This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.ABSTRACTPhotosynthesis is a fundamental process upon which the majority of Earths 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.References1. 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.

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