ULTRASTRUCTURAL ANALYSIS AND TOMOGRAPHY OF BRACHIOLA ALGERAE

海藻臂形藻的超微结构分析和断层扫描

• 批准号: 8172282
• 负责人: PETER M TAKVORIAN
• 金额: $ 1.08万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-07 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8172282
• 关键词: 3-Dimensional; Agaricales; Anterior; Area; Buffers; Caliber; Cells; Citrates; Complex; Computer Graphics; Computer Retrieval of Information on Scientific Projects Database; Computers; Cytoplasm; Data Collection; Dimethylarsinate; Disease; Electron Microscope; Electrons; Ethanol; Event; Filament; Funding; Generations; Goals; Grant; Hour; Human; Image; Imagery; Immunocompetent; Immunocompromised Host; Infection; Institution; Investigation; Knowledge; Laboratories; Lead; Learning; Membrane; Methodology; Methods; Microsporidia; Modeling; Organelles; Organism; Parasites; Plant Resins; Play; Positioning Attribute; Process; Production; Protocols documentation; Publications; Reporting; Reproduction spores; Research; Research Personnel; Resolution; Resources; Role; Series; Shapes; Slice; Solid; Source; Specimen; Staining method; Stains; Structural Models; Structure; Surface; System; Technology; Thick; Time; Tomogram; Tube; Tubular formation; United States National Institutes of Health; Visit; Work; abstracting; araldite; base; cellular imaging; epon; high voltage electron microscopy; image processing; improved; movie; pathogen; propylene oxide; reconstruction; three-dimensional modeling; tomography; transmission process; voltage

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT: Background -The Microsporidia are "emerging" human and well-documented veterinary pathogens that cause disease in both immunocompromised and immunocompetent hosts. These spore-forming intracellular protistan parasites, enter their host by the extrusion of a hollow polar tube. The polar tube serves as a unique vehicle for transmission of infection by discharging from the spore, piercing a host cell, and inoculating its infective sporoplasm directly into that cell's cytoplasm. In the non-activated spore (approx. 2 X 4 ¿m), this hollow polar "tube" is a solid filament composed of a series of concentric "circles" of electron lucent and dense material when viewed in cross section. This single polar filament (approx. 100- 150 nm diameter) is coiled around the inner periphery of the spore and the infective sporoplasm. The PF becomes straight in the anterior portion of the spore where it terminates in a "mushroom" shaped anchoring disc. The straight or manubroid portion of the PF is surrounded by a series of elaborate membranes and "tubules" termed the lamellar and tubular polaroplast, respectively. Upon activation, this "solid" PF everts through the anterior portion of the spore, becomes hollow, and is thought to turn "inside out" , thus becoming the polar tube, and serving as the sporoplasm transfer mechanism. Longitudinal sections of the spore reveal cross sections of the PF, and extensive membrane systems that surround the filament. The mechanism of eversion, the process of sporoplasm transfer, and the role these complex membrane systems play, are poorly understood. In addition, the structure of the sporoplasm, both inside the spore and after discharge also need extensive investigation. Goals -While the polar filament and its unique function were described almost 100 years ago, the structure and mechanism of its formation, position in the spore, attachment to the sporoplasm, and method of discharge, remain to be definitively determined. Several aspects of our knowledge of this complex series of events can be greatly advanced by the generation of 3D models of: 1) the non-activated spore structure and its contents, 2) the translocation of the spore contents during activation, 3) the structure of the extruded polar tube and sporoplasm. Project Description -Collection of data for 3-D reconstruction with the high-voltage or intermediate voltage electron microscope will greatly increase our knowledge of the microsporidian spore and its contents. Results from our recent studies have revealed several previously unknown aspects of these organisms, but we have not been able to obtain sufficient images to produce structural models. It is hoped, that images of material in "thick" sections, will enable the production of three dimensional models by computer graphics. Based on your publications list and my discussions with your staff, I believe your facility's technologies to image cells at high resolution, using computer-facilitated image processing to reconstruct and visualize organelles from a wide range of organisms, should work with the microsporidian spores. The structural details of spore and sporoplasm organelles, obtained by electron microscope tomography, will enable us to finally have a true representation of this important entity. Suggested Methodology  I will provide your facility with Epon-Araldite embedded non-activated and activated spore blocks. These will be the same or similar to the material used in our recent publication (Brachiola algerae - Cali et al. 2002). The material in the blocks has been fixed in 2.5 % cacodylate buffered gluteraldehyde (2-4 hours), post fixed in 1% buffered OsO4 , en block stained with UA for 1 hr in 70% ethanol, dehydrated in graded ethanols, transitioned through several rinses of propylene oxide, varying ratios of PO /epon, several transfers in pure resin, and then embedded. If this initial material is inadequate for your needs, I have growing cultures of infected cells in our laboratories and can provide an ample supply of material if necessary. After some experimentation, it was found that when these specimens are sectioned at 250 or 500 nm thickness, post-staining with lead citrate alone gave the best results. In the previous reporting period, two double-tilt and one single-tilt tomographic reconstructions were made. Dr Takvorian visited the RVBC three times, to use the HVEM to evaluate staining protocols, to choose specimens for tomography, and to learn about and discuss visualization methods. In the previous reporting period, Dr. Takvorian visited the resource twice. He gave a seminar on the first visit, and worked with computer visualization on both visits. Sterecon was implemented in the Takvorian lab, so that segmentation of tomograms could be done locally. This helped with the interpretation of the structure observed. The two double-tilt reconstructions from the previous reporting period were re-computed using improved image processing, and surface models of portions of the reconstructions were made, one showing a membranous area in the sporoplasm, another showing extruded membranes, another focusing on polar tubes. Animated movies were made of these models. Images of tomographic slices and surface-rendered models were prepared for presentation. Additional 250 and 500-nm-thick specimens were examined in order to obtain an optimal profile of the sporoplasm. New 250-nm-thick sections were cut from an "activated" spore block. Suitable specimens were located, a tomographic tilt-series was collected on the HVEM, and a reconstruction was made.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 抽象的： 背景 - 微孢子虫是“新兴”的人类和有据可查的兽医病原体，在免疫功能低下和免疫能力的宿主中引起疾病​​。这些散射的细胞内protistan寄生虫，通过延伸空心极性管进入其宿主。极性管通过从孢子中排出，刺穿宿主细胞并将其感染性孢子体直接进入该细胞的细胞质来充当传播感染的独特车辆。 在未激活的孢子（大约2 x 4€）中，该中空极性“管”是一种固体灯丝，当时在横截面中观察时，由电子和密集材料的一系列同心“圆”组成。这个单个极性细丝（大约100-150 nm的直径）围绕孢子和感染性孢子质的内部周围盘绕。 PF在孢子的前部直接终止在“蘑菇”形锚固盘中。 PF的笔直或手感部分被一系列精心设计的膜和“ tubulles”围绕，分别称为层状和块茎层状。激活后，这种“固体” PF穿过酱汁的前部，变成空心，被认为会变成“内而外”，从而成为极性管，并用作孢子质转移机制。孢子的纵向截面显示了PF的横截面以及围绕细丝的广泛膜系统。对这些复杂的膜系统发挥的作用，远处的机制，孢子质转移的过程以及这些作用知之甚少。此外，在孢子内部和放电后，孢子质的结构也需要大量投资。 目标 - 极性细丝及其独特的功能大约在100年前描述了其形成的结构和机制，散射中的位置，对孢子质的附着以及排放方法，但仍有待确定的确定。我们对这一复杂事件的知识的几个方面可以通过以下3D模型的产生：1）非激活的散射结构及其内容物的生成，2）激活过程中散点内容的易位，3）挤出的极性管和孢子质的结构。 项目描述 - 使用高压或中间电压电子显微镜的3-D重建数据集合将大大增加我们对微孢子虫酱及其含量的了解。我们最近的研究的结果揭示了这些生物的几个以前未知的方面，但是我们无法获得足够的图像来产生结构模型。希望“厚”部分中的材料图像能够通过计算机图形来生产三维模型。根据您的出版物列表和与您的员工的讨论，我相信您的设施的技术可以使用计算机采用的图像处理来进行高分辨率，以重建和可视化各种细胞器的细胞器，应与微孢子孢子孢子一起使用。通过电子显微镜断层扫描获得的孢子和孢子形细胞器的结构细节将使我们最终能够对该重要实体具有真实的表示。 建议的方法我将为您的设施提供嵌入的非激活和活性调味料块的epon-阿拉尔德石。这些将与我们最近出版物中使用的材料相同或相似（Brachiola Algerae -Cali等，2002）。 The material in the blocks has been fixed in 2.5 % cacodylate buffered glutaraldehyde (2-4 hours), post fixed in 1% buffered OsO4 , en block stained with UA for 1 hr in 70% ethanol, dehydrated In graded ethanols, transitioned through several routes of propylene oxide, varying ratios of PO /epon, several transfers in pure resin, and then embedded.如果这种最初的材料不足以满足您的需求，那么我在实验室中具有越来越多的感染细胞培养物，并且可以在必要时提供大量的材料供应。 经过一些实验后，发现这些样品以250 nm或500 nm的厚度切片时，单独使用柠檬酸铅染色后会产生最佳效果。在上一个报告期间，进行了两次双倾斜和一个单倾斜断层扫描重建。 Takvorian博士访问了RVBC三次，使用HVEM评估染色方案，选择用于层析成像的标本，并了解和讨论可视化方法。 在上一个报告期间，Takvorian博士两次访问了资源。他对第一次访问进行了开创性，并在两次访问中都进行了计算机可视化。 Sterecon是在Takvorian Lab中实施的，因此可以在本地进行断层图的分割。这有助于解释观察到的结构。 使用改进的图像处理重新计算了上一个报告期的两次双倾斜重建，并进行了部分重建的表面模型，该模型显示出孢子质中的膜面积，另一个显示挤压膜，另一个侧重于极性管。动画电影是由这些模型制作的。准备层析成像切片和表面渲染模型的图像用于演示。为了获得孢子质的最佳特征，还检查了另外250 nm厚的标本。 从“活化”的酱汁块中切开了新的250 nm厚的部分。找到合适的标本，在HVEM上收集了层析成像倾斜系列，并进行了重建。