Internal Dynamics of the Postsynaptic Density

产生、维持和调节突触的机制是人类行为的基本组成部分。 这些机制的破坏与异常行为和疾病有着千丝万缕的联系， 抑郁症和精神分裂症到成瘾和阿尔茨海默病。因此，这笔赠款的长期目标是 追求对突触传递和可塑性的分子组织的深入理解。 我们之前的工作利用了单分子成像的极高分辨率 方法，并确定，在突触，关键蛋白的活动区和突触后 密度在突触下纳米结构域（<100 nm）中富集。最令人惊讶的是， 突触前活动区中的融合调节蛋白RIM和Munc13在整个突触区中以高精度对齐。 来自突触后谷氨酸受体富集的纳米结构域的突触间隙。利用单囊泡融合 映射，我们确定了活动区子区域内RIM的局部密度预测了 动作电位诱发的囊泡融合。这一引人注目的建筑布局具有重要的意义， 突触是如何运作的这种释放位点和受体之间的纳米排列可以调节突触 传递并潜在地影响细胞内信号传导。这里的初步数据和发表的工作， 其他人则认为跨突触纳米排列是突触结构的重要元素， 存在于不同的突触类型中。此外，我们的数据提供了有力的证据表明，突触下纳米结构和 纳米排列在突触可塑性期间被动态调节，并通过持续的 分子间相互作用这些观察结果强烈地激发了对参与的机制的理解， 产生并维持跨突触排列。因此，我们将测试一组相关但独立的 关于跨突触纳米排列的起源和维持的假说。我们将测试1）是否两个关键 neurexin伴侣，神经连接蛋白和LRRTM，合作提供跨突触排列的结构基础， 2)谷氨酸受体本身是否是影响纳米级蛋白质的必要条件或足够条件 活动区的组织3），活动区RIM复合体是否向 建立突触后纳米图案，以及4）肌动蛋白细胞骨架如何对突触施加持续控制 纳米结构。为了回答这些问题，我们已经建立和应用了几个新的 广泛实用的技术。我们利用一种新的超分辨率成像方法来可视化细胞 在体内以纳米分辨率进行亚结构，应用多路复用单分子成像来映射许多 蛋白质，并开发新的光学和生物化学工具，以急性控制肌动蛋白 细胞骨架、粘附复合物和受体分布，具有高时空分辨率和脑内 切片这些实验的结果将回答关于一个重要的新事物起源的核心问题。 方面的突触架构和测试的生理作用突触nanoalignment在脑回路。

Mechanisms that create, maintain, and modulate synapses are essential building blocks of human behavior. Disruptions to these mechanisms are inextricably linked to aberrant behavior and diseases ranging from depression and schizophrenia to addiction and Alzheimer’s Disease. Thus, the long-term goal of this grant is to pursue a deep understanding of the molecular organization underlying synaptic transmission and plasticity. Our previous work took advantage of the extremely high-resolution enabled by single-molecule imaging methods and determined that at glutamatergic synapses, key proteins in the active zone and the postsynaptic density are enriched in subsynaptic nanodomains (<100 nm). Most surprisingly, nanodomains of the critical fusion-regulatory proteins RIM and Munc13 in the presynaptic active zone align with high precision across the synaptic cleft from nanodomains enriched in postsynaptic glutamate receptors. Using single-vesicle fusion mapping, we determined that the local density of RIM within active zone subregions predicts the probability of action potential-evoked vesicle fusion. This striking architectural arrangement has important implications for how synapses function. This nano-alignment between release sites and receptors can modulate synaptic transmission and potentially influence intracellular signaling. Preliminary data here and published work from others establishes that transsynaptic nanoalignment is an important element of synaptic architecture, widely present in diverse synapse types. Further, our data provide firm evidence that subsynaptic nanostructure and nanoalignment are dynamically modulated during synaptic plasticity and actively maintained by ongoing molecular interactions. These observations strongly motivate understanding the mechanisms involved in creating and maintaining transsynaptic alignment. Therefore, we will test a set of related but independent hypotheses about the origin and maintenance of transsynaptic nanoalignment. We will test 1) whether two key neurexin partners, neuroligin and LRRTM, cooperate to provide the structural basis of transsynaptic alignment, 2) whether glutamate receptors themselves are necessary or sufficient to influence the nanoscale protein organization of the active zone 3), whether the active zone RIM complex conveys instructive information to establish postsynaptic nanopatterning, and 4) how the actin cytoskeleton exerts ongoing control over synapse nanoscale architecture. To answer these questions, we have worked to establish and apply several new broadly useful technologies. We utilize a new super-resolution imaging methodology to visualize cellular substructure at nanometer resolution in vivo, apply multiplexed single-molecule imaging to map numerous proteins in the same sample, and develop new optical and biochemical tools to acutely control the actin cytoskeleton, adhesion complexes, and receptor distribution with high spatiotemporal resolution and in brain slices. The outcomes of these experiments will answer core questions about the genesis of an important new aspect of synaptic architecture and test the physiological role of synaptic nanoalignment in brain circuits.