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; 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上的突触后密度（PSD）是一种大于十亿达尔顿的分子量的大分子机。已知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，我们开发了一种冻结亚种海马培养物的方法，然后通过EM层析成像在薄层中检查它们，以在PSD内显示其自然环境中的单个蛋白质复合物。采用断层扫描的最初作品表明，PSD的核心是垂直定向的细丝，其中包含脚手架蛋白PSD-95，以扩展的构型和偏振方向，其N末端位于后突触膜上。这一发现提供了对PSD的整体组织的见解，因为脚手架蛋白（例如PSD-95家族Maguk蛋白）具有沿其长度阵列的其他蛋白质的多种多种，不同的结合位点。因此，PSD-95的常规阵列可能与其他家庭成员一起对许多其他PSD蛋白（包括谷氨酸受体）施加订购，并为PSD结构提供了总体计划。 将FRET结构用于研究与绿色实验室（U芝加哥）合作调节PSD-95 MAGUK构象的可能机制。两个荧光团（RFP和YFP）融合到PSD-95或其他家庭成员的相对末端。这些标签允许家庭成员的构象通过免疫原-EM和对活细胞进行FRET测量来确定（如果分子FRET的两端，它们必须处于封闭构型中）。到目前为止，结果表明PSD-95在PSD中采用了扩展的构象，但在非突触部位的封闭构型中采用了构象。相比之下，SAP-97，另一个MAGUK具有开放式配置，但与后突触膜平行。 PSD在PSD上的开放构象必须与NMDAR和AMPAR-Stargazin复合物相互作用。 EM断层扫描还显示，垂直丝的C末端末端与水平方向的细丝有关。命令一类水平细丝与垂直丝形成六边形的交联，并集中在NMDA受体下方。现在，免疫金标记暂时将一类水平细丝鉴定为GKAP，已知在PSD-95的C末端与GK结构域结合。免疫金标记还用于定位另一个主要的脚手架分子尚克，该分子已知可以直接结合GKAP。 PSD的新兴结构模型显示了PSD-95基质如何稳定谷氨酸受体，同时允许在PSD边缘添加新受体的空间。识别PSD的组件是耗时的，并且识别蛋白质的方法需要改进。表达的探针Minisog已可以使用，我们使用它来确认垂直丝是PSD-95。现在，我们正在准备使用Minisog肯定识别PSD中的GKAP和小腿的探针。 通过使用EM断层扫描来确定RNAi敲击Maguks的效果，已经探索了PSD-95依赖性支架稳定PSD的想法。最近，我们研究了同时敲击三种主要MAGUK蛋白的影响：PSD-95，PSD-93和SAP102，EM层析成像首次揭示了PSD中心核心的显着损失，包括NMDA受体结构，垂直细丝和AMPA受体。 NICOLL实验室（UCSF）的合作者的电生理测量表征了同一敲门的影响，显示NMDAR和AMAPR型EPSP的显着功能损失与结构损失兼容。电子显微镜表现出垂直丝的消耗以及PSD的外围区域的AMPAR类型结构以及PSD中间的NMDAR簇的大小显着降低。这些结构数据表明，垂直丝与Maguks锚定AMPAR相对应，也是组织NMDAR的一个因素。 NMDAR和AMPAR的差异化组织为静音突触提供了结构性基础，其中PSD包含一个被少数AMPAR包围的NMDAR群集。因此，PSD-95 Maguk被证明是PSD处谷氨酸受体的必不可少的组织者。 使用高压茎断层扫描（HVST）的新开发的电子显微镜方法（Leapman Lab，Nibib）与多达两个微米厚的部分兼容，并且正在揭示许多整个突触的详细重建。我们在1-2个UM部分上使用了HVST，这些HVST包含突触处的整个PSD，以证明同时击倒了三个主要的PSD-95家族Maguks，从而显着降低了整个PSD区域，从而使许多突触只有很小的PSD。由于突触的亚群完全失去了其电子密集的PSD材料，因此三重敲击实际上导致了许多无声突触，如电生理学报道。 与美国国家标准技术研究所合作，采用了一种新型的定量质谱法来评估PSD组件的石化元素。随后，使用PSD-95的拷贝数的共识文献值估算了PSD内目标蛋白的拷贝数。 NMDA受体与AMPA受体拷贝数比率分别为34 10 NMDA通道和68 36 36 AMPA通道的估计值分别与其他方法的估计相一致。 AMPAR四聚体与TARP辅助亚基的比率为≈1：2，支持大多数AMPA受体通过TARP亚基锚定在PSD上的断言。 该研究还首次对几种密钥PSD蛋白的拷贝数产生了估计，并确认或挑战了某些其他PSD组件的先前估计。 这些拷贝数提供了有价值的指南，以绘制PSD的定量分子体系结构。