Functional nanoscopy of the synaptic active zone

对生物过程的基本了解需要关于结构如何编码功能的信息。尽管逐渐出现了一个全面的蛋白质目录，我们仍然缺乏基本的信息来描述蛋白质在突触处的纳米级组织如何引起神经传递。从本质上讲，这是由于传统光学显微镜的衍射有限的分辨率，这阻碍了进入空间nanodomain在生理学相关context.The目前的研究建议集中在突触活性区（AZ），高度专业化的突触前神经递质释放的网站。AZ的精确分子结构产生了不同的结构和功能AZ状态，这些状态形成化学神经传递并从根本上影响大脑功能。本项目将利用最新的技术创新来验证一个假设，即AZ的功能状态可以通过特定分子的数量和精确的空间组织来预测。为了进入分子域，将使用遗传上可接近的生物果蝇。在第一个项目部分，突触蛋白的表达将被操纵，以改变AZ的分子组织。功能纳米显微镜，电生理学和超分辨率荧光显微镜的相关应用，将集中在关键AZ蛋白，建立定量的，因果关系的分子超微结构和AZ的功能，即AZ状态的分子蓝图。在第二个项目部分，新的光遗传学工具将被用来触发活性依赖的可塑性的AZ在体内。本研究计划将测试AZ功能如何与超微结构联系，并研究关键分子的纳米级重组如何在体内不同时间尺度上介导不同的可塑性机制。为此，电生理学，超分辨率显微镜和光遗传学的协同组合旨在确定大脑功能组织的基本原则。

Fundamental insight into biological processes demands information on how structure encodes function. Despite a gradually emerging comprehensive protein catalogue, we still lack basic information describing how the nanoscopic organisation of proteins at synapses gives rise to neurotransmission. In essence, this is due to the diffraction-limited resolution of conventional light microscopy, which has hindered access to the spatial nanodomain in a physiologically relevant context.The present research proposal focuses on the synaptic active zone (AZ), the highly specialised presynaptic site of neurotransmitter release. The precise molecular architecture of AZs gives rise to different structural and functional AZ states, which shape chemical neurotransmission and fundamentally influence brain function. By engaging recent technological innovations, this project will test the hypothesis that the functional status of the AZ can be predicted by the number and the precise spatial organisation of specific molecules.To gain access to the molecular domain, the genetically accessible organism Drosophila melanogaster will be employed. In the fist project section, synaptic protein expression will be manipulated to alter the molecular organisation of AZs. Functional nanoscopy, the correlative application of electrophysiology and super-resolution fluorescence microscopy, will then focus on key AZ proteins to establish quantitative, causal relationships between the molecular ultrastructure and the function of AZs, i.e. molecular blueprints of AZ states.In the second project section, novel optogenetic tools will be utilised to trigger activity-dependent plasticity of the AZ in vivo. The molecular mechanisms underlying these physiological changes in AZ function will then be interpreted using the established blueprints.This research proposal will test how function is linked to the ultrastructure of AZs and examine how nanoscopic reorganisations of key molecules mediate distinct mechanisms of plasticity over different time scales in vivo. To this end, the synergistic combination of electrophysiology, super-resolution microscopy and optogenetics aims to identify fundamental principles of organisation underlying brain function.