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.

兴奋性谷氨酸能突触上的突触后密度（PSD）是一种大分子机器，已知是信息处理和存储的关键位置。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。 调查了PSD-95 MAGUK构象的机制与William Green实验室合作研究了。采用了一种新技术，荧光共鸣能传递（FRET）用于测量不同功能构型中PSD-95分子的构型。 PSD-95在PSD中采用了扩展的构象，但在非突触部位保持封闭状态。 相比之下，另一个Maguk SAP-97采用开放式配置，但与邮政突触膜平行。 现在确定了PSD-95在PSD-95上的开放构象，是它与NMDAR和AMPAR-Stargazin复合物相互作用的要求。 EM断层扫描还显示，PSD-95垂直丝的C末端末端与水平定向细丝有关。 免疫元标记将一类水平丝标识为GKAP，已知在PSD-95的C末端与GK结构域结合。 PSD的新兴结构模型显示了PSD-95基质如何稳定形成精心的分子复合物的谷氨酸受体，同时允许在PSD边缘添加新受体的空间。 我们正在继续使用EM层析成像进行这项工作，以解开GKAP-SHANK和HOMER脚手架系统如何有助于谷氨酸受体的正确功能。直接识别PSD的组件很困难，我们试图开发一种改进的方法来使用表达探针，Minisog识别蛋白质，证实垂直细丝是PSD-95，但是，Minisog产生的染色是为止，到目前为止，太差异了，无法与20 Nm的分子进行差异化，因此可以进一步解决这个方法，因此可以很好地调整这个方法，因此可以很好地调整这个问题。 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，我们检查了同时击倒三种主要MAGUK蛋白的影响：PSD-95，PSD-93和SAP102以及EM层析成像显示PSD中心核心的损失显着损失，包括NMDA受体结构，垂直丝和AMPA受体。罗杰·尼科尔（Roger Nicoll）实验室的合作者的电生理测量表征了同一敲门的影响，显示与结构损失兼容的NMDAR和AMAPR型EPSP的功能损失显着。电子显微镜还显示出垂直丝的耗竭以及PSD的外围区域的AMPAR类型结构，以及PSD中心的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。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。 继续进行这项工作，我们试图通过结合NMDAR的CRISPR-CAS9敲除和重建突触后密度（PSD）与深色场扫描Emhyproghich的重建，以更直接地识别完整的海马突触中的NMDAR。现在，我们有新的证据表明，包含较大球形细胞质谱的跨膜结构可能包含NMDAR，而具有较小和平坦的细胞质剖面的结构可能包含AMPAR。 能够直接识别NMDAR和AMPARS为可视化突触可塑性基础受体分布的变化铺平了道路。 与M. constantine-patton实验室的新合作旨在确定拖鞋小鼠突变体中突触的结构，其中许多基于肌动蛋白的肌球蛋白V电动机都是不活跃的。这种缺陷导致树突状棘中的PSD-95 Maguks以及Gkap和柄 - 大分子的显着降低，并大大降低了突触传播。到目前为止，我们已经检查了Flailer小鼠的分离培养物，并将继续进行串行部分EM，以比较对照和突变小鼠的树突状刺和PSD的结构差异。最终，通过EM断层扫描分析将分析该突变体中的实际分子缺陷。我们还与科罗拉多大学的M. Dellacqua开发了一项合作，以研究海马突触中激酶锚定蛋白（AKAP）的构象和分布。这些分子是膜相关的蛋白质，已知可与PSD-95 Maguks相互作用，并锚定了几类激酶（PKA，PKC）和钙调蛋白，对突触可塑性（LTP和LTD）很重要。这项工作表明，PSD中AKAP的构象变化与外膜外膜不同。这种区别可能在理解AKAP在调节PSD的AMPAR中的作用方面具有重要的功能含义。 与罗杰·尼科尔（Roger Nicoll）实验室合作，我们正在研究过度表达组成型激活的CAMKII对突触结构和功能的影响。电生理测量表明，活性CAMKII表达增强了突触传播，我们计划使用串行部分EM分析脊柱大小和PSD结构的变化。最后，我们使用深色田间茎断层扫描与理查德·莱夫曼（Richard Leapman）合作开发了一种新的电子显微镜方法，该方法最高为300-400 nm厚，以提供整个PSD的详细重建。 Darkfield成像可增强PSD上最小结构的可视化，这为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。 一项新的计划是与卡罗琳·史密斯（Carolyn Smith）在Ninds轻型显微镜设施中进行的一项持续合作。史密斯博士培养了一种原始的动物，这是显着的，因为它缺乏突触，但表明了表明神经功能的行为。这些结果与不断发展的神经系统的早期阶段兼容，在突触发展之前，它利用了肽信号传导途径取决于高等动物突触中发现的许多相同蛋白质。也发现了一种感知重力和介导行为方向的细胞，但使用哪些控制系统尚不清楚。确切地了解这些非常规的，非突触系统如何控制行为，以提供先前被忽视的有关哺乳动物大脑中非突触信号机制的信息。