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.We are investigating the process of cell wall synthesis in diatoms, unicellular eukaryotic phytoplankton that make cell walls of nanostructured silica. Electron and fluorescence microscopy is being applied to monitor subcellular structures and their dynamics during cell wall synthesis. In addition, the intracellular location of specific proteins in relation to silica structures is being determined, with a goal of correlating formation of specific structures with the proteins involved.We are interested in applying advanced imaging techniques to understand how diatoms, unicellular eukaryotic algae, make their cell walls out of silica. We discussed with Mark Ellisman, Guido Gaietta, and Tom Deerinck at NCMIR three pilot scale projects and one longer term investigation. One pilot project is to generate high-resolution 3D images of the diatom Thalassiosira pseudonana during cell wall synthesis using electron tomography. We have developed a synchronized culture technique that enables enrichment of cells forming particular silica structures, which facilitates this approach. Because we can incorporate fluorescent dyes into diatom silica, examination of these cells using two-photon confocal scanning fluorescence microscopy could also provide valuable information. A second pilot project is to attempt real-time confocal imaging of cell wall formation, monitoring silica incorporation and membrane dynamics with specific fluorescent dyes. By analyzing a reasonable number of cells, we should be able to identify those in specific stages in cell wall synthesis. We propose to initially look at Nitzschia alba, a non-photosynthetic diatom on which we have done preliminary work in this regard, but would like to extend this to T. pseudonana. Examination of cytoskeletal dynamics during this process would also be valuable. A third pilot level project is to investigate protein localization approaches using tags that enable sequential fluorescence and electron microscopy to fine map proteins during formation of specific structures. Our collaborators, Nicole Poulsen and Nils Kr ger at Georgia Tech, have developed transformation and GFP tagging techniques for T. pseudonana, and have shown that one protein isolated from the silica cell wall is specifically localized to a particular silica substructure. We propose to generate a fusion construct with this gene, GFP, and a tetracysteine tag that would enable biarsenical dye labeling of the localized protein. After evaluating fluorescence localization, we would then photobleach the dye to facilitate staining for TEM to more precisely map location. The longer term investigation would involve localizing, in relationship to silica structure and larger scale dynamic events, sets of proteins we have identified using microarrays that are suspected to be involved in cell wall synthesis. These approaches have not been previously applied to diatoms, and are likely to change our fundamental understanding of the components and dynamics involved in silica structure formation.
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