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: 7357273
• 负责人: CLAIRE S TING
• 金额: $ 7.31万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7357273
• 关键词: ELECTRON; MICROSCOPE; TOMOGRAPHY; ENVIRONMENTALLY; CRITICAL

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.

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。光合作用是地球上大部分生物赖以生存的基本过程。it’这要视情况而定。光合作用的核心是独特的蛋白质复合物，它们已经进化到可以收集光能并将其转化为化学能。这些蛋白质复合物的结构完整性、组织和正常功能依赖于光合作用原核生物的胞质内片层和叶绿体的类囊体膜。该项目将包括对海洋中两种最丰富的光合作用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珠球藻胞质内膜的三维组织，并在细胞分裂过程中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促进我们对胞质内片层内部组织的基本认识，从而对我们理解光合作用和光合生物膜的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藻球菌的分子结构。具体来说，我们试图定义这些密切相关的蓝藻的细胞质的超分子组织，特别关注胞质内片层的结构、组织和接触。这些内部薄片是主要代谢过程的场所，如光合作用，在这些细胞中。我们特别感兴趣的是在膜生物发生过程中以及细胞暴露于非生物胁迫后，这些光合片层的结构和组织是如何变化的。虽然原绿球藻和聚球藻关系密切，被认为有共同的祖先，但常规电子显微镜显示，它们在胞浆内片层的组织结构上进化出了显著的差异。这些差异部分是由于它们光合作用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这些细胞的胞浆内片层的三维结构和组织，并确定内部片层与细胞质膜之间是否存在接触。此外，对不同生理状态的细胞进行比较研究（即，经历二元裂变与静止阶段，暴露于正常生长条件下与非生物胁迫下）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非生物应激的敏感性增加是否部分归因于关键细胞结构（如内膜）的损伤的见解。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的生物发生的基本理解。电子显微镜层析成像提供的结构信息有可能为光合作用原核生物的内部组织，特别是光合作用片层的三维结构、组织和接触提供基本的见解。原绿球藻和聚球菌（图1A和1B）是电子显微镜断层扫描研究的理想候选者。首先，作为开放海洋初级生产的主要贡献者，它们是环境上重要的微生物，是已知数量最多的光合生物之一。其次，它们的小尺寸（原绿球藻：直径0.5至0.9 m；聚球藻：0.6 um（宽度）x 1.8 um（长度））使它们适合于电子显微镜断层扫描，其中样品厚度是优化图像质量的主要限制。第三，在过去的几年里，我一直在使用化学固定、薄切片和透射电子显微镜技术来表征这些蓝藻的超微结构。作为这项工作的一部分，我开发了一种改进的方法，用于在透射电子显微镜下固定原绿球藻，使这些细胞的胞浆内片层可视化（Ting等人，手稿正在准备中）。通过原绿球藻薄片的常规电子显微镜，我已经确定了胞浆内片层的结构与大多数其他蓝藻不同，其中光合作用装置的蛋白质位于胞浆内片层。胞浆内片层是原绿球藻细胞的主要特征。与许多蓝藻不同的是，这些膜在原绿球藻中紧密地贴附在细胞周围。单个磷脂双分子层的片层宽度约为6 ~ 7nm，由两个磷脂双分子层和膜内空间组成的单个片层的宽度为15nm ~ 19nm。在我们的切片中经常可以看到由内膜形成的囊状结构。在纵剖面上，这些薄片经常延伸细胞的长度，并且紧密地贴压在一起。它们通常在细胞的一端不连续，在那里增加的细胞质空间将单个膜分开。电子显微镜断层扫描可以表征原绿球藻和聚珠球藻胞质内膜的三维组织，并可以在细胞分裂过程中可视化整个细胞的内膜系统。这反过来将使我们能够识别分裂过程中独特的特定膜构象和接触。此外，电子显微镜断层扫描将使我们能够确定在暴露于特定环境变化和压力后蓝藻细胞的超分子组织的变化。从我之前对原绿球藻的超微结构的研究中，很明显，在高和低辐照水平下生长的细胞之间最显著的区别是细胞的细胞膜含量。我们的初步结果表明，与传统的化学固定方法相比，低温电子断层扫描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所提供的结构信息将促进我们对胞质内片层内部组织的认识，从而对我们理解光合作用和光合作用原核生物的膜生物发生具有重要意义。这项工作的第一阶段将包括获得在不同环境条件下生长的原绿球藻和聚球藻细胞的三维重建，这些细胞被化学固定并嵌入到Spurr或Epon中。我们的初步数据是有希望的，我们已经能够获得使用这些技术保存的原绿球藻细胞的层析重建。然而，由于膜对制备技术特别敏感，我们需要用更“原生”的方法来证实我们的发现。方法。因此，在这项工作的第二阶段，整个细胞将被冷冻，并从冷冻的水合细胞中记录层析图，而不需要化学固定，脱水或染色。这将使整个膜系统可视化。这些海洋蓝藻的小尺寸使它们特别适合使用这种技术进行检查。冻水原绿球藻和聚球藻细胞的初步结果看起来非常令人鼓舞。为了获得更高分辨率的断层扫描，需要更薄的制备材料，因此颗粒状细胞将被高压冷冻并通过冷冻-超显微切开术进行切片。这些冷冻固定细胞的超微结构将与使用上述常规方法制备的细胞进行比较。这个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现有的中压和高压电子显微镜，以及该设施现有的其他设备（高压和低温冷冻机、低温超低温组）和分析工具。需要400kV能量过滤的EM才能获得高质量的冷冻水合蓝藻整体层析成像，而冷冻水合切片的层析成像可以对同一标本进行更高分辨率的比较研究。这在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中是有用的。引用1。Ubrach E, Robertson， DL, Chisholm SW（1992）蓝藻辐射中原绿藻的多重进化起源。自然355:267 - 269。2. 丁志强，王志强，王志强（2002）海洋蓝藻光合作用：不同光收集策略的起源和意义。微生物学趋势：10:134-142。3. （1992）电子断层扫描：电子显微镜的三维成像。全体会议出版社，纽约。Mannella CA, Marko M, Penczek P, Barnard D, Frank J（1994）大鼠肝脏线粒体内部区隔：高压透射电子显微镜的层析研究。显微镜研究技术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。生物化学进展22:37-38。6. Frey TG, Mannella CA（2000）线粒体的内部结构。生物化学趋势，25:319-324。7. 张晓明，张晓明，张晓明（2002）生物复杂性的三维成像。结构生物学杂志138:85-91。8. Murk J, Humbel BM, Ziese U, Griffith JM, Posthuma G, Slot JW, Koster AJ, Verkleij AJ, Geuze HJ, kleimeer J .（2003）膜融合的三维内体区隔化。美国国家科学院学报，100:13332-13337。9. Partensky F, Hess WR, Vaulot D（1999）原绿球藻，一种具有全球意义的海洋光合原核生物。微生物学与分子生物学，63:106-127。