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 拟议的研究应用生物物理学方法来阐明 在化学突触的信号传递过程中，Ca 2+触发神经递质的胞吐。 神经递质（例如，谷氨酸，GABA，儿茶酚胺）在未受刺激的神经元内， 被隔离在停靠在突触前末梢质膜上的分泌囊泡内。 当轴突末端的动作电位到达时，电压依赖性钙通道打开 由此产生的钙的涌入引发了生化级联反应，导致神经递质- 含有囊泡，与质膜融合，将其内容物释放到突触间隙中。 这一过程是由质膜上表达的SNARE蛋白介导的， 囊泡表面的对应物。神经元v-SNARE（VAMP 2）和t-SNARE的反式配对 （syntaxin和SNAP-25）已被证明形成必要的分子机制， 在突触前末端诱导囊泡融合。SNARE的融合功能受到调节， 相关的辅助分子，包括突触结合蛋白和复合蛋白。虽然已经 在识别聚变机器的各个组成部分方面取得了相当大的进展， 近年来，我们对SNARE介导的膜融合的理解仍有许多空白 以及这个过程是如何被监管的。我们研究的中心假设是， SNARE蛋白产生一种机械力，使贴壁膜不稳定， 降低了膜融合所需的能量。钙结合突触结合蛋白促进融合 通过进一步降低这种能量需求，而复合蛋白通过阻止细胞融合来抑制融合。 SNARE复合物完全退火。为了验证这一假设，拟议的研究将 采用最先进的原子力显微镜技术来测量由 同源陷阱的相互作用。结果将确定是否足以使膜达到 紧密靠近以启动SNARE促进的膜融合过程。而且还要 通过直接力测量确定SNARE，沿着复合蛋白和突触结合蛋白， 改变膜融合过程的能量学，从而揭示SNARE的机制- 介导的膜融合。