Macromolecular Architecture Of The Synapse

突触的大分子结构

• 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
• 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

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.

兴奋性突触的突触后密度（PSD）是一个分子量大于10亿道尔顿的大分子机器。PSD是信息处理和存储的关键场所。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断层扫描在薄切片中检查它们，以显示PSD内自然环境中的单个蛋白质复合物。最初的工作采用断层扫描显示，PSD的核心是一个阵列的垂直取向的细丝，包含支架蛋白，PSD-95，在一个扩展的配置和极化方向，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）具有沿着其长度排列的其他蛋白质的独特的多个不同的结合位点。因此，PSD-95可能与其他家族成员的规则阵列对许多其他PSD蛋白质（包括谷氨酸受体）施加了排序，并为PSD的结构提供了总体规划。 与绿色实验室（U芝加哥）合作，制备FRET构建体以研究调节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-EM和通过对活细胞进行FRET测量来确定家族成员的构象（如果分子FRET的两端，则它们必须处于闭合构型）。到目前为止的结果表明，PSD-95在PSD中采用伸展构象，但在非突触位点处采用闭合构象。相比之下，SAP-97，另一种MAGUK具有开放构型，但与突触后膜平行取向。PSD-95在PSD处的开放构象是其与NMDAR和AMPAR-Stargazin复合物相互作用的必要条件。 EM断层扫描还显示，垂直细丝的C-末端与水平取向的细丝相关。一类水平丝与垂直丝形成六角交联体，集中在NMDA受体下方。免疫金标记现在暂时将一类水平细丝鉴定为GKAP，已知其在PSD-95的C末端结合GK结构域。免疫金标记也被用来定位另一个主要的支架分子，SHANK，这是已知的直接结合GKAPs。PSD的新兴结构模型显示了PSD-95基质如何稳定谷氨酸受体，同时为在PSD边缘添加新受体提供了空间。PSD组分的鉴定是耗时的，并且用于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细丝是PSD-95。我们现在准备使用miniSOG探测以明确识别PSD中的GKAP和SHANK。 PSD-95依赖性支架稳定PSD的想法已经通过使用EM断层摄影术来确定MAGUKS的RNAi敲低的效果而被探索。 最近，我们研究了同时敲除三种主要MAGUK蛋白：PSD-95，PSD-93和SAP 102的影响，并且第一次，EM断层扫描显示PSD的中央核心，包括NMDA受体结构，垂直细丝和AMPA受体的显著损失。来自Nicoll实验室（UCSF）的合作者的电生理学测量表征了相同敲除的影响，其显示在与结构损失相容的水平下NMDAR和AMAPR型EPSP的显著功能损失。 一种新开发的电子显微镜方法（Leapman实验室，NIBIB）使用高压STEM断层扫描（HVST）与厚达2 m的切片兼容，并揭示了许多完整突触的详细重建。我们在突触处含有完整PSD的1-2 μ m切片上使用HVST，以证实三种主要PSD-95家族MAGUK的同时敲除导致总体PSD面积的显著减少，使得许多突触仅具有小PSD。同样，结构损失与通过生理记录测量的功能损失密切相关。这种抑制作用实际上导致了沉默的突触。 一种用于孤立PSD的高分辨率EM断层扫描的方法随着与断层扫描兼容的阴性染色剂的发现而变得可用。使用这种染色剂可以准确地绘制孤立PSD中CaMK II的分布。第二池的CaMKII被发现嵌入在基质的PSD，这可能是功能上不同的可溶性池的CaMKII结合到PSD在活动期间。该池已不可见的光显微镜分析，因此恢复的想法，在PSD中的CaMKII可能是功能上重要的池。