Internal Dynamics of the Postsynaptic Density

创建、维持和调节突触的机制是人类行为的重要组成部分。 这些机制的中断与异常行为和疾病有着千丝万缕的联系 抑郁症、精神分裂症、成瘾和阿尔茨海默氏症。因此，这笔赠款的长期目标是 深入了解突触传递和可塑性背后的分子组织。 我们之前的工作利用了单分子成像带来的极高分辨率 方法并测定了谷氨酸能突触活动区和突触后的关键蛋白 密度丰富于突触下的纳米结构域(&lt；100 nm)。最令人惊讶的是，关键的纳米域 突触前活动区的融合调节蛋白RIM和Munc13在突触前活动区 富含突触后谷氨酸受体的纳米结构域形成突触裂隙。使用单囊泡融合 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对 突触是如何运作的。释放部位和受体之间的这种纳米排列可以调节突触 传递，并潜在地影响细胞内信号。这里的初步数据和从 其他人证实，跨突触的纳米排列是突触结构的一个重要元素，广泛 存在于不同的突触类型中。此外，我们的数据提供了确凿的证据，表明突触下的纳米结构和 在突触可塑性过程中，纳米排列被动态地调制，并由正在进行的 分子相互作用。这些观察结果强烈地促进了对涉及到的机制的理解 创建和维护跨突触对齐。因此，我们将测试一套相关但独立的 关于跨突触纳米排列起源和维持的假说。我们将测试1)是否有两个密钥 神经连接素和LRRTM合作伙伴共同提供跨突触排列的结构基础， 2)谷氨酸受体本身是否必须或足以影响纳米级蛋白质 活动区的组织3)，活动区RIM复合体是否向 建立突触后纳米调控，以及4)肌动蛋白细胞骨架如何对突触进行持续控制 纳米级架构。为了回答这些问题，我们已经努力建立和应用了几个新的 广泛适用的技术。我们利用一种新的超分辨率成像方法来可视化细胞 在体内纳米分辨率的亚结构，应用多路复用单分子成像来映射众多 同一样本中的蛋白质，并开发新的光学和生化工具来精确控制肌动蛋白 高时空分辨率的细胞骨架、黏附复合体和受体在脑内的分布 切片。这些实验的结果将回答关于一个重要的新的 并测试突触纳米排列在大脑回路中的生理作用。

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.