Macromolecular Architecture Of The Synapse

突触的大分子结构

• 批准号: 8940051
• 负责人: Thomas S Reese
• 金额: $ 118.87万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8940051
• 关键词: AMPA Receptors; Adopted; Affinity; Architecture; Area; Binding; Binding Sites; C-terminal; Cells; Chemicals; Chicago; Collaborations; Complex; Consensus; Data; Development; Electron Microscopy; Electrons; Electrophysiology (science); Family; Family member; Filament; Floor; Fluorescence Resonance Energy Transfer; Freezing; Glutamate Receptor; Glutamates; Goals; Guidelines; Hippocampus (Brain); Individual; Information Storage; Institutes; Label; Laboratories; Lead; Left; Length; Life; Literature; Maps; Mass Spectrum Analysis; Measurement; Memory; Methods; Microscopic; Microtomy; Molecular; Molecular Conformation; Molecular Machines; Molecular Probes; Molecular Weight; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; National Institute of Biomedical Imaging and Bioengineering; Neurons; Pattern; Peripheral; Positioning Attribute; Postsynaptic Membrane; Protein Array; Proteins; RNA Interference; Reporting; Resolution; Scaffolding Protein; Signal Transduction; Site; Structural Models; Structure; Synapses; Synaptic Membranes; Technology; Thick; Time; Vertebral column; Work; base; dalton; density; fluorophore; functional loss; information processing; innovation; insight; knock-down; novel; postsynaptic; postsynaptic density protein; presynaptic density protein 95; protein complex; receptor; reconstruction; scaffold; stargazin; stoichiometry; synaptic function; tomography; voltage

The postsynaptic density (PSD) at excitatory glutamatergic synapses is a large molecular machine of molecular weight greater than one billion Daltons. The PSD is known to be a key site of information processing and storage. In order to explore the detailed molecular organization of the PSD, we developed a method to freeze-substitute hippocampal cultures and then examine them in thin sections by EM tomography to show individual protein complexes in their natural setting within the PSD. The initial work employing tomography revealed that the core of the PSD is an array of vertically oriented filaments that contain the scaffold protein, PSD-95, in an extended configuration and a polarized orientation, with its N-terminus positioned at the postsynaptic membrane. This finding provides insight into the overall organization of the PSD because scaffolding proteins such as PSD-95 family MAGUK proteins have distinct multiple, diverse binding sites for other proteins arrayed along their length. Thus, the regular arrays of PSD-95, perhaps with other family members, impose an ordering on many other PSD proteins, including the glutamate receptors, and provide an overall plan for the structure of the PSD. FRET constructs were made to study possible mechanisms that regulate PSD-95 MAGUK conformations in collaboration with the Green laboratory (U Chicago). Two fluorophores (RFP and YFP) are fused to the opposite termini of PSD-95 or other family members. The labels allow the conformations of the family members to be determined both by immunogold-EM and by making FRET measurements on living cells (if the two ends of the molecule FRET, they must be in a closed configuration). So far, results suggest that PSD-95 adopts an extended conformation in PSDs, but in closed conformation at non-synaptic sites. In contrast, SAP-97, another MAGUK has an open configuration, but is oriented parallel with the post synaptic membrane. Open conformation of PSD-95 at the PSD is a requirement for it to interact with NMDAR and AMPAR-Stargazin complexes. EM tomography also revealed that the C-terminal ends of the vertical filaments are associated with horizontally oriented filaments. One class of horizontal filament is ordered to form hexagonal cross-linkers with the vertical filaments, and is concentrated beneath the NMDA receptors. Immunogold labeling now tentatively identifies a class of horizontal filaments as GKAP, which is a known to bind to the GK domain at the C-terminal end of PSD-95. Immunogold labeling is also being used to locate another major scaffolding molecule, SHANK, which is known to bind GKAPs directly. The emerging structural model of the PSD shows how the PSD-95 matrix can stabilize glutamate receptors, and at the same time allows room for the addition of new receptors at the edges of the PSD. Identification of the components of the PSD is time consuming and the methods for identifying the proteins need improvement. An expressible probe, miniSOG, has become available and we use it to confirm that the vertical filaments are PSD-95. We are now preparing probes to use miniSOG to definitely identify GKAP and SHANK in the PSD. The idea that the PSD-95 dependent scaffold stabilizes the PSD has been explored by using EM tomography to determine the effects of RNAi knock down of MAGUKs. Recently, we examined the effects of knocking down simultaneously three major MAGUK proteins: PSD-95, PSD-93 and SAP102, and for the first time, EM tomography revealed significant loss from the central core of the PSD, including NMDA receptor structures, vertical filaments, and AMPA receptors. Electrophysiology measurements by collaborators from the Nicoll laboratory (UCSF) characterizing the effects of the same knock down show significant functional loss of NMDAR and AMAPR type EPSPs at levels compatible with the structural losses. Electron microscopy, showed depletion of vertical filaments along with AMPAR type structures at the peripheral region of the PSD and significant reduction of size of NMDAR cluster in the middle of the PSD. These structural data indicate that vertical filaments corresponding to MAGUKs anchor AMPARs and are also a factor in organizing NMDARs. The differentiated organization of NMDARs and AMPARs provides a structural basis for silence synapses, in which the PSD contains an NMDAR cluster surrounded by few AMPARs. Thus, PSD-95 MAGUKs are demonstrated to be the essential organizer of glutamate receptors at the PSD. A newly developed electron microscopic method (Leapman Lab, NIBIB) using high voltage STEM tomography (HVST) is compatible with sections up to two micrometers thick and is revealing detailed reconstructions of many whole synapses. We used HVST on 1-2 um sections that contain entire PSDs at synapses to demonstrate that simultaneous knock down of the three major PSD-95 family MAGUKs results in significant reduction in the overall PSD area, leaving many synapses with only small PSDs. Since a subpopulation of synapses completely loses their electron dense PSD material, the triple knock down in effect results in many silent synapses as reported by electrophysiology. In collaboration with the National Institute of Standards and Technology, a novel type of quantitative mass spectrometry was applied to evaluate stoichiometries of PSD components. Copy numbers for targeted proteins within the PSD were subsequently estimated using a consensus literature value for the copy number of PSD-95. The NMDA receptor to AMPA receptor copy number ratio was determined to be ≈ 1:2, yielding an estimate of 34 10 NMDA channels and 68 36 AMPA channels per average PSD, respectively, in line with estimates from other methods. A ratio for AMPAR tetramers to TARP auxiliary subunits was ≈ 1:2 supporting the assertion that most AMPA receptors anchor to the PSD via TARP subunits. The study also generated estimates for the for the copy number of several key PSD proteins for the first time, and confirmed or challenged, previous estimates for certain other PSD components. These copy numbers provide valuable guidelines to map quantitatively molecular architecture of the PSD.

兴奋性谷氨酸能突触的突触后密度（PSD）是一个分子量大于十亿道尔顿的大型分子机器。众所周知，PSD 是信息处理和存储的关键站点。为了探索 PSD 的详细分子组织，我们开发了一种方法来冷冻替代海马培养物，然后通过 EM 断层扫描在薄片中检查它们，以显示 PSD 内自然环境中的各个蛋白质复合物。采用断层扫描的初步工作表明，PSD 的核心是一系列垂直定向的细丝，其中含有支架蛋白 PSD-95，呈扩展结构和极化方向，其 N 末端位于突触后膜。这一发现提供了对 PSD 整体组织的深入了解，因为支架蛋白（例如 PSD-95 家族 MAGUK 蛋白）对于沿其长度排列的其他蛋白具有独特的多个、多样化的结合位点。因此，PSD-95 的规则阵列（可能与其他家族成员一起）对许多其他 PSD 蛋白（包括谷氨酸受体）施加了排序，并为 PSD 结构提供了总体规划。 与 Green 实验室（芝加哥大学）合作，构建 FRET 结构来研究调节 PSD-95 MAGUK 构象的可能机制。两个荧光团（RFP 和 YFP）融合到 PSD-95 或其他家族成员的相对末端。标签允许通过免疫金电镜和对活细胞进行 FRET 测量来确定家族成员的构象（如果分子的两端有 FRET，则它们必须处于闭合构型）。到目前为止，结果表明 PSD-95 在 PSD 中采用扩展构象，但在非突触位点采用闭合构象。相比之下，另一种 MAGUK SAP-97 具有开放配置，但方向与突触后膜平行。 PSD-95 在 PSD 处的开放构象是它与 NMDAR 和 AMPAR-Stargazin 复合物相互作用的必要条件。 EM 断层扫描还显示，垂直丝的 C 末端与水平定向的丝相关。一类水平丝有序地与垂直丝形成六角形交联剂，并集中在 NMDA 受体下方。免疫金标记现在暂时将一类水平丝鉴定为 GKAP，已知它与 PSD-95 C 末端的 GK 结构域结合。免疫金标记也被用来定位另一种主要的支架分子 SHANK，已知它可以直接结合 GKAP。 PSD 的新兴结构模型展示了 PSD-95 基质如何稳定谷氨酸受体，同时为 PSD 边缘添加新受体留出空间。 PSD 成分的鉴定非常耗时，并且鉴定蛋白质的方法需要改进。一种可表达的探针 miniSOG 已经可用，我们用它来确认垂直丝是 PSD-95。我们现在正在准备探针，使用 miniSOG 来明确识别 PSD 中的 GKAP 和 SHANK。 通过使用 EM 断层扫描来确定 PSD-95 依赖性支架稳定 PSD 的想法，以确定 MAGUK 的 RNAi 敲低的效果。最近，我们检查了同时敲除三种主要 MAGUK 蛋白：PSD-95、PSD-93 和 SAP102 的影响，并且 EM 断层扫描首次显示 PSD 中央核心的显着损失，包括 NMDA 受体结构、垂直丝和 AMPA 受体。 Nicoll 实验室 (UCSF) 的合作者进行的电生理学测量表征了相同击倒的影响，结果显示 NMDAR 和 AMAPR 型 EPSP 的显着功能损失，其水平与结构损失相一致。电子显微镜显示，PSD 外围区域的垂直细丝以及 AMPAR 型结构被耗尽，PSD 中部的 NMDAR 簇尺寸显着减小。这些结构数据表明，与 MAGUK 相对应的垂直丝锚定 AMPAR，也是组织 NMDAR 的一个因素。 NMDAR 和 AMPAR 的差异化组织为沉默突触提供了结构基础，其中 PSD 包含一个被少量 AMPAR 包围的 NMDAR 簇。因此，PSD-95 MAGUK 被证明是 PSD 谷氨酸受体的重要组织者。 新开发的电子显微方法（Leapman Lab，NIBIB）使用高压 STEM 断层扫描 (HVST)，与厚度达两微米的切片兼容，并揭示了许多整个突触的详细重建。我们在突触处包含整个 PSD 的 1-2 um 切片上使用 HVST，以证明同时敲除三个主要 PSD-95 家族 MAGUK 会导致整体 PSD 面积显着减少，从而使许多突触仅具有较小的 PSD。由于突触亚群完全失去了电子致密的 PSD 材料，因此根据电生理学的报告，三重击倒实际上会导致许多沉默的突触。 与美国国家标准与技术研究所合作，应用了一种新型定量质谱法来评估 PSD 成分的化学计量。随后使用 PSD-95 拷贝数的共识文献值估计 PSD 内目标蛋白的拷贝数。 NMDA 受体与 AMPA 受体拷贝数比率确定为 ≈ 1:2，得出每个平均 PSD 分别有 34×10 个 NMDA 通道和 68×36 个 AMPA 通道的估计值，与其他方法的估计值一致。 AMPAR 四聚体与 TARP 辅助亚基的比率约为 1:2，支持大多数 AMPA 受体通过 TARP 亚基锚定到 PSD 的论点。 该研究还首次对几种关键 PSD 蛋白的拷贝数进行了估计，并证实或挑战了之前对某些其他 PSD 成分的估计。 这些拷贝数为定量绘制 PSD 分子结构提供了宝贵的指导。