Macromolecular Architecture Of The Synapse

兴奋性谷氨酸能突触的突触后密度(PSD)是一个分子量大于10亿道尔顿的大分子机器。众所周知，PSD是信息处理和存储的关键场所。为了探索PSD的详细分子结构，我们开发了冷冻替代海马区培养的方法，然后用EM断层扫描对其进行薄层扫描，以显示PSD内各个蛋白质复合体的自然环境。断层扫描的初步工作显示，PSD的核心是一系列垂直定向的细丝，其中包含支架蛋白PSD-95，呈扩展配置和极化方向，其N端位于突触后膜。这一发现为PSD的整体组织提供了洞察，因为支架蛋白，如PSD-95家族Maguk蛋白，与沿其长度排列的其他蛋白质具有不同的多个不同的结合位点。因此，PSD-95的规则排列可能与其他家族成员一起对包括谷氨酸受体在内的许多其他PSD蛋白施加了顺序，并为PSD的结构提供了一个整体计划。 与绿色实验室(芝加哥大学)合作，制作了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反末端。这些标记允许通过免疫金-EM和对活细胞进行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中采用延伸构象，但在非突触部位采用闭合构象。相反，SAP-97是另一种Maguk，具有开放的结构，但与突触后膜平行定向。PSD-95在PSD处的开放构象是它与NMDAR和AMPAR-Stargazin络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有关。一类水平纤维有序地与垂直纤维形成六角形交联剂，并集中在NMDA受体的下方。免疫金标记法现在初步鉴定了一类水平细丝为GKAP，它是一种已知与PSD-95 C末端的GK结构域结合的蛋白。免疫金标记法也被用来定位另一个主要的支架分子，Shank，它被认为是直接结合GKAP的。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的边缘添加新的受体的空间。鉴定PSD的成分是耗时的，鉴定蛋白质的方法也需要改进。一种可表达的探针mini SOG已经问世，我们使用它来确认垂直细丝是PSD-95。我们现在准备用微型SOG来明确识别PSD中的GKAP和Shank。 用EM断层扫描技术研究了PSD-95依赖支架稳定PSD的想法，以确定MAGUKS的RNAi击倒效应。最近，我们研究了同时击倒三种主要的Maguk蛋白：PSD-95、PSD-93和SAP102的效果，并首次通过EM断层扫描发现PSD的中央核心显著丢失，包括NMDA受体结构、垂直丝和AMPA受体。尼科尔实验室(UCSF)的合作者对相同击穿效应进行的电生理学测量表明，NMDAR和AMAPR型EPSP的功能损失与结构损失相容。 一种新开发的电子显微镜方法(Leapman Lab，NIBIB)使用高压STEM断层扫描(HVST)，可以兼容厚度达2米的切片，并显示许多完整突触的详细重建。我们在突触包含整个PSD的1-2微米切片上使用HVST来证实，同时击倒三个主要的PSD-95家族MAGUK导致整个PSD面积显著减少，使许多突触只有很小的PSD。同样，结构损失与生理记录测量的功能损失密切相关。这种击倒实际上导致了沉默的突触。 随着发现与断层扫描相容的负性染色，一种对孤立的PSD进行高分辨率EM断层扫描的方法成为可能。使用这种染色可以准确地定位CaMKII在分离的PSD中的分布。在PSD的基质中发现了第二个CaMKII池，它可能在功能上不同于在活性过程中与PSD结合的可溶性CaMKII池。这个池在光学显微镜分析中是看不见的，因此再次提出了PSD中的CaMKII可能是功能上重要的池的想法。

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.