AFM studies of SNARE-mediated membrane fusion

描述(申请人提供)：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神经递质胞吐的潜在机制。未受刺激的神经元内的神经递质(如谷氨酸、GABA、儿茶酚胺)被隔离在停靠在突触前终末质膜上的分泌小泡中。当动作电位到达轴突末端时，电压依赖的钙通道打开，由此产生的钙离子流入触发了一系列生化反应，导致含有神经递质的小泡与质膜融合，将其内容物释放到突触间隙。这一过程是由质膜上表达的SNARE蛋白和小泡表面表达的SNARE蛋白介导的。神经元的v-SNARE(VAMP2)和t-SNARE(Synaxin和SNAP-25)的反式配对已被证明形成了在突触前终末诱导小泡融合所必需的基本分子机制。SNARs的融合功能受相关辅助分子的调节，包括突触凝集素和复合蛋白。尽管近年来在识别融合机制的各个组件方面取得了相当大的进展，但在我们对SNARE介导的膜融合及其调控过程的理解方面仍存在许多空白。我们研究的中心假设是，SNARE蛋白的相互作用产生一个机械力，使相对的膜不稳定，从而降低膜融合的能量需求。钙结合的突触素通过进一步降低这一能量需求促进融合，而复合蛋白通过防止SNARE复合体完全退变而抑制融合。为了验证这一假设，这项拟议的研究将使用最先进的原子力显微镜技术来测量同源陷阱相互作用产生的力。结果将决定是否足以使膜接近，以启动诱捕促进膜融合过程。此外，我们将通过直接测力来确定SNARs如何与络合素和突触素一起改变膜融合过程的能量学，从而揭示SNARE介导的膜融合的机制。公共卫生相关性：这项拟议的研究应用生物物理方法来阐明在神经网络化学突触的信号传递过程中钙离子引发神经递质胞吐的潜在机制。需要解决的根本问题是神经元如何调节囊泡与质膜的融合。对钙离子引发的胞吐作用的详细分子理解是重要的，因为囊泡机械的组件是许多治疗药物的潜在靶点，用于治疗大量的神经疾病。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: The proposed research applies biophysical methods toward elucidating the underlying mechanism of Ca2+ triggered exocytosis of neurotransmitters during signal transmission at chemical synapses of neural networks. The fundamental problem to be addressed is how do neurons regulate the fusion of vesicles with the plasma membrane. A detailed molecular understanding of Ca2+ triggered exocytosis is important because components of vesicle machinery are potential targets of many therapeutic reagents for a plethora of neurological disorders.