Proposed is a plan of research designed to determine the mechanism of microtubule nucleation by the centrosome in molecular detail. A combination of hierarchical structural approaches (EM Tomography, EM single particle reconstruction, and x-ray crystallography) and biochemical dissection and reconstitution methods will be utilized to determine the structures of gamma-tubulin and its complexes in vivo and in situ. Spanning size scales from the atomic to the entire organelle, our goal is to synthesize an atomic resolution picture of all the relevant structural and functional interactions between tubulin, gamma-tubulin complexes, and the centrosomal matrix. This is a continuation and expansion of a well-established research program that has made dramatic progress in the analysis of large, complex, supramolecular assemblies through a combination of light and electron microscopies. In addition, we plan to continue our efforts to determine the three-dimensional fine structure of mitotic chromosomes in a variety of biologically well-defined functional states. The long range goal is to understand the structural complexities that underlie DNA condensation and its organization into higher-order structures that can support as well as modulate transcriptional activity, and that change throughout the cell cycle. Current efforts focus on the structural analysis of Xenopus sperm chromatic condensed in vitro using Xenopus extracts as pioneered by Professor T. Mitchison (Harvard). By providing a reproducible and well controlled sampling of haploid chromosome condensation states, this will allow us to understand the condensation process as well as to determine the 3D organization of key protein components being discovered by other laboratories using this system to understand function. Furthermore, the structural consequences of immuno-depletion of such key protein components will be determined. In addition, we continue to pursue structural studies on HeLa telophase chromosomes. While ultimately a less powerful system than the Xenopus vitro condensation approach, for now it offers several important technical advances for structural analysis that makes it worthwhile. Key will be the use of High Pressure Freezing, cyro embedding, DNA-specific staining and cryo, low dose automated Intermediate Voltage Electron Microscope Tomography (IVEM-T) to allow tracing the 3D chromatin paths.
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