Proposed is a plan of research designed to determine the mechanism of microtubule (MT) nucleation by the centrosome in molecular detail. A combination of hierarchical structural approaches (EM Tomography, EM single particle reconstruction, and x-ray crystallography) will be utilized to determine the structures of gamma-tubulin and its complexes in vitro and in situ and the structure of intact centrosomes. Detailed analysis of MT nucleation kinetics will reveal how gamma-tubulin and its larger assemblies alter the mechanism and rate of MT-nucleus formation. The role of guanine nucleotide binding and hydrolysis in the nucleation process will also be determined. We will extend these studies to assay effects of a broad array of kinases including known regulators of centrosome function. Cellular and embryonic extracts will be screened for novel factors that regulate MT nucleation by the purified gamma-tubulin ring complex. Through mass spectrometry, we will determine the protein components of intact and extracted centrosomes, proteins that interact with the gamma-tubulin ring complex, and seek to discover their functions. siRNA knockdowns in Drosophila S2 cells will be used as a first screen for phenotypes. This will be complemented by in vivo and in situ localization using 3D fluorescence microscopy and 3D electron microscopic tomography. The results from structural, proteomics, and siRNA approaches will be integrated with our in vitro functional studies and in vivo analyses to build an understanding of the mechanisms by which MT nucleation is regulated within the cell. Spanning size scales from the atomic to the entire organelle, our long-term goal is to synthesize an atomic resolution picture of all the relevant structural and functional interactions between tubulin, gamma-tubulin complexes, regulatory proteins 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 biochemical analyses and light and electron microscopic studies. To facilitate these scientific directions, we will continue to pioneer the development of new software technologies for the collection, processing, reconstructing and understanding of EM data. In addition, we continue our efforts to build a new class of light microscopes that provide dramatically improved resolution.
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