The goal of this proposal is to employ a combination of cryofixation/freeze-substitution and dual-axis high voltage electron microscope tomographic techniques to produce a high resolution, three-dimensional (3-D) structural database of the cytokinetic apparatii of somatic and syncytial types of Arabidopsis cells, tobacco BY-2 suspension cultured cells and onion epidermal cells. Cytokinesis in plants involves the fusion of Golgi-derived vesicles into a cell plate that undergoes a series of complex changes, which transform it into a new cell wall. These transformations are mediated by a cytoskeletal array known as the phragmoplast. The site of fusion of the cell plate with the mother cell wall is defined prior to prophase by another cytoskeletal array, the preprophase band. We will tomographically reconstruct, model and quantitatively analyze all of these cytokinesis- related structures with a resolution of about 6 nm and then place the data into a new, internet accessible 3-D structural database that is being set up by the Boulder Laboratory for 3-D Fine Structure, a NIH Research Resource facility. This new database will be unique in that the tomographic data will be made available to all researchers in a form that will allow them to both check the accuracy of the original modeling as well as carry out additional modeling and quantitative analyses. For example, the tomograms will provide quantitative 3-D information on all of the microtubules, microfilaments, Golgi stacks, Golgi-derived vesicles, clathrin-coated buds and different types of cell plate membrane configurations within substantial cell volumes (up to 4x3x1 mum) containing preprophase bands, phragmoplasts and adjacent cytoplasm, and cell plates. In addition, these tomograms will provide information on the state of the end(s) (capped, flared and blunt) of every single preprophase or phragmoplast MT, the distribution of microtubule- associated proteins, and show whether MTs at the inner edge of an expanding phragmoplast differ on average in their state from those at the outer edge. Together these studies will not only yield new models of the cytokinetic apparatus of plants with a better than 10 fold improvement in 3-D resolution over current models, but also provide a new baseline for cell biologists to plan new functional studies and for geneticists and molecular biologists to evaluate cytokinesis mutants.
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