AFM studies of SNARE-mediated membrane fusion

描述（由申请人提供）：拟议的研究应用生物物理学方法来阐明化学突触信号传递期间Ca 2+触发神经递质胞吐的潜在机制。神经递质（例如，未受刺激的神经元内的谷氨酸、GABA、儿茶酚胺）被隔离在停靠在突触前末梢的质膜上的分泌囊泡内。当动作电位到达轴突末端时，电压依赖性钙通道打开，由此产生的钙内流触发生化级联反应，导致含有神经递质的囊泡融合到质膜上，将其内容物释放到突触间隙中。这个过程是由在质膜上表达的SNARE蛋白及其在囊泡表面上的对应物介导的。神经元v-SNARE（VAMP 2）和t-SNARE（syntaxin和SNAP-25）的反式配对已被证明形成在突触前末端诱导囊泡融合所必需的基本分子机制。SNARE的融合功能受相关辅助分子（包括突触结合蛋白和复合蛋白）的调节。虽然近年来在识别融合机制的各个组成部分方面取得了相当大的进展，但我们对SNARE介导的膜融合以及该过程如何调节的理解仍然存在许多差距。我们研究的中心假设是，SNARE蛋白的相互作用产生了一种机械力，这种机械力使相对的膜不稳定，从而降低了膜融合的能量需求。钙结合的突触结合蛋白通过进一步降低这种能量需求来促进融合，而复合蛋白通过阻止SNARE复合物完全退火来抑制融合。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同源SNARE相互作用产生的力。结果将确定是否足以使膜紧密接近以启动SNARE促进的膜融合过程。此外，我们将通过直接力测量来确定SNARE沿着复合蛋白和突触结合蛋白如何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：拟议的研究应用生物物理学方法来阐明在神经网络的化学突触的信号传递过程中Ca 2+触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Ca 2+触发的胞吐作用的详细分子理解是重要的，因为囊泡机制的组分是许多治疗剂用于过多神经系统疾病的潜在靶点。

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.