Macromolecular Architecture Of The Synapse

突触的大分子结构

• 批准号: 9563108
• 负责人: Thomas S Reese
• 金额: $ 130.45万
• 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/9563108
• 关键词: A kinase anchoring protein; AMPA Receptors; Actins; Adopted; Affinity; Alpha Cell; Animals; Architecture; Area; Behavior; Behavior Control; Binding; Binding Sites; Brain; C-terminal; CRISPR/Cas technology; Calcineurin; Chemicals; Collaborations; Colorado; Complex; Cyclic AMP-Dependent Protein Kinases; Darkness; Data; Defect; Dendritic Spines; Development; Diffuse; Dimensions; Electron Microscopy; Electrons; Electrophysiology (science); Energy Transfer; Family; Family member; Filament; Force of Gravity; Freeze Substitution; Glutamate Receptor; Glutamates; Goals; Hippocampus (Brain); Image; Imagery; Individual; Information Storage; Knock-out; Label; Laboratories; Length; Maps; Mass Spectrum Analysis; Measurement; Measures; Mediating; Membrane; Membrane Proteins; Memory; Methods; Microscopic; Microtomy; Molecular; Molecular Conformation; Molecular Machines; Molecular Probes; Motor; Mus; Mutant Strains Mice; Myosin Type V; N-Methyl-D-Aspartate Receptors; National Institute of Neurological Disorders and Stroke; Nervous system structure; Neurons; Neurophysiology - biologic function; Optics; Peptide Signal Sequences; Peripheral; Phosphotransferases; Positioning Attribute; Postsynaptic Membrane; Protein Array; Proteins; RNA Interference; Resolution; Role; Scaffolding Protein; Scanning; Signal Pathway; Signal Transduction; Site; Staining method; Stains; Structural Models; Structure; Synapses; Synaptic Membranes; Synaptic Transmission; Synaptic plasticity; System; Techniques; Thick; Time; Universities; Vertebral column; Work; base; calmodulin-dependent protein kinase II; density; functional loss; improved; information processing; innovation; insight; interest; knock-down; light microscopy; mutant; postsynaptic; postsynaptic density protein; presynaptic density protein 95; protein complex; receptor; reconstruction; scaffold; stargazin; synaptic function; tomography; trafficking

The postsynaptic density (PSD) at excitatory glutamatergic synapses is a large molecular machine that 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 provided 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. The mechanisms that regulate PSD-95 MAGUK conformations were investigated in collaboration with the William Green laboratory. A new technique, Fluorescent Resonate Energy Transfer (FRET) was applied to measure configurations of PSD-95 molecules in different functional configurations. PSD-95 adopts an extended conformation in PSDs, but remains in closed conformation at non-synaptic sites. In contrast, SAP-97, another MAGUK, adopts an open configuration, but is oriented parallel with the post synaptic membrane. The open conformation of PSD-95 at the PSD is now established as a requirement for it to interact with NMDAR and AMPAR-Stargazin complexes. EM tomography also revealed that the C-terminal ends of the PSD-95 vertical filaments are associated with horizontally oriented filaments. Immunogold labeling identifies one class of horizontal filaments as GKAP, which is a known to bind to the GK domain at the C-terminal end of PSD-95. The emerging structural model of the PSD shows how the PSD-95 matrix can stabilize glutamate receptors forming elaborate molecular complexes, and at the same time allows room for the addition of new receptors at the edges of the PSD. We are continuing this line of work with EM tomography to unravel how the GKAP-Shank and perhaps Homer scaffolding system might contribute to the proper functions of glutamate receptors. Direct identification of the components of the PSD is difficult and we attempted to develop an improved method for identifying the proteins using an expressible probe, miniSOG, confirming that the vertical filaments are PSD-95, however, the staining generated by miniSOG is, so far, too diffuse to localize molecules with better than 20 nm precision, so further fine tuning of this approach is necessary to overcome this problem. 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 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 Roger Nicoll laboratory 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, also showed depletion of vertical filaments along with AMPAR type structures at the peripheral region of the PSD, and significant reduction in size of NMDAR clusters in the center of the PSD. These structural data indicate that vertical filaments corresponding to MAGUKs anchor AMPARs and are also a factor in organizing NMDARs. Thus, PSD-95 MAGUKs are demonstrated to be the essential organizer of glutamate receptors at the PSD. Continuing on this line of work, we are trying to identify NMDARs more directly in intact hippocampal synapses by combining CRISPR-Cas9 knockout of NMDARs and reconstruction of the postsynaptic density (PSD) with dark field scanning EM tomography. We now have new evidence that the class of transmembrane structures containing larger globular cytoplasmic profile likely contain NMDARs whereas the structures with smaller and flat cytoplasmic profile likely contain AMPARs. Being able to directly identify NMDAR and AMPARs paves the way to visualizing the changes in receptor distributions underlying synaptic plasticity. A new collaboration with the M. Constantine-Patton lab seeks to determine the structure of synapses in a Flailer mouse mutant where many of actin based myosin V motors are inactive. This defect leads to significant reduction in PSD-95 MAGUKs, as well as GKAPs and Shank-Homer molecules in the dendritic spines, and greatly reduced synaptic transmission. So far, we have examined disassociated cultures of flailer mice, and will proceed with serial section EM to compare the structural differences in dendritic spines and PSDs of control and mutant mice. Ultimately, analysis by EM tomography will sort out the actual molecular deficits in this mutant. We have also developed a collaboration with M. DellAcqua, University of Colorado, to study conformations and distribution of A Kinase Anchoring Proteins (AKAPs) at hippocampal synapses. These molecules are membrane associated proteins known to interact with PSD-95 MAGUKs and anchor several classes of kinases (PKA, PKC) and calcineurin, important for synaptic plasticity (LTP and LTD). This work is showing that there is a conformational change in AKAPs in the PSD, different from that at the extrasynaptic membrane. This distinction may have important functional implications in understanding the role of AKAPs in regulating AMPARs at the PSDs. In collaboration with the Roger Nicoll lab, we are studying the effects of over expressing constitutively activated CaMKII on synaptic structure and function. Electrophysiology measurements show that activated CaMKII expression enhances synaptic transmission, and we plan to analyze changes in spine sizes and PSD structure, using serial section EM. Finally, we have developed a new electron microscopic method in collaboration with Richard Leapman using dark field STEM tomography for sections up to 300-400 nm thick to provide detailed reconstructions of whole PSDs. The darkfield imaging, which provides enhanced visualization of the smallest structures at the PSDs provides an opportunity to reconstruct detailed molecular organization of more or less complete PSDs in intact neurons. A new initiative is an ongoing collaboration with Carolyn Smith in the NINDS Light Microscopy Facility. Dr. Smith has cultured a primitive animal that is remarkable in that it lacks synapses but shows behavior indicative of neural function. These results are compatible with an early stage in evolving nervous systems, prior to the development of synapses, that utilizes peptide signaling pathways dependent on many of the same proteins found at synapses in higher animals. A cell that senses direction of gravity and mediates behavior accordingly has also been discovered, but what control systems are utilized is not yet clear. Knowing exactly how these unconventional, nonsynaptic systems function to control behaviors are expected to provide previously overlooked information on non-synaptic signaling mechanisms in mammalian brains.

兴奋性谷氨酸突触的突触后密度（PSD）是一个大型分子机器，被认为是信息处理和存储的关键部位。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 William Green 实验室合作研究了调节 PSD-95 MAGUK 构象的机制。应用荧光共振能量转移 (FRET) 新技术来测量不同功能构型的 PSD-95 分子的构型。 PSD-95 在 PSD 中采用扩展构象，但在非突触位点保持闭合构象。 相比之下，另一种 MAGUK SAP-97 采用开放配置，但方向与突触后膜平行。 PSD-95 在 PSD 处的开放构象现已确定为它与 NMDAR 和 AMPAR-Stargazin 复合物相互作用的要求。 EM 断层扫描还显示 PSD-95 垂直细丝的 C 末端与水平方向的细丝相关。 免疫金标记将一类水平丝识别为 GKAP，已知它与 PSD-95 C 末端的 GK 结构域结合。 PSD 的新兴结构模型展示了 PSD-95 基质如何稳定谷氨酸受体，形成复杂的分子复合物，同时为 PSD 边缘添加新受体留出空间。 我们正在利用 EM 断层扫描继续开展这一工作，以揭示 GKAP-Shank 以及 Homer 支架系统如何有助于谷氨酸受体的正常功能。直接鉴定 PSD 的成分很困难，我们尝试开发一种改进的方法，使用可表达探针 miniSOG 来鉴定蛋白质，确认垂直丝是 PSD-95，然而，到目前为止，miniSOG 生成的染色是，过于分散，无法以优于 20 nm 的精度定位分子，因此需要进一步微调此方法来克服此问题。 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。 Roger Nicoll 实验室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。电子显微镜还显示 PSD 外围区域的垂直细丝和 AMPAR 型结构的耗尽，以及 PSD 中心 NMDAR 簇的尺寸显着减小。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。因此，PSD-95 MAGUK 被证明是 PSD 谷氨酸受体的重要组织者。 继续这一工作，我们试图通过结合 CRISPR-Cas9 敲除 NMDAR 和用暗场扫描 EM 断层扫描重建突触后密度 (PSD)，更直接地识别完整海马突触中的 NMDAR。我们现在有新的证据表明，含有较大球状细胞质轮廓的跨膜结构类别可能含有 NMDAR，而具有较小且平坦细胞质轮廓的结构可能含有 AMPAR。 能够直接识别 NMDAR 和 AMPAR 为可视化突触可塑性受体分布的变化铺平了道路。 与 M. Constantine-Patton 实验室的一项新合作旨在确定 Flailer 小鼠突变体的突触结构，其中许多基于肌动蛋白的肌球蛋白 V 马达不活跃。这种缺陷导致树突棘中的 PSD-95 MAGUK、GKAP 和 Shank-Homer 分子显着减少，并大大减少突触传递。到目前为止，我们已经检查了连枷小鼠的分离培养物，并将继续进行连续切片电镜来比较对照和突变小鼠的树突棘和 PSD 的结构差异。最终，电磁断层扫描分析将找出该突变体中实际的分子缺陷。我们还与科罗拉多大学 M. DellAcqua 开展合作，研究海马突触中 A 激酶锚定蛋白 (AKAP) 的构象和分布。这些分子是已知与 PSD-95 MAGUK 相互作用的膜相关蛋白，并锚定几类激酶（PKA、PKC）和钙调神经磷酸酶，对突触可塑性（LTP 和 LTD）很重要。这项工作表明，PSD 中的 AKAP 存在构象变化，与突触外膜处的构象变化不同。这种区别对于理解 AKAP 在调节 PSD 的 AMPAR 中的作用可能具有重要的功能意义。 我们与 Roger Nicoll 实验室合作，研究过度表达组成型激活的 CaMKII 对突触结构和功能的影响。电生理学测量表明，激活的 CaMKII 表达增强了突触传递，我们计划使用连续切片 EM 分析脊柱大小和 PSD 结构的变化。最后，我们与 Richard Leapman 合作开发了一种新的电子显微方法，使用暗场 STEM 断层扫描来拍摄厚度达 300-400 nm 的切片，以提供整个 PSD 的详细重建。暗场成像增强了 PSD 最小结构的可视化，为重建完整神经元中或多或少完整的 PSD 的详细分子组织提供了机会。 一项新举措是与 Carolyn Smith 在 NINDS 光学显微镜设施中持续合作。史密斯博士培养了一种原始动物，这种动物的非凡之处在于它缺乏突触，但表现出表明神经功能的行为。这些结果与进化神经系统的早期阶段相一致，在突触发育之前，神经系统利用依赖于高等动物突触中发现的许多相同蛋白质的肽信号传导途径。还发现了一种能够感知重力方向并相应调节行为的细胞，但使用什么控制系统尚不清楚。确切地了解这些非常规的非突触系统如何发挥控制行为的作用，有望提供以前被忽视的有关哺乳动物大脑中非突触信号机制的信息。