Macromolecular Architecture Of The Synapse

突触的大分子结构

• 批准号: 10018402
• 负责人: Thomas S Reese
• 金额: $ 177.55万
• 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/10018402
• 关键词: A kinase anchoring protein; AMPA Receptors; Affinity; Animals; Antibodies; Architecture; Area; Behavior; Behavior Control; Binding; Binding Sites; Brain; CRISPR/Cas technology; Calcineurin; Cells; Chemicals; Collaborations; Colorado; Complex; Cyclic AMP-Dependent Protein Kinases; Data; Development; Diffuse; Diffusion; Dimensions; Distal; Electron Microscopy; Electrons; Electrophysiology (science); Evolution; Excitatory Postsynaptic Potentials; Family; Filament; Force of Gravity; Freeze Substitution; Glutamate Receptor; Glutamates; Goals; Hippocampus (Brain); Image; Imagery; Individual; Information Storage; Knock-out; Label; Laboratories; Length; Light; Lighting; Maps; Mass Spectrum Analysis; Measurement; Mediating; Membrane; Membrane Proteins; Memory; Methods; Microscopic; Microtomy; Molecular; Molecular Conformation; Molecular Machines; Molecular Probes; N-Methyl-D-Aspartate Receptors; NMDA receptor A1; National Institute of Neurological Disorders and Stroke; Nervous system structure; Neurons; Neurophysiology - biologic function; Optics; Oxides; Peptide Signal Sequences; Peripheral; Phosphotransferases; Polymers; Positioning Attribute; Postsynaptic Membrane; Primates; Process; Protein Array; Proteins; RNA Interference; Research; Resolution; Role; Scaffolding Protein; Scanning; Signal Pathway; Signal Transduction; Singlet Oxygen; Site; Stains; Structure; Sucrose; Synapses; Synaptic Transmission; Synaptic plasticity; System; Techniques; Thick; Tomogram; Universities; Vertebral column; Work; calmodulin-dependent protein kinase II; density; functional loss; improved; innovation; insight; interest; knock-down; light microscopy; macromolecule; overexpression; postsynaptic; postsynaptic density protein; presynaptic density protein 95; protein complex; receptor; reconstruction; scaffold; 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 memory information 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 impose an ordering on many other PSD proteins, including the glutamate receptors, and provide an overall plan for the structure of the PSD. We have used antibodies to label proteins in tomograms, but the antibody complexes appearing as filamentous structures in tomograms confounds the identification of the target protein. An indirect approach combining knockdown with tomography has proved additional, but provided limited information for identifying components of protein complexes in tomograms. We attempted to develop an additional alternative method for identifying the proteins using an expressible probe, miniSOG, which generates singlet oxygen upon blue light illumination, and then oxidizes diaminobenzidine (DAB) to form electron dense polymers visualized by EM. We overexpressed miniSOG-PSD-95 in neurons and found that the staining generated by miniSOG is too diffuse to localize molecules with better than 20 nm precision. We therefore used various dilutions of sucrose solution in the photoconversion process to slowdown molecular diffusion. Eventually, with the help of EM tomography, we found evidence that electron dense structures developed at the distal ends of some of the membrane associated vertical filaments known to contain PSD-95, thus allowed identification of miniSOG tagged PSD-95. We plan to expand this work by making CaMKII-miniSOG construct to see if the similar results will provide a specific marker for the large macromolecule CaMKII. 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. 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. We also developed another line of research recently with the Rumbaugh Lab to characterize structural role of SynGAP, which negatively regulates AMPAR binding to the PDZ domain of PAS-95 MAGUKs at the PSD. We are also identifying s NMDARs more directly in intact hippocampal synapses by using CRISPR-Cas9 construct developed in the Nicoll Lab to knockout the obligated GluN1 subunit of NMDARs and reconstruction of the postsynaptic density (PSD) with dark field scanning EM tomography. We now have confirmation that the class of transmembrane structures containing large globular cytoplasmic profiles contain NMDARs, whereas the structures with smaller and flat cytoplasmic profile contain AMPARs. We are completing with M. DellAcqua, University of Colorado, study of the conformations and distribution of A Kinase Anchoring Proteins (AKAPs) 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 or thick section STEM tomography. Finally, we have developed a new electron microscopic method in collaboration with Richard Leapman using darkfield 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, Trichoplax, that is remarkable in that it lacks synapses, but shows behavior indicative of neural function. These results appear to signify 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 a 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 is 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，被认为是记忆信息和存储的关键部位。 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、不同的结合位点。 因此，PSD-95 的规则阵列对许多其他 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。 我们已经使用抗体来标记断层照片中的蛋白质，但断层照片中显示为丝状结构的抗体复合物混淆了目标蛋白质的识别。 结合敲除与断层扫描的间接方法已被证明是额外的，但为识别断层图像中蛋白质复合物的成分提供了有限的信息。 我们尝试开发另一种替代方法，使用可表达探针 miniSOG 来识别蛋白质，该探针在蓝光照射下产生单线态氧，然后氧化二氨基联苯胺 (DAB) 形成电子致密聚合物，通过 EM 可视化。 我们在神经元中过表达 miniSOG-PSD-95，发现 miniSOG 产生的染色过于分散，无法以优于 20 nm 的精度定位分子。 因此，我们在光转换过程中使用各种稀释的蔗糖溶液来减慢分子扩散。 最终，在 EM 断层扫描的帮助下，我们发现证据表明，在一些已知含有 PSD-95 的膜相关垂直丝的远端形成了电子致密结构，从而可以识别带有 PSD-95 的 miniSOG。 我们计划通过构建 CaMKII-miniSOG 构建来扩展这项工作，看看类似的结果是否能为大分子 CaMKII 提供特异性标记。 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。 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 外围区域垂直细丝和 AMPAR 型结构的耗尽，以及 PSD 中心 NMDAR 簇尺寸的显着减小。 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。 我们最近还与 Rumbaugh 实验室合作开展了另一项研究，以表征 SynGAP 的结构作用，它负向调节 AMPAR 与 PSD 处 PAS-95 MAGUK 的 PDZ 结构域的结合。 我们还使用 Nicoll 实验室开发的 CRISPR-Cas9 构建体敲除 NMDAR 的必需 GluN1 亚基，并使用暗场扫描 EM 断层扫描重建突触后密度 (PSD)，从而更直接地在完整海马突触中识别 s NMDAR。 我们现在已经确认，含有大球状细胞质轮廓的跨膜结构类别包含 NMDAR，而具有较小且平坦细胞质轮廓的结构包含 AMPAR。 我们正在与科罗拉多大学的 M. DellAcqua 合作完成对 A 激酶锚定蛋白 (AKAP) 海马突触的构象和分布的研究。 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 PSD 调节 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 表达可增强突触传递，我们计划使用连续切片 EM 或厚切片 STEM 断层扫描来分析脊柱大小和 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，Trichoplax，它的引人注目之处在于它缺乏突触，但表现出表明神经功能的行为。 这些结果似乎标志着神经系统进化的早期阶段，在突触发育之前，其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。 准确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。