AFM studies of SNARE-mediated membrane fusion

项目总结 这项拟议的研究应用生物物理方法来阐明 在化学突触的信号传递过程中，钙离子触发了神经递质的胞吐。 未受刺激的神经元内的神经递质(如谷氨酸、GABA、儿茶酚胺)是 被隔离在停靠在突触前终末质膜上的分泌小泡内。 当动作电位到达轴突末端时，电压依赖性钙通道开放 由此产生的钙离子流入触发了一系列生化反应，导致神经递质- 含有融合到质膜的囊泡，将其内容释放到突触裂隙中。 这一过程是由质膜上表达的SNARE蛋白和它们的 囊泡表面的对应物。神经元V-SNARE(VAMP2)和T-SNARES的反式配对 (Synaxin和SNAP-25)已被证明形成必要的必要分子机制 诱导突触前末端的囊泡融合。圈套的融合功能受到调节 由相关的辅助分子，包括突触素和络合素。尽管有 在识别核聚变机械的各个部件方面取得了相当大的进展 近年来，我们对SNARE介导的膜融合的认识还存在许多空白。 以及这一过程是如何监管的。我们研究的中心假设是 SNARE蛋白产生的机械力破坏了相对的膜的稳定性，因此 降低了膜融合的能量需求。钙结合突触素促进融合 通过进一步降低这一能量需求，而复合素通过阻止 完全退火制得复杂的陷阱。为了验证这一假设，拟议的研究将 使用最先进的原子力显微镜技术来测量由 同源圈套的相互作用。结果将决定是否足以将膜带到 为了启动诱捕器促进的膜融合过程，我们需要更近的距离。此外，我们还将 通过直接测力确定圈套以及络合蛋白和突触聚集蛋白， 改变了膜融合过程的能量学，从而揭示了陷阱的机制-- 介导膜融合。

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.