Browsing by Subject "Membrane Fusion"
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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 Measurement and Analysis of Calcium-Dependent Exocytosis in Giant Excised Membrane Patches(2008-09-19) Wang, Tzu-Ming; Hilgemann, Donald W.Ca2+-dependent exocytosis was studied in both excised and whole-cell patch clamp with emphasis on the rat secretory cell line, RBL. Capacitance and amperometric recordings show that secretory granules (SGs) containing serotonin are mostly lost from excised patches. Small vesicles that are retained (non-SGs) do not contain substances detected by amperometry. Non-SG fusion is reduced by tetanus toxin light chain treatment, however, it is unaffected by N-ethylmaleimide, implying that SNARE cycling is not required for non-SG fusion in excised patches. Although non-SG fusion is ATP-dependent and blocked by PI-kinase inhibitors, wortmannin and adenosine, the dependency is not neutralized by the PI(3)-kinase inhibitor LY294002, PI(4,5)P2 ligands, such as neomycin, a PI-transfer protein that can remove PI from membranes, and PI(4,5)P2, PI(3)P and PI(4)P antibodies etc. In whole-cell recording, non-SG fusion is strongly reduced by osmotically-induced cell swelling, and subsequent recovery after shrinkage is inhibited by wortmannin, indicating that membrane stretch occurring during patch formation may be a major cause of the ATP-dependency in excised patches. Syt7 and several PLCs are not required for non-SG fusion because fusion remains robust in mouse embryonic fibroblasts deficient of Syt7, PLC(delat)1, PLC(delta)1/(delta)4, or PLC(gamma)1. Furthermore, the Ca2+ dependence of non-SG fusion reflects a lower Ca2+ affinity (KD ~71 uM) than expected for these C2-domain-containing proteins. I also developed a program for measuring and analyzing membrane capacitance. The program uses either sine waves or square waves to estimate cell parameters. Phase-sensitive detection is utilized in both cases. For square wave perturbation, either integrated charges or direct current trace is used for calculating cell parameters. Other functions like digital filtering, pulse stimulation, offline phase angle adjustment, baseline subtraction, and data normalization are also implemented. In summary, using the software I developed, non-SG fusion were characterized and found to be regulated substantially differently from SG fusion. An ATP-dependent process is probably required for restoring non-SG fusion capability after it is perturbed by membrane stretch and dilation.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 Studies on the Structure and Interactions of Synaptotagmin-1 and SNARE Proteins in Neurotransmitter Release(2014-11-14) Brewer, Kyle Daniel; Rosen, Michael K.; Rizo-Rey, José; Yu, Hongtao; Jiang, YouxingThe SNARE complex and synaptotagmin-1 are essential for Ca²⁺-evoked neurotransmitter release, yet the mechanism of how these proteins work together in membrane fusion is unclear. Dozens of studies performed over two decades have described different types of synaptotagmin-1/SNARE interactions and reported the individual structures of the SNAREs, the SNARE complex, and the C₂ domains that form most of the cytoplasmic region of synaptotagmin-1. However, a high-resolution structure of a synaptotagmin-1/SNARE complex, which is crucial to understand the mechanism of release, has not been reported. In this work, we explore methods to examine the biophysical properties of synaptotagmin-1 and the SNAREs, primarily using NMR. We first examine the conformation of synaptobrevin on nanodisc bilayers and find that that the N-terminal portion of the SNARE domain has a high propensity to remain unfolded on membranes. We next look at the conformation of synaptotagmin-1 on nanodiscs and demonstrate that although both C₂ domains primarily bind to the same membrane, a small population of antiparallel conformers also exist. Finally and most importantly, we look at the structure of synaptotagmin-1/SNARE complex. After overcoming many obstacles and failed approaches, we were able to obtain intermolecular restraints for this 66 kDa machinery by introducing lanthanide tags for measurement of pseudocontact shifts (PCSs). Computational analyses incorporating these restraints show that a static structure cannot fully explain all the PCS measurements, but the data can be fit with a dynamic ensemble of structures whereby a polybasic region of the synaptotagmin-1 C₂B domain binds to a polyacidic region formed by the syntaxin-1 and SNAP-25 SNARE motifs. The orientation of the synaptotagmin-1 C₂B domain with respect to the SNARE complex within the ensemble is expected to allow quick, simultaneous interaction with lipids on both membranes upon Ca²⁺ binding to bring the membranes together. Distinct mutations in the C₂B domain polybasic region caused differential disruptions of SNARE complex-binding that correlate with the impairment of neurotransmitter release caused by these mutations. Overall, these results and the architecture of the synaptagmin-1/SNARE complex revealed by our NMR data support the hypothesis that synaptotagmin-1 cooperates with the SNAREs by bringing membranes together to trigger fast fusion upon Ca²⁺ influx.