Macromolecular Architecture Of The Synapse

突触的大分子结构

基本信息

批准号：
8746782
负责人：
Thomas S Reese
金额：
$ 106.81万
依托单位：
NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
依托单位国家：
美国
项目类别：
财政年份：
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/8746782
关键词：
AMPA Receptors Adopted Affinity Architecture Area Binding Binding Sites C-terminal Cells Chemicals Chicago Collaborations Complex Development Electrons Electrophysiology (science)Family Family member Filament Floor Fluorescence Resonance Energy Transfer Freezing Glutamate Receptor Glutamates Goals Hippocampus (Brain)Individual Information Storage Label Laboratories Lead Left Length Life Light Maps Measurement Measures Methods Microscopic Microtomy Molecular Molecular Conformation Molecular Machines Molecular Weight N-Methyl-D-Aspartate Receptors National Institute of Biomedical Imaging and Bioengineering Negative Staining Neurons Pattern Physiological Positioning Attribute Postsynaptic Membrane Protein Array Proteins RNA Interference Resolution Scaffolding Protein Signal Transduction Site Staining method Stains Structural Models Structure Synapses Synaptic Membranes Thick Time Work calmodulin-dependent protein kinase II dalton density fluorophore functional loss information processing innovation insight knock-down postsynaptic postsynaptic density protein presynaptic density protein 95 protein complex receptor reconstruction scaffold stargazin study characteristics synaptic function tomography voltage

项目摘要

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 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 suggests 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 now preparing probed 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. A newly developed electron microscopic method (Leapman Lab, NIBIB) using high voltage STEM tomography (HVST) is compatible with sections up to two m 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 confirm 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. Again the structural loss correlates closely well with functional loss measured by physiological recordings. The knock-down in effect results in silent synapses. A method for high resolution EM tomography of isolated PSDs became available with the discovery of a negative stain compatible with tomography. Using this stain makes it possible accurately to map the distribution of CaMKII in isolated PSDs. A second pool of CaMKII is found embedded in the matrix of the PSD, which may be functionally distinct from the soluble pool of CaMKII that binds to the PSD during activity. This pool has been invisible to light microscopic analyses and so revives the idea that the CaMKII in the PSD may be the functionally important pool.

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，我们开发了冷冻替代海马培养物的方法，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）施加排序，并为 PSD 结构提供总体规划。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）。迄今为止的结果表明，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 具有开放结构，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。我们现在准备探讨用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 MAGUKS 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。新开发的电子显微方法（Leapman Lab，NIBIB）使用高压 STEM 断层扫描 (HVST)，与厚达 2 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，以确认同时敲除三个主要 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。同样，结构损失与生理记录测量的功能损失密切相关。击倒实际上会导致沉默的突触。随着与断层扫描兼容的负染色的发现，一种对分离的 PSD 进行高分辨率电磁断层扫描的方法成为可能。使用这种染色剂可以准确地绘制孤立 PSD 中 CaMKII 的分布图。发现第二个 CaMKII 池嵌入 PSD 基质中，其功能可能与在活动期间与 PSD 结合的可溶性 CaMKII 池不同。这个池在光学显微镜分析中是不可见的，因此重新提出了 PSD 中的 CaMKII 可能是功能上重要的池的想法。