ELECTRON MICROSCOPE TOMOGRAPHY OF ENVIRONMENTALLY CRITICAL CYANOBACTERIA:

环境临界蓝细菌的电子显微镜断层扫描：

• 批准号: 7721697
• 负责人: CLAIRE S TING
• 金额: $ 2.22万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7721697
• 关键词: Affect; Architecture; Area; Biochemical; Biogenesis; Biological; Caliber; Cell Division Process; Cell membrane; Cell model; Cells; Cellular Structures; Chemicals; Chloroplasts; Comparative Study; Complex; Computer Retrieval of Information on Scientific Projects Database; Condition; Cyanobacterium; Cytoplasm; Data; Dehydration; Dextrans; Dimensions; Electron Microscope; Electron Microscopy; Employee Strikes; Equipment; Exposure to; Freezing; Frozen Sections; Funding; Grant; Growth; Harvest; Heart; Image; Imagery; Individual; Institution; Journals; Knowledge; Learning; Length; Life; Light; Liver Mitochondria; Localized; Manuscripts; Marines; Membrane; Membrane Fusion; Membrane Lipids; Metabolism; Methods; Microbiology; Microscopy; Microtomy; Mitochondria; Modeling; Molecular; Molecular Biology; Molecular Conformation; Morphologic artifacts; N(delta)-acetylornithine, -isomer; N-dodecanoylglutamic acid, -isomer, sodium salt; Nature; New York; Oceans; Organelles; Organism; Peripheral; Phase; Phospholipids; Photosynthesis; Physiological; Preparation; Process; Prochlorococcus; Prochlorophytes; Production; Prokaryotic Cells; Proteins; Publishing; Purpose; Radiation; Range; Rattus; Reporting; Research; Research Personnel; Research Technics; Resolution; Resources; Science; Silicon; Site; Source; Specimen; Staining method; Stains; Stress; Structure; Students; Surface; Synechococcus; System; Techniques; Testing; Thick; Thinking; Three-Dimensional Image; Thylakoid Membranes; Tomogram; Transmission Electron Microscopy; Ultramicrotomy; United States National Academy of Sciences; United States National Institutes of Health; Visit; Width; Work; abstracting; alpha-difluoromethyl-DOPA, -isomer; alpha-methylornithine dihydrochloride, -isomer; comparative; dextran; electron tomography; environmental change; epon; improved; insight; interest; membrane biogenesis; microorganism; pressure; protein function; reconstruction; research study; response; sample fixation; size; spurr resin; structural biology; three dimensional structure; tomography; tool; transmission process; trend; 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 Photosynthesis is a fundamental process upon which the majority of Earth's life depends. At the heart of photosynthesis are unique protein complexes that have evolved to harvest light energy and transform it into chemical energy. The structural integrity, organization, and proper functioning of these protein complexes are dependent on a surrounding lipid membrane called the intracytoplasmic lamellae in photosynthetic prokaryotes and thylakoid membranes in chloroplasts. This project will involve a comparative study of the molecular architecture of two of the most abundant photosynthetic prokaryotes in the oceans. The use of electron microscope tomography will enable us to characterize the supramolecular organization of these cyanobacterial cells and establish how environmental stress affects cell architecture. In particular, electron microscope tomography will allow us to characterize the three dimensional organization of the intracytoplasmic membranes in Prochlorococcus and Synechococcus, and visualize the internal membrane system throughout the cell during the cell division process. The structural information provided by electron microscope tomography will advance our fundamental knowledge of the internal organization of the intracytoplasmic lamellae, and thus will have important implications for our understanding of photosynthesis and membrane biogenesis in photosynthetic organisms. The aim of this project is to characterize the molecular architecture of the globally important cyanobacteria Prochlorococcus and Synechococcus. Specifically, we seek to define the supramolecular organization of the cytoplasm of these closely related cyanobacteria, focusing particularly on the structure, organization, and contacts of the intracytoplasmic lamellae. These internal lamellae are the sites of major metabolic processes, such as photosynthesis, in these cells. We are especially interested in establishing how the structure and organization of these photosynthetic lamellae change during membrane biogenesis and following the exposure of cells to abiotic stress. Although Prochlorococcus and Synechococcus are closely related and are thought to share a common ancestor1, conventional electron microscopy indicates that they have evolved striking differences in the organization of their intracytoplasmic lamellae. These differences are due in part to dissimilarities in the protein composition of their photosynthetic apparatus2. Electron microscope tomography will enable us to characterize the three-dimensional structure and organization of the intracytoplasmic lamellae in these cells and establish whether contacts exist between the internal lamellae and the cytoplasmic membranes. Furthermore, comparative studies on cells at different physiological states (i.e., undergoing binary fission vs. stationary phase, exposed to normal growth conditions vs. abiotic stress) will enable us to define changes that occur in the photosynthetic lamellae and other internal structures under different growth conditions. These studies will also provide insights on whether the increased sensitivity of Prochorococcus to abiotic stress is due in part to damage of key cellular structures such as the internal membranes. The structural information provided by electron microscope tomography will contribute to our fundamental understanding of photosynthesis and the biogenesis of photosynthetic membranes. The structural information provided by electron microscope tomography3-8 has the potential to provide fundamental insights into the internal organization of photosynthetic prokaryotes, and in particular on the three-dimensional structure, organization, and contacts of the photosynthetic lamellae. Prochlorococcus and Synechococcus (Figures 1A and 1B) are ideal candidates for electron microscope tomography studies. First, as major contributors to primary production in the open oceans, they are environmentally important microorganisms and are among the most abundant photosynthetic organisms known9. Second, their small size (Prochlorococcus: 0.5 to 0.9 ¿m, diameter; Synechococcus 0.6 um (width) x 1.8 um (length)) make them suitable for electron microscope tomography, where specimen thickness is a major limitation in optimizing image quality. Third, in these past years, I have been characterizing the ultrastructure of these cyanobacteria using chemical fixation, thin sectioning, and transmission electron microscopy techniques. As part of this work, I developed an improved method for the fixation of Prochlorococcus for transmission electron microscopy that has permitted visualization of the intracytoplasmic lamellae of these cells (Ting et al., manuscript in preparation). Through conventional electron microscopy of Prochlorococcus thin sections, I have established that the structure of the intracytoplasmic lamellae, where the proteins of the photosynthetic apparatus are localized, differ from the majority of other cyanobacteria. The intracytoplasmic lamellae are a dominant feature of the Prochlorococcus cell. In contrast to many cyanobacteria, these membranes are tightly appressed in Prochlorococcus, and are located near the cell periphery. A single phospholipid bilayer of the lamella is approximately 6 to 7 nm, and the width of an individual lamella consisting of two phospholipids bilayers and an intramembrane space ranges from 15 nm to 19 nm. The sac-like structure formed by the internal membranes was often visible in our sections. In longitudinal sections, these lamellae frequently extend the length of the cell and are tightly appressed. They are generally discontinuous at one end of the cell, where an increased amount of the cytoplasmic space separates the individual membranes. Electron microscope tomography will permit the characterization of the three dimensional organization of these intracytoplasmic membranes in Prochlorococcus and Synechococcus, and will allow the visualization of the internal membrane system throughout the cell during the cell division process. This in turn will enable us to identify specific membrane conformations and contacts that are unique to the division process. Furthermore, electron microscope tomography will allow us to define alterations in the supramolecular organization of cyanobacterial cells following exposure to specific environmental changes and stresses. From my previous work on the ultrastructure of Prochlorococcus, it is clear that the most notable difference between cells grown at high and low irradiance levels is the internal membrane content of the cells. Our preliminary results suggest that the use of cryoelectron tomography, which introduces fewer structural artifacts than conventional chemical fixation approaches6,7, should be particularly effective in these studies. The structural information provided by electron microscope tomography will advance our knowledge of the internal organization of the intracytoplasmic lamellae, and thus will have important implications for our understanding of photosynthesis and membrane biogenesis in photosynthetic prokaryotes. The first phase of this work will involve obtaining three-dimensional reconstructions of Prochlorococcus and Synechococcus cells that have been grown under different environmental conditions and are chemically-fixed and embedded in Spurr or Epon. Our preliminary data have been promising and we have been able to obtain tomographic reconstructions of Prochlorococcus cells that were preserved using these techniques. Since membranes are particularly sensitive to preparative techniques, however, we need to confirm our findings with a more "native" method. Thus, in the second phase of this work, whole cells will be plunge-frozen, and tomograms will be recorded from frozen-hydrated cells, without the need for chemical fixation, dehydration, or stains. This will allow visualization of the membrane system as a whole. The small size of these marine cyanobacteria renders them particularly suitable for examination using this technique. Preliminary results with frozen-hydrated Prochlorococcus and Synechococcus cells look very encouraging. For higher resolution tomography, a thinner preparation will be needed, so pelleted cells will be high-pressure frozen and sectioned by cryo-ultramicrotomy. The ultrastructure of these cryofixed cells will be compared with those prepared using the conventional approaches described above. This project requires the expertise and facilities available at the Resource for the Visualization of Biological Complexity. The proposed experiments will involve both the intermediate and high voltage electron microscopes available at the RVBC, as well as other equipment (high-pressure and plunge freezers, cryo-ultramicrotome) and analysis tools available at this facility. The 400kV energy-filtered EM is required to obtain high quality tomograms of frozen-hydrated whole-mounts of cyanobacteria, and tomography of frozen-hydrated sections allows higher-resolution comparative study of the same specimen. This should be useful in tests aimed at refining techniques of whole-cell and frozen-hydrated section cryo-tomography. References 1. Ubrach E, Robertson, DL, Chisholm SW (1992) Multiple evolutionary origins of prochlorophytes within the cyanobacterial radiation. Nature 355:267-269. 2. Ting CS, Rocap G, King J, Chisholm SW (2002) Cyanobacterial photosynthesis in the oceans: the origins and significance of divergent light-harvesting strategies. Trends inMicrobiology 10:134-142. 3. Frank J (1992) Electron Tomography: Three-Dimensional Imaging with the Electron Microscope. Plenum Press, New York. 4. Mannella CA, Marko M, Penczek P, Barnard D, Frank J (1994) The internal compartmentalization of rat-liver mitochondria: tomographic study using the high-voltage transmission electron microscope. Microscopy Research Technique 27:278-283. 5. Mannella CA, Buttle K, Marko M (1997) Reconsidering mitochondrial structure: new views of an old organelle. Trends in Biochemical Sciences 22:37-38. 6. Frey TG, Mannella CA (2000) The internal structure of mitochondria. Trends in Biochemical Sciences 25:319-324. 7. Frank J, Wagenknecht T, McEwen BF, Marko M, Hsieh CE, Mannella CA (2002) Three- dimensional imaging of biological complexity. Journal of Structural Biology 138:85-91. 8. Murk J, Humbel BM, Ziese U, Griffith JM, Posthuma G, Slot JW, Koster AJ, Verkleij AJ, Geuze HJ, Kleijmeer MJ (2003) Endosomal compartmentalization in three dimensions: Implications for membrane fusion. Proceedings of the National Academy of Science, USA 100:13332-13337. 9. Partensky F, Hess WR, Vaulot D (1999) Prochlorococcus, a marine photosynthetic prokaryote of global significance. Microbiology and Molecular Biology Reviews 63:106-127. In the previous reporting period, several tomograms of plunge-frozen, intact cyanobacteria were made. Three strains were represented, Prochlorococcus, Synechococcus, and MED4A. Dr. Ting visited the RVBC with student Sesh Sundararaman, who learned how to make surface-rendered models using Sterecon. A Silicon Graphics workstation was loaned and set up in the Ting lab for this purpose. A representative model of each strain was made. New insights on the comparative structure of both peripheral and internal membrane systems in the different strains were gained, and the work was reported by Dr. Ting in a platform talk at the Microscopy and Microanalysis 2005 Meeting, Honolulu, HI, July 29  August 4, 2005: + Ting, C.S., Hsieh, C., Sundararaman, S., Mannella, C. and Marko, M. (2005) Comparative three-dimensional imaging of environmentally critical cyanobacteria through cryo-electron tomography. Microsc. Microanal. 11 (Suppl 2): 332CD. Cyanobacteria were concentrated and high-pressure frozen in 20% dextran. Frozen-hydrated sections were cut from this material, and five tomographic reconstructions from Prochlorocccus strain 9313 were made. One of these was shown at the Microscopy and Microanalysis 2005, as a comparison with the plunge-frozen whole mounts. One of the reconstructions represented a cross-section along the long axis of the cell, and revealed a spiral arrangement of the internal membranes, not previously known. Since the membrane surface area is known to change in response to light level, such a spiral arrangement of membrane growth would seem to be advantageous. Modeling of cells from sections is underway. A study of light response with strains 9313 and MED4A was carried out, adding short-light and long-light conditions to the normal light cycle condition studied so far. Material was both plunge-frozen for whole mounts and high-pressure frozen for frozen-hydrated sections. The following abstract was published: + Ting, C.S., Hsieh, C., Sundararaman, S., Frost, A., Mannella, C., Marko, M. (2006) Visualizing molecular responses to environmental stress in marine cyanobacteria. Microsc. Microanal. 12 (Suppl 2): 40-41.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 对于中心来说，它不一定是研究者的机构。 抽象的 光合作用是地球上大多数生命所依赖的一个基本过程，光合作用的核心是独特的蛋白质复合物，这些蛋白质复合物的结构完整性、组织和正常功能已进化为收集光能并将其转化为化学能。依赖于周围的脂质膜，在光合原核生物中称为胞质内片层，在叶绿体中称为类囊体膜。 该项目将对海洋中两种最丰富的光合原核生物的分子结构进行比较研究，使用电子显微镜断层扫描将使我们能够表征这些蓝藻细胞的超分子组织，并确定环境压力如何影响细胞结构。特别是，电子显微镜断层扫描将使我们能够表征原绿球藻和聚球藻细胞质内膜的三维组织，并可视化整个细胞的内膜系统。电子显微镜断层扫描提供的结构信息将增进我们对细胞质内片层内部组织的基础了解，从而对我们理解光合生物的光合作用和膜生物发生具有重要意义。 该项目的目的是表征全球重要的蓝藻原绿球藻和聚球藻的分子结构，具体而言，我们寻求定义这些密切相关的蓝藻细胞质的超分子组织，特别关注蓝藻的结构、组织和接触。这些内部片层是这些细胞中主要代谢过程（例如光合作用）的场所。这些光合片层在膜生物发生过程中以及细胞暴露于非生物胁迫后发生变化，尽管原绿球藻和聚球藻密切相关并且被认为具有共同的祖先1，但传统电子显微镜表明它们在细胞质内片层的组织中进化出了显着的差异。这些差异部分是由于其光合装置的蛋白质组成不同，电子显微镜断层扫描将使我们能够表征三维结构和特征。这些细胞中胞质内片层的组织，并确定内部片层和细胞质膜之间是否存在接触。此外，对不同生理状态下的细胞进行比较研究（即，经历二元裂变与稳定期、暴露于正常生长条件与正常生长条件）。非生物胁迫）将使我们能够定义在不同生长条件下光合片层和其他内部结构发生的变化，这些研究还将提供关于原绒球菌对光合作用的敏感性是否增加的见解。非生物胁迫部分是由于关键细胞结构（例如内膜）的损坏造成的，电子显微镜断层扫描提供的结构信息将有助于我们对光合作用和光合膜生物发生的基本理解。 电子显微镜断层扫描3-8提供的结构信息有可能为光合原核生物的内部组织提供基础见解，特别是原绿球藻和聚球藻的三维结构、组织和接触（图1A）。和 1B）是电子显微镜断层扫描研究的理想候选者，首先，作为公海初级生产的主要贡献者，它们是对环境重要的微生物。是已知最丰富的光合生物之一9。 其次，它们体积小（原绿球藻：直径 0.5 至 0.9 微米；聚球藻 0.6 微米（宽）x 1.8 微米（长）），使其适合电子显微镜断层扫描，其中样本厚度为第三，在过去的几年里，我一直在使用化学方法来表征这些蓝藻的超微结构。作为这项工作的一部分，我开发了一种用于透射电子显微镜固定原绿球藻的改进方法，该方法可以使这些细胞的胞质内片层可视化（Ting 等人，手稿于准备）。 通过原绿球藻薄片的传统电子显微镜，我确定了细胞质内片层的结构（光合机构的蛋白质位于其中）与大多数其他蓝细菌不同。胞质内片层是原绿球藻细胞的主要特征。与许多蓝藻相反，原绿球藻中的这些膜被紧密地压住，并且位于细胞附近片层的单个磷脂双层约为6至7nm，由两个磷脂双层和膜内空间组成的单个片层的宽度为15nm至19nm。在我们的切片中经常可见，这些薄片经常延伸到细胞的长度并且紧密地压在一起，它们通常是不连续的。细胞的末端，细胞质空间的增加将各个膜分开。 电子显微镜断层扫描将允许对原绿球藻和聚球藻中这些胞质内膜的三维组织进行表征，并将允许在细胞分裂过程中观察整个细胞的内部膜系统，这反过来将使我们能够识别特定的膜。此外，电子显微镜断层扫描将使我们能够确定蓝藻细胞在暴露于特定环境变化和环境变化后的超分子组织的变化。从我之前对原绿球菌超微结构的研究来看，很明显，在高辐照度和低辐照度下生长的细胞之间最显着的差异是细胞的内膜含量，我们的初步结果表明，冷冻电子断层扫描的使用与传统的化学固定方法相比，引入的结构伪影更少6,7，在这些研究中应该特别有效，电子显微镜断层扫描提供的结构信息将增进我们对细胞质内片层内部组织的了解。因此，这对于我们理解光合作用原核生物的光合作用和膜生物发生具有重要意义。 这项工作的第一阶段将涉及获得在不同环境条件下生长并经过化学固定并嵌入 Spurr 或 Epon 中的原绿球藻和聚球藻细胞的三维重建。我们的初步数据很有希望，我们已经能够做到这一点。然而，由于膜对制备技术特别敏感，因此我们需要使用更“天然”的方法来确认我们的发现。在这项工作的第二阶段，整个细胞将被深度冷冻，并且将从冷冻水合的细胞中记录断层图，而不需要化学固定、脱水或染色，这将使整个膜系统可视化。这些海洋蓝细菌的大小使得它们特别适合使用这种技术进行检查，冷冻水合原绿球藻和聚球藻细胞的初步结果看起来非常令人鼓舞，对于更高分辨率的断层扫描来说，更薄的制剂将是非常令人鼓舞的。由于需要，因此将沉淀的细胞高压冷冻并通过冷冻超薄切片术将这些冷冻细胞的超微结构与使用上述常规方法制备的细胞进行比较。 该项目需要生物复杂性可视化资源提供的专业知识和设施。拟议的实验将涉及 RVBC 提供的中压和高压电子显微镜，以及其他设备（高压和投入式冷冻机、冷冻机）。 -超薄切片机）和该设施提供的分析工具需要 400kV 能量过滤 EM 才能获得冷冻水合蓝藻整体的高质量断层图和断层扫描。冷冻水合切片允许对同一样本进行更高分辨率的比较研究，这对于旨在完善全细胞和冷冻水合切片冷冻断层扫描技术的测试很有用。 参考 1. Ubrach E, Robertson, DL, Chisholm SW (1992) 蓝藻辐射中原绿藻的多重进化起源 355:267-269。 2. Ting CS、Rocap G、King J、Chisholm SW (2002) 海洋中的蓝藻光合作用：不同的光捕获策略的起源和意义 10：134-142。 3. Frank J (1992) 电子断层扫描：电子显微镜三维成像，纽约。 4. Mannella CA、Marko M、Penczek P、Barnard D、Frank J (1994) 大鼠肝脏线粒体的内部区室化：使用高压透射电子显微镜的断层扫描研究 27：278-283。 5. Mannella CA、Buttle K、Marko M (1997) 重新考虑线粒体结构：旧细胞器的新观点 22:37-38。 6. Frey TG，Mannella CA (2000)《生物化学科学趋势》25：319-324。 7. Frank J、Wagenknecht T、McEwen BF、Marko M、Hsieh CE、Mannella CA (2002) 生物复杂性的三维成像 138:85-91。 8. Murk J, Humbel BM, Ziese U, Griffith JM, Posthuma G, Slot JW, Koster AJ, Verkleij AJ, Geuze HJ, Kleijmeer MJ (2003) 三个维度的内体区室化：对膜融合的影响。科学，美国 100：13332-13337。 9. Partensky F、Hess WR、Vaulot D (1999) 原绿球藻，具有全球意义的海洋光合原核生物，微生物学和分子生物学评论 63：106-127。 在上一个报告期间，制作了几张冷冻的完整蓝细菌断层图，其中包括原绿球藻、聚球藻和 MED4A。Ting 博士与学生 Sesh Sundararaman 一起参观了 RVBC，后者学习了如何制作表面渲染模型。为此目的，在 Ting 实验室中借用了一台 Silicon Graphics 工作站，并建立了每种菌株的代表性模型。获得了不同菌株的外周和内部膜系统的结构，Ting 博士在 2005 年显微镜和微量分析会议（夏威夷州檀香山，2005 年 7 月 29 日至 8 月 4 日）的平台演讲中报告了这项工作： + Ting, C.S.、Hsieh, C.、Sundararaman, S.、Mannella, C. 和 Marko, M. (2005) 通过冷冻电子断层扫描对环境至关重要的蓝藻进行比较三维成像（Suppl 2）。 ）：332CD。 将蓝藻浓缩并高压冷冻在 20% 葡聚糖中。从该材料中切下冷冻水合切片，并对原绿球菌菌株 9313 进行五次断层扫描重建，其中一个在 2005 年显微镜和微量分析中进行了比较。其中一个重建物代表了沿着细胞长轴的横截面，并揭示了内部的螺旋排列。由于已知膜表面积会随光照水平而变化，因此细胞切片的这种螺旋排列似乎是有利的。 对菌株 9313 和 MED4A 的光响应进行了研究，在迄今为止研究的正常光循环条件中添加了短光和长光条件，材料对整个安装进行了骤冷，对冷冻材料进行了高压冷冻。水合切片。 发表了以下摘要： + Ting, C.S.、Hsieh, C.、Sundararaman, S.、Frost, A.、Mannella, C.、Marko, M. (2006) 海洋蓝细菌对环境应激的分子反应可视化（Suppl 2）。 ）：40-41。