AFM studies of SNARE-mediated membrane fusion

SNARE 介导的膜融合的 AFM 研究

• 批准号: 8011155
• 负责人: VINCENT T MOY
• 金额: $ 1.38万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-24 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8011155
• 关键词: Action Potentials; Address; Arts; Atomic Force Microscopy; Binding; Binding Proteins; Biochemical; Biological Neural Networks; Calcium; Calcium Binding; Calcium Channel; Catecholamines; Cell membrane; Charge; Chemical Synapse; Collaborations; Complex; Coupled; Coupling; Cytoplasmic Tail; Docking; Ethylmaleimide; Exocytosis; Glutamates; Hydration status; Individual; Laboratories; Leucine Zippers; Lipid Bilayers; Measurement; Measures; Mechanics; Mediating; Membrane; Membrane Fusion; Methods; Molecular; Neurons; Neurotransmitters; Phospholipids; Play; Presynaptic Terminals; Procedures; Process; Property; Proteins; Reagent; Research; Role; SNAP receptor; Secretory Vesicles; Signal Transduction; Surface; Synaptic Cleft; Techniques; Testing; Therapeutic; Transmembrane Domain; Universities; Vesicle; Wisconsin; Work; gamma-Aminobutyric Acid; nervous system disorder; neurotransmitter release; prevent; receptor; reconstitution; research study; response; single molecule; stem; synaptotagmin; syntaxin; target SNARE proteins; transmission process

PROJECT SUMMARY The proposed research applies biophysical methods toward elucidating the underlying mechanism of Ca2+ triggered exocytosis of neurotransmitters during signal transmission at chemical synapses. Neurotransmitters (e.g., glutamate, GABA, catecholamine) within unstimulated neurons are sequestered within secretory vesicles docked at the plasma membrane of the presynaptic terminal. Upon the arrival of an action potential at the axon terminal, voltage-dependent calcium channels open and the resulting influx of calcium triggers a biochemical cascade that causes the neurotransmitter- containing vesicles to fuse to the plasma membrane, releasing their contents into the synaptic cleft. This process is mediated by SNARE proteins expressed on the plasma membrane and their counterparts on the vesicle's surface. Trans pairing of the neuronal v-SNARE (VAMP2) and t-SNAREs (syntaxin and SNAP-25) has been shown to form the essential molecular machinery necessary to induce vesicle fusion at the presynaptic terminal. The fusogenic function of the SNAREs is regulated by associated accessory molecules, including synaptotagmin and complexin. Although there have been considerable advances toward identifying the individual components of the fusion machinery in recent years, there is still numerous gaps in our understanding of SNARE-mediated membrane fusion and how the process is regulated. The central hypothesis of our research is that the interaction of the SNARE proteins generates a mechanical force that destabilizes the apposing membranes and thus lowers the energy requirement for membrane fusion. Calcium bound synaptotagmin promotes fusion by further lowering this energy requirement, whereas complexin inhibits fusion by preventing the SNARE complex from completely annealing. To test this hypothesis, the proposed research will employ state-of-the-art atomic force microscopy techniques to measure the force generated by the interactions of the cognate SNAREs. Results will determine if it is sufficient to bring the membranes to close proximity in order to initiate the SNARE-facilitated membrane fusion process. Moreover, we will determine by direct force measurements how the SNAREs, along with complexin and synaptotagmin, alter the energetics of the membrane fusion process, hence revealing the mechanism of SNARE- mediated membrane fusion.

项目摘要 拟议的研究将生物物理方法应用于阐明 CA2+在化学突触时信号传输过程中神经递质的胞吞作用。 神经递质（例如谷氨酸，GABA，儿茶酚胺）未刺激的神经元是 在突触前末端的质膜上停靠的分泌囊泡中。 在轴突终端的动作电位到达后，电压依赖性钙通道打开 钙的涌入引发了导致神经递质的生化级联 含有囊泡将其融合到质膜中，将其含量释放到突触裂隙中。 该过程是由在质膜上表达的核心蛋白介导的 囊泡表面上的对应物。神经元V-SNARE（VAMP2）和T-SNARES的反式配对 （语法和SNAP-25）已显示出形成必需的分子机制 在突触前末端诱导囊泡融合。 SNARES的融合功能受到调节 通过相关的辅助分子，包括突触蛋白和复合蛋白。虽然那里有 在确定融合机械的各个组件方面取得了长足的进步 近年来，我们对媒介介导的膜融合的理解仍然存在许多差距 以及如何调节过程。我们研究的中心假设是 网罗蛋白会产生一种机械力，使膜不稳定，因此 降低膜融合的能量需求。钙结合突触蛋白促进融合 通过进一步降低这种能量需求，而复合蛋白通过防止融合来抑制融合 完全退火而圈套复合物。为了检验这一假设，拟议的研究将 采用最先进的原子力显微镜技术来测量 同源网罗的相互作用。结果将确定是否足以将膜带到 为了启动圈养的膜融合过程，紧密的接近度。而且，我们会的 通过直接测量来确定贪食素如何与复合蛋白和突触抗体一起确定 改变膜融合过程的能量学，因此揭示了圈套的机制 介导的膜融合。