Dynamic Structural Properties of Synapses

突触的动态结构特性

• 批准号: 10708615
• 负责人: Thomas S Reese
• 金额: $ 125.25万
• 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/10708615
• 关键词: 3-Dimensional; A kinase anchoring protein; AMPA Receptors; Acidic Region; Animals; Antibodies; Behavior; Behavior Control; Binding Sites; Brain; CRISPR/Cas technology; Cell Differentiation process; Cell physiology; Cells; Chemical Synapse; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Colorado; Complex; Cyclic AMP-Dependent Protein Kinases; Data; Electrophysiology (science); Endosomes; Evolution; Filament; Freeze Substitution; Genetic; Glean; Glutamate Receptor; Glutamates; Goals; Gravity Perception; Hippocampus (Brain); Image; Immunoelectron Microscopy; Individual; Knock-in; Knock-out; Label; Length; Light; Link; Location; Macromolecular Complexes; Maps; Measurement; Membrane; Memory; Methods; Microtomy; Modeling; Molecular; Molecular Conformation; Molecular Machines; Morphologic artifacts; Muscle; NMDA receptor A1; National Institute of Neurological Disorders and Stroke; Nerve; Nervous System; Neurons; Neuropeptides; Neurophysiology - biologic function; Neurotransmitters; Organelles; Organism; Peroxides; Phosphotransferases; Plastic Embedding; Population; Positioning Attribute; Problem Solving; Process; Property; Proteins; Publications; Publishing; Recycling; Regulation; Research; Resolution; Role; Scaffolding Protein; Scanning; Secretory Cell; Shapes; Signal Pathway; Signal Transduction; Site; Structural Models; Structure; Synapses; Synaptic Transmission; Synaptic plasticity; System; Thick; Tomogram; Universities; Vertebral column; Water; Work; calmodulin-dependent protein kinase II; cell type; density; information processing; insight; intercellular communication; interest; light microscopy; macrophage; molecular dynamics; nanobodies; nanocluster; neuroregulation; overexpression; palmitoylation; postsynaptic; postsynaptic density protein; presynaptic density protein 95; protein complex; receptor binding; reconstruction; success; 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 processing, and storage. To map the molecular organization of the PSD, we freeze-substitute hippocampal cultures and examine them in plastic embedded sections by EM tomography. This reveals individual protein complexes within the PSD. Our early tomography work revealed that the core of the PSD is an array of membrane-associated, vertically oriented filaments like PSD-95. This finding provided insight into the overall organization of the PSD. For instance, scaffolding proteins like PSD-95 have multiple common binding sites arrayed along their length, such that, regular arrays of vertically oriented PSD-95 filaments impose order on other PSD proteins, including the glutamate receptors, and provide an overall plan for the core structure of the PSD. The projects outlined below use and refine our fundamental insight into the organization of synaptic structures to explore the dynamics of molecular changes in the PSD and understand how these molecules contribute to synaptic function. We have several lines of ongoing research and collaboration to investigate specific synaptic proteins. Recently, we collaborated with Rumbaugh Lab to characterize the structural role of SynGAP, which negatively regulates the glutamate AMPA receptor binding to the PDZ domain of PSD-95 at the PSD. We have used immunoEM to map the location, orientation, and conformation of SynGAP at the PSD to arrive at a structural model of how SynGAP might regulate and control synaptic excitability. In collaboration with the Roger Nicoll Lab, we are studying the effects of overexpressing constitutively activated CaMKII on synaptic structure and function. To ameliorate potential artifacts due to overexpressing of CaMKII, we are also using a newly developed CaMKII CRISPR knock-in construct which allows expression and localization of endogenous CaMKIIs in neurons. 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. We are finishing up the work directly identifying NMDARs in the PSD in intact hippocampal synapses by using CRISPR-Cas9 construct developed in the Nicoll Lab. The knockout eliminates the required GluN1 subunit of NMDARs. We made 3Dreconstructions of the resulting PSDs with dark field scanning EM tomography. As result, we now have evidence that individual NMDARs and AMPARs can be identified by EM tomography, and their organization and connections with other molecules can be delineated. We are in the process of finishing up this work and preparing it for publication. Also, we just published a major study with M. DellAcqua's lab at the University of Colorado on the conformations and distribution of Anchoring Proteins (AKAPs) in the hippocampal synapse. In this work, we used immunoEM on thick sections and visualized them using STEM tomography. The results showed palmitoylation effects on AKAP150/79 membrane organization, trafficking, and mobility. Membrane-associated AKAPs are known to interact with PSD-95 MAGUKs and anchor several classes of kinases (PKA and PKC) important for synaptic plasticity (LTP and LTD). This work demonstrates that there is a conformational change in AKAPs in the PSD, different than that at the extrasynaptic membrane, and this distinction may have important functional implications in understanding the role of AKAPs in regulating AMPARs at the PSDs. We also found extensive AKAP association with recycling endosomes and that depalmitolylation appeared to diminish such association. This project opened an exciting new front in imaging AMPAR trafficking. EM tomography has allowed the creation of 3D reconstructions to delineate the organization of subsynaptic organelles, key synaptic proteins, and macromolecular complexes at synapses. Reconstructions provide the size, shape, and location of structures at 2-4 nm resolution but cannot guarantee unambiguous molecular identification of the individual structures. While we had success using immunogold to label endogenous and overexpressed GFP-tagged PSD-95, the large antibody complexes that also manifest as filamentous structures in tomograms confound the identification of the target PSD proteins. Now we have a major technical breakthrough with APEX2 and nanobody labeling to solve this problem. In one localization project, we are using the genetic tag APEX2 to localize CaMKII. The CaMKII-APEX2 construct in the presence of DAB and peroxide has revealed individual CaMKIIs. We are studying CaMKII in the spine and membrane in basal and high K stimulated conditions by EM tomography, and we are in the process of finishing up the work and preparing a publication. In the meantime, we are expanding the APEX2 work to Shank and Homer. Furthermore, another localization project uses an advance in nanobody labeling. In this parallel method, we developed a method to use nanobodies with EM tomography to directly identify PSD proteins in spines. This is a swift method, and we are expecting exciting new results in the coming years on a slew of synaptic molecules. Recently, a major issue has cropped up in the field regarding PSD-95. Several super-resolution light microscopy studies have suggested that PSD-95 forms 100 nm subsynaptic nanoclusters at the PSD. This is significantly smaller than the average size of a PSD, yet we considered PSD-95 to be uniformly distributed. Currently, we are re-examining the endogenous PSD-95 distribution at the PSD with thick section tomography to further study PSD-95 distribution and clustering at the PSD. We are also further analyzing the distribution of all vertical filaments in tomograms using the methods outlined above to see if any clustering of the vertical filaments ever exists within the PSD and if we can reconcile the super-resolution findings with immuno-labeling and tomographic EM. Outside our work in mammalian neurons, an ongoing collaboration with Carolyn Smith in the NINDS Light Microscopy Facility and Adriano Senatore (University of Toronto Mississauga) sheds light on the evolution of cell types and pre-neural regulations in a primitive animal, Trichoplax. Although lacking muscles, nerves, and synapses, Trichoplax demonstrates different types of behaviors indicative of neural function. We identified a cell that senses gravity and described the consequence of stages, which occurs during the differentiation of this cell. We also described a population of cells functioning as macrophages. Our collaborative effort provided evidence that Trichoplax can sense the pH of ambient water and demonstrates avoidance of low pH (acidic) regions. However, the regulatory mechanisms as well as the ways Trichoplax cells communicate with each other are not yet clear. Our previous results and elsewhere obtained data showed that this organism utilizes neuropeptide signaling pathways dependent on many of the same proteins found at synapses in higher animals. In our next steps, we will characterize different types of secretory cells in Trichoplax, which presumably regulate and integrate functional activities of the cells in the nerveless animal. 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.

兴奋性谷氨酸能突触上的突触后密度（PSD）是一台大分子机器，已知是记忆，信息处理和存储的关键位置。为了绘制PSD的分子组织，我们冻结了海马培养物，并通过EM层析成像在塑料嵌入的切片中检查它们。这揭示了PSD内的单个蛋白质复合物。我们的早期断层扫描工作表明，PSD的核心是膜相关的，垂直方向的细丝，如PSD-95。这一发现提供了对PSD整体组织的见解。例如，像PSD-95这样的脚手架蛋白具有沿其长度阵列的多个常见结合位点，因此，常规的垂直定向的PSD-95细丝对其他PSD蛋白（包括谷氨酸受体）施加了顺序，并为PSD的核心结构提供了整体计划。以下概述的项目使用了我们对突触结构组织的基本见解，以探索PSD分子变化的动力学，并了解这些分子如何对突触功能产生贡献。 我们有几条正在进行的研究和协作来研究特定的突触蛋白。最近，我们与Rumbaugh Lab合作，表征了Syngap的结构作用，该结构作用负责调节PSD上PSD-95的PDZ域的谷氨酸AMPA受体结合。我们已经使用免疫系统来绘制Syngap在PSD处的位置，方向和构象，以达到Syngap如何调节和控制突触兴奋性的结构模型。与Roger Nicoll实验室合作，我们正在研究过表达Camkii对突触结构和功能的过表达的影响。为了改善由于Camkii过表达而导致的潜在伪影，我们还使用了新开发的Camkii CRISPR敲入结构，该结构允许内源性CAMKII在神经元中的表达和定位。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或厚截面茎断层扫描来分析脊柱大小和PSD结构的变化。 我们正在通过使用Nicoll Lab中开发的CRISPR-CAS9构建体来完成直接识别完整海马突触中PSD中NMDAR的工作。敲除消除了NMDAR所需的Glun1亚基。我们通过深色场扫描EM层析成像对由此产生的PSD进行了3型结构。结果，我们现在有证据表明，可以通过EM层析成像来识别单个的NMDAR和AMPAR，并且可以划定它们的组织和与其他分子的联系。我们正在完成这项工作并准备出版。此外，我们刚刚与科罗拉多大学的Dellacqua M. Dellacqua的实验室发表了一项重大研究，涉及海马突触中锚定蛋白（AKAP）的构象和分布。在这项工作中，我们在厚部分上使用了免疫原子，并使用STEM断层扫描对其进行了可视化。结果表明，棕榈酰化对AKAP150/79膜组织，贩运和活动性的影响。已知膜相关的AKAP与PSD-95 Maguks相互作用，并锚定了几类激酶（PKA和PKC）对于突触可塑性（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的构象变化不同，并且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意义。我们还发现了与回收内体的广泛的AKAP关联，并且去山膜膜层基因似乎减少了这种关联。该项目在成像AMPAR贩运方面开辟了一个令人兴奋的新阵线。 EM层析成像允许创建3D重建，可以描绘突触下突触的细胞器，关键突触蛋白和大分子复合物的组织。重建以2-4 nm分辨率提供结构的大小，形状和位置，但不能保证单个结构的明确分子鉴定。尽管我们成功地使用免疫金来标记内源性和过表达的GFP标记的PSD-95，但大型抗体复合物也表现为在断层图中表现为丝状结构，使靶PSD蛋白的鉴定混淆了。现在，我们在APEX2和纳米型标签上取得了重大的技术突破，以解决此问题。 在一个本地化项目中，我们使用遗传标签Apex2来定位CAMKII。在DAB和过氧化物存在下，CAMKII-APEX2构建体揭示了单个CAMKII。我们正在通过EM层析成像在基底和高k刺激条件下研究CAMKII，我们正在完成工作并准备出版物。同时，我们将APEX2的工作扩展到了Shank和Homer。 此外，另一个本地化项目在纳米型标签中使用了预付款。在这种并行方法中，我们开发了一种使用具有EM断层扫描的纳米生物的方法来直接识别棘中的PSD蛋白。这是一种迅速的方法，我们期望在未来几年通过一系列突触分子获得令人兴奋的新结果。 最近，有关PSD-95的现场已经出现了一个主要问题。几项超分辨率光学显微镜研究表明，PSD-95在PSD处形成100 nm突触的纳米簇。这明显小于PSD的平均大小，但我们认为PSD-95均匀分布。目前，我们正在以较厚的截面断层扫描为PSD的内源性PSD-95分布进行重新检查，以进一步研究PSD的PSD-95分布和聚类。我们还使用上面概述的方法进一步分析了在断层图中所有垂直丝的分布，以查看PSD内是否存在任何垂直丝的聚类，以及我们是否可以通过免疫标记和层析成像和层析成像和层析成像和层析成像来调和超分辨率的发现。 在我们在哺乳动物神经元中的工作之外，与卡罗琳·史密斯（Carolyn Smith）在Ninds光学显微镜设施中进行的一项持续合作和Adriano Senatore（多伦多密西沙加大学）阐明了对原始动物Trichoplax中细胞类型和神经前法规的演变。尽管缺乏肌肉，神经和突触，但Trichoplax表现出不同类型的行为，表明了神经功能。我们确定了一个感受重力的细胞，并描述了阶段的结果，该细胞发生在该细胞的分化过程中。我们还描述了作用为巨噬细胞的细胞群。我们的协作努力提供了证据，表明毛lax可以感觉到环境水的pH值，并证明避免了低pH（酸性）区域。但是，调节机制以及三斑藻细胞彼此通信的方式尚不清楚。我们先前的结果以及其他地方获得的数据表明，该生物利用神经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。在下一步中，我们将表征毛lax中不同类型的分泌细胞，这大概可以调节和整合Nerveless动物中细胞的功能活性。确切地了解这些非常规的非智能系统在控制行为方面的功能如何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。