3 MUN domain and a fragment containing the Munc13-1 C1, C2B and MUN domains. These crystals will be further optimized to enable structure determination. On the other hand, I have tried to identify the binding partners of the MUN domain using various methods. Cross-linking experiments revealed an interaction between the Munc13-1 MUN domain and endogenous Munc18-1. In addition, cofloatation assays revealed an interaction between MUN and reconstituted SNARE complex. Detailed analysis using cofloatation assays suggested both MUN and complexin can compete with Munc18-1 for SNARE complex binding in a membrane environment. Our studies also suggested that the membrane environment can modulate the strength of protein-protein interactions remarkably, which emphasize the importance to include membranes in the studies of protein-protein interactions involved in neurotransmission. I have also analyzed the structural and biochemical properties of the RIM1 C2B domain. NMR spectroscopy and FRET experiments demonstrated no interaction between the RIM1 C2B domain and Ca2+, phospholipids, or its putative binding partners, synaptotagmin 1 and liprins. X-ray crystallography revealed the existence of a RIM1 C2B domain homodimer, which was confirmed by analytical ultracentrifugation and NMR spectroscopy. Our results suggested a model that RIM1 C2B dimerization might facilitate the Munc13 C2A homodimer to Munc13 C2A/RIM zinc finger heterodimer switch during synaptic vesicle priming.Item Structural Studies of Complexin/SNARE Interactions(2008-09-18) Lee, Daeho; Rizo-Rey, JoséVesicular neurotransmitter release is mediated by exocytosis of synaptic vesicles at the presynaptic active zone of nerve terminals. The Ca2+-triggered release process is extremely fast, lasts less than half a millisecond, and is tightly regulated by Ca2+. Action potentials cause Ca2+ influx through voltage-gated Ca2+ channels, which in turn triggers synaptic vesicle fusion. The typical sub-millisecond latency between an action potential and neurotransmitter release and the precise timing of the release process are crucial for information processing in the nervous system. To achieve this exquisite regulation, many proteins are involved. The goal of our investigations was to delineate interactions between complexin and SNARE components that lead to the formation of a primed minimal fusion machinery. We have generated and used new constructs of short forms of synaptobrevin and complexin, as well as constructs of SNAP-25 and syntaxin that have previously been shown to be part of the minimal fusion machinery. With NMR spectroscopy, the use of the short synaptobrevin constructs has led to experimental results suggesting at least two key intermediates during the docking/priming process that are independent of complexin. We found evidence for a modular assembly of the full SNARE complex. In the absence of Syb2-CT, the N-terminal half of SNARE complex forms a four-helix bundle, while the C-terminal half, starting just after the polar layer, is disordered. In the presence of the Syb2-CT, however, both halves of the SNARE complex form a four-helix bundle. It is interesting to note that Syb2 residues 29-84 are sufficient for formation of the fully assembled SNARE complex. This evidence strongly suggests the existence of at least two intermediates during the docking priming reaction. Furthermore, with NMR spectroscopy, we have found new evidence that complexin can bind to the t-SNARE complex, in contrast to earlier evidence suggesting that complexin regulates the fully assembled SNARE complex. We demonstrated that Cpx-FL binds the t-SNARE complex SN1/SN3/Syx1a(188-259) in solution, as wa suggested for membrane-bound t-SNAREs. Note, however, that the t-SNARE complex does not contain the large complexin-binding interface provided by Syb2. Furthermore, we found that Cpx-FL also binds t-SNARE sub-complexes formed by SN1/SN3, and SN1/Syx1a(188-259), while very little binding was observed between Cpx-FL and Syx1a(188-259) alone. This finding is particularly interesting, because the cryst structure of the fully assembled SNARE complex does not suggest any binding between Cpx26-83 and either SN1 or SN3, whereas the only common component in all of the above experiments was SN1 domain.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.