Macromolecular Architecture Of The Synapse

突触的大分子结构

• 批准号: 10915958
• 负责人: Thomas S Reese
• 金额: $ 175.43万
• 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/10915958
• 关键词: Acceleration; Affect; Algorithm Design; Architecture; Automation; Binding; Brain; Classification; Collaborations; Complex; Computer software; Cryo-electron tomography; Cryoelectron Microscopy; DLG4 gene; Darkness; Data; Data Set; Dendritic Spines; Development; Electrons; Elements; Failure; Filament; Freeze Substitution; Freezing; Future; Genetic; Glutamate Receptor; Goals; Gold; Hand; Hippocampus; Horseradish Peroxidase; Hour; Hydration status; Hydrogen Peroxide; Image; Imaging Techniques; Individual; Investigation; Ions; Label; Link; Machine Learning; Manuals; Maps; Membrane; Memory; Methods; Microscope; Molecular; Molecular Probes; Molecular Structure; Morphology; Nature; Neurons; Noise; Organelles; Phosphotransferases; Positioning Attribute; Postsynaptic Membrane; Preparation; Presynaptic Terminals; Procedures; Process; Proteins; Publications; Publishing; Rattus; Resolution; Shapes; Sonication; Specificity; Stains; Structure; Subcellular structure; Synapses; Synaptic Cleft; Synaptic Membranes; Synaptic Vesicles; Synaptosomes; Techniques; Technology; Thick; Time; Tomogram; United States National Institutes of Health; Use of New Techniques; Vertebral column; Vesicle; Visualization; Work; algorithmic methodologies; artificial intelligence algorithm; automated segmentation; calmodulin-dependent protein kinase II; classification algorithm; data pipeline; density; design; electron optics; electron tomography; image processing; improved; innovation; man; nano; nanobodies; nanoscale; oxidation; particle; postsynaptic; pressure; presynaptic; reconstruction; response; synaptic function; tomography; tool; trafficking

Progress Summary: A key priority for this project is to develop methods of determining molecular identity within tomograms of the PSD. First, we developed a new technique using nanobody labeling for tomograms. A nanobody binds directly to the target protein with high specificity which allows us to use gold particle labels on synaptic proteins and identify them directly in tomograms. We have data on PSD-95, CaMKII, and Homer1b, and we are in the process of finishing this work and preparing an initial publication for this type of work. Second, we succeeded in fine-tuning a genetic labeling procedure for EM tomography using APEX2, a cloneable horseradish peroxidase, which catalyzes the oxidation of DAB into electron-dense material in the presence of hydrogen peroxide. CaMKII is a kinase required for LTP and is the most abundant protein in the brain. Using this APEX2 method in rat hippocampal neurons imaged by dark-field STEM tomography, individual APEX2 labeled CaMKII are readily identified in tomographic reconstructions of dendritic spines. As a result, we are beginning to understand the distribution of CaMKII in spines, on the membrane, and in the PSD, their self-association, and their response to synaptic activity. We are preparing this work for publication. We have made great strides in characterizing synaptic structures using high-pressure freezing and freeze substitution (HPF/FS). However, HPF/FS is time intensive with many points of failure and relies on stain to visualize the structure. With cryo-EM tomography (cryo-ET), we can trade a slight degradation in resolution for ease in preparation and pure visualization of structure. We are experimenting with three cryo-focused methods in collaboration with NIH cryo-EM facilities (NICE and MICEF). First, we acquired over 150 tomograms on the NCI 300 kV Krios cryo-ET microscope, specifically on frozen-hydrated isolated PSDs from rat brains or sonicated PSD fragments. Second, we are also using the cryo-focused ion beam (FIB) milling. With FIB milling we can shave neuronal processes and synapses to obtain 200-300 nm thick lamellar. Third, we are freezing synaptosomes isolated from the brain. Our goal with each cryo method is to develop the method to a point where we achieve results comparable to our HPF/FS techniques. Our transsynaptic assembly project investigates intracellular structures linked by cleft-spanning structures. In this project, transcleft structures and all connected transmembrane and intracellular structures are segmented and analyzed in tomograms of synapses from high-pressure frozen, freeze-substituted neuronal cultures. In renderings, cleft-spanning structures typically make continuous connections from one intracellular compartment to the other, forming what we call transsynaptic assemblies. This project has yielded several clear findings. First, nearly all transcleft objects have some intracellular component. Second, transsynaptic assemblies with large intracellular volumes and more than one intracellular component are very likely to be associated with synaptic vesicles. Third, transsynaptic assemblies share intracellular components and produce large domains of associated assemblies or just association domains. We believe association domains explain the underpinnings of the nanodomain phenomena and reveal a more complex picture of their composition and function, as our results show that less than half of assemblies associate with synaptic vesicles. Further, we classified and enumerated over three thousand structures. We designed an algorithm to display a structure at random, prompt the user for a description, and then parse descriptions for common morphological elements. We were able to use this information to find common structures associated within assemblies. This work was published this year. We hypothesize that there are functionally critical transsynaptic combinations of presynaptic, postsynaptic, and cleft molecules. Unfortunately, we do not have the technology to analyze the number of synapses necessary to confidently identify these with satisfying specificity. To get the number of structures necessary, we are pairing this project with the automated segmentation project. Automated segmentation is crucial for the future of previously discussed projects and electron tomography in all forms. With our automated segmentation project, our goal is to accelerate the segmentation and visualization of synaptic structures with automation. We developed an automatic segmentation optimization method (ASOM). With ASOM, we are processing many large tomograms. For one project, ASOM segmented detailed structures of fragments isolated from sonicated and control PSDs imaged by cryo-EM. However, many structures within PSDs segmented by ASOM are still interconnected in complicated ways. To examine those structures more in detail, we improved ASOM further by adding watershed segmentation, widely used to separate connected structures automatically. This enabled the automatic segmentation of hundreds of tightly packed granular structures in intact PSDs into individual modules. These results were recently published. Recently, we improved ASOM to automatically segment filaments connected to only the postsynaptic membrane in one step, revealing that PSD-95-like filaments can be segmented by automation. The improved ASOM automatically segments other distinct classes such as those connected to the presynaptic membrane, postsynaptic membranes, and vesicle membranes. Further, the automatic segmentation of transsynaptic components are consistent with those assemblies obtained by painstaking manual segmentation, demonstrating that this approach will contribute to expediting the segmentation of the assemblies. We need a platform for users to apply ASOM algorithms. Most segmentation tools do not have the most basic functions of ASOM. Therefore, we have been developing our own software package that streamlines the entire tomography data pipeline, from image alignment to visualization. In addition to ASOM, we will integrate object classification algorithms based on machine learning and AI. Our goal is to decrease the time needed to fully analyze a tomogram from several months to a few days. Recently, we successfully implemented an advanced reconstruction method called the Simultaneous Iterative Reconstruction Technique (SIRT), widely used for generating tomograms. Our method has improved accuracy and reduced noise for both conventional ET and cryo-ET. Our SIRT method produces EM tomograms more efficiently than IMOD while qualities of the tomograms were found to be equal to or better than those generated by IMOD. Also, we combined ASOM with skeletonization and found that ASOM automatically segmented distinct transsynaptic structures similar to those segmented by hand. More work is required to provide the same level of detail as hand segmentation. Currently, we are combining the advanced ASOM algorithm with new AI algorithms that are potentially applicable to various subcellular structures. This project will make our software package a more efficient and robust segmentation platform for more varied structures, thus expanding the scope of our investigations to include large amounts of datasets and various experimental conditions.

进度摘要： 该项目的关键优先级是开发确定PSD断层图内分子身份的方法。首先，我们使用用于断层图的纳米病标记开发了一种新技术。纳米病毒直接与具有高特异性的靶蛋白结合，这使我们能够在突触蛋白上使用金颗粒标签，并直接在断层图中识别它们。我们拥有有关PSD-95，CAMKII和HOMER1B的数据，我们正在完成这项工作并为此类工作准备首次出版物。其次，我们成功地使用了使用可克性的辣根过氧化物酶Apex2微调EM断层扫描的遗传标记程序，该过程将DAB氧化催化为电子密集的材料，在存在过氧化氢的情况下。 CAMKII是LTP所需的激酶，是大脑中最丰富的蛋白质。在树突状刺的断层扫描重建中，使用这种APEX2方法在黑暗场茎断层扫描中成像的大鼠海马神经元中使用。结果，我们开始了解Camkii在刺，膜上和PSD中的分布，它们的自我关联以及它们对突触活动的反应。我们正在准备这项出版作品。 我们在使用高压冻结和冻结替代（HPF/FS）表征突触结构方面取得了长足的进步。但是，HPF/FS是时间密集的，有很多故障点，并且依赖于污点来可视化结构。借助Cryo-EM层析成像（Cryo-ET），我们可以在分辨率方面轻微降解，以便于制备和纯粹的结构可视化。我们正在与NIH Cryo-EM设施（NICE和MICEF）合作尝试三种以低温为中心的方法。首先，我们在NCI 300 kV Krios Cryo-ET显微镜上获得了150多个断层图，特别是从大鼠大脑或超声的PSD片段中的冷冻水合分离的分离的PSD上。其次，我们还使用了以低温为中心的离子束（FIB）铣削。使用Fib铣削，我们可以剃光神经元过程和突触以获得200-300 nm厚的层状。第三，我们是从大脑中分离出来的冻结突触体。我们使用每种冷冻方法的目标是将方法开发到与HPF/FS技术相当的结果。 我们的透射性装配项目研究了由跨裂结构连接的细胞内结构。在该项目中，在来自高压冷冻，冷冻冻结成立的神经元培养的突触的突触中进行了分割和分析，对transcleft结构和所有连接的跨膜和细胞内结构进行了分割和分析。在效果图中，跨裂裂结构通常会连续连接从一个细胞内隔室到另一个隔室，从而形成了我们所谓的跨色素组件。 该项目提出了一些明确的发现。首先，几乎所有的transcleft对象都有一些细胞内组件。其次，具有较大细胞内体积和一个以上细胞内成分的透射性集成很可能与突触囊泡有关。第三，经突触组件共享细胞内组件并产生相关组件或仅关联域的大域。我们认为，关联域解释了纳米域现象的基础，并揭示了其组成和功能的更复杂的图片，因为我们的结果表明，与突触囊泡相关的小于一半的组装。 此外，我们对超过三千个结构进行了分类和列举。我们设计了一种算法来随机显示结构，提示用户进行描述，然后解析对常见形态元素的描述。我们能够使用这些信息来查找组件中相关的共同结构。这项工作今年出版了。 我们假设存在突触前，突触后和裂口分子的功能上关键的透射性组合。不幸的是，我们没有技术来分析以满意的特异性确定这些突触的数量。为了获得必要的结构数量，我们将该项目与自动分割项目配对。 自动分割对于以前讨论的各种形式的项目和电子断层扫描的未来至关重要。借助我们的自动分割项目，我们的目标是通过自动化加速突触结构的分割和可视化。我们开发了一种自动分割优化方法（ASOM）。使用ASOM，我们正在处理许多大型断层图。对于一个项目，ASOM分割了从Cero-EM成像的超声波和控制PSD中分离出的片段的详细结构。但是，由ASOM分割的PSD中的许多结构仍然以复杂的方式互连。为了更详细地检查这些结构，我们通过添加流域分割进一步改善了ASOM，该分割被广泛用于自动分离连接的结构。这使得完整PSD中数百个紧密堆积的颗粒结构的自动分割为单个模块。这些结果最近发表了。 最近，我们改进了ASOM，以自动将仅连接到突触后膜的丝段一步，表明可以通过自动化对PSD-95样细丝进行分割。改进的ASOM自动将其他不同的类别段，例如连接到突触前膜，突触后膜和囊泡膜的类别。此外，经跨性别成分的自动分割与通过艰苦的手动分割获得的组件一致，这表明这种方法将有助于加快组件的分割。 我们需要一个平台，以便用户应用ASOM算法。大多数分割工具没有ASOM的最基本功能。因此，我们一直在开发自己的软件包，该软件包简化了整个层析成像数据管道，从图像对齐到可视化。除ASOM外，我们还将基于机器学习和AI集成对象分类算法。我们的目标是将完全分析断层图从几个月降低到几天所需的时间。 最近，我们成功实施了一种称为同时迭代重建技术（SIRT）的高级重建方法，该方法广泛用于生成断层图。我们的方法提高了常规ET和Cryo-ET的精度和噪声降低。我们的SIRT方法比IMOD更有效地产生了EM层图，而pogracton的质量则与IMOD产生的质量相等或更好。同样，我们将ASOM与骨架化结合在一起，发现ASOM自动分割了与手动分割的不同的跨突触结构。需要更多的工作来提供与手部分割相同的细节。 当前，我们将先进的ASOM算法与可能适用于各种亚细胞结构的新的AI算法相结合。该项目将使我们的软件包成为更有效，更强大的分割平台，以实现更多种的结构，从而扩大我们的调查范围，以包括大量数据集和各种实验条件。

项目成果

Identifying individual scaffolding molecules in the postsynaptic density.

识别突触后密度中的单个支架分子。

• DOI: 10.1017/s1431927608085449
• 发表时间: 2008
• 期刊: Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada
• 作者: Chen,X;Winters,C;Azzam,R;Crocker,V;Li,X;Galbraith,J;Leapman,Rd;Reese,Ts
• 通讯作者: Reese,Ts

Electron tomography on γ-aminobutyric acid-ergic synapses reveals a discontinuous postsynaptic network of filaments.

• DOI: 10.1002/cne.23453
• 发表时间: 2014-03
• 期刊: JOURNAL OF COMPARATIVE NEUROLOGY
• 影响因子: 2.5
• 作者: Linsalata, Alexander E.;Chen, Xiaobing;Winters, Christine A.;Reese, Thomas S.
• 通讯作者: Reese, Thomas S.

Neuropeptidergic integration of behavior in Trichoplax adhaerens, an animal without synapses.

• DOI: 10.1242/jeb.162396
• 发表时间: 2017-09-15
• 期刊: The Journal of experimental biology
• 作者: Senatore A;Reese TS;Smith CL
• 通讯作者: Smith CL

PSD-95 is required to sustain the molecular organization of the postsynaptic density.

• DOI: 10.1523/jneurosci.5968-10.2011
• 发表时间: 2011-04-27
• 期刊: The Journal of neuroscience : the official journal of the Society for Neuroscience
• 作者: Chen X;Nelson CD;Li X;Winters CA;Azzam R;Sousa AA;Leapman RD;Gainer H;Sheng M;Reese TS
• 通讯作者: Reese TS

Coordinated Feeding Behavior in Trichoplax, an Animal without Synapses.

• DOI: 10.1371/journal.pone.0136098
• 发表时间: 2015
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Smith CL;Pivovarova N;Reese TS
• 通讯作者: Reese TS