The fundamental function of centrosomes is to nucleate and stabilize microtubules that serve to segregate chromosomes (the spindle microtubules) or position the spindle (the aster microtubules). Microtubule nucleation is a critical event in the cell cycle and cells regulate the nucleation capacity of the centrosome to increase at the start of mitosis when more microtubules are required. This project focuses on the nucleation process with a combination of both in vitro and in vivo experimental approaches. Gamma-tubulin is a conserved essential element of microtubule nucleation. With two other conserved proteins it forms the gamma-tubulin small complex. Multimers of the small complex can form a ring that is thought to template the assembly of alpha-beta tubulin dimers into a ring of protofilaments and thereby form a nascent microtubule (2). However, the control of small complex ring formation, the activation of the nucleation capacity of the ring complex, the mechanism by which alpha-beta tubulin dimers are captured and stabilized, and the feedback mechanisms that control the nucleation capacity are still not well understood. The first two aims address the activation of the y-tubulin ring complex. The 6.5 A structure of filaments of the y- tubulin complex showed the y-tubulins are held in a ring of 13. However they are positioned too far apart to template the 13 protofilaments found in microtubules (1). This result suggested the current hypothesis: the y- tubulin small complex could be activated to form a template for nucleation by a structural transformation that positions the y-tubulins to match the orientation and geometry of the alpha-beta tubulin dimers that form the microtubule. This transformation requires both closure of the gamma-tubulin small complex and allosteric activation (1) (unpublished data). We will use genetic, biochemical and cell biological approaches to perform functional analyses of the y-tubulin complex in vitro and in vivo. Our work is directly complementary to structural and biophysical approaches taken by the Agard lab. Together, we will provide a detailed understanding of the physical and biological basis of microtubule nucleation. In the third aim we step back from the mechanism of nucleation and examine the control of the overall nucleation capacity of the centrosome. The nucleation capacity of the centrosome expands several fold in preparation for mitosis (3). This expansion has been termed centrosome maturation. For the centrosome of higher eukaryotes, a model is beginning to emerge that the assembly and maturation of the pericentriolar material involves the enrichment of core proteins driven by protein phosphorylation by the mitotic kinases PIkl and Aurora-A (4). However the complexity of the pericentriolar material both in ultrastructure and composition has hampered progress. The full complement of proteins that are involved is not known, the upstream signals that trigger phosphorylation are not known, and the consequences of phosphorylation on maturation remains to be discovered. In yeast nucleation capacity is regulated by cell cycle events and cell ploidy. Notably expansion of the spindle pole body occurs upon activation of the mitotic checkpoint. This provides a very simple method to experimentally control expansion and thereby study its requirements (5-7). In yeast, all the structural proteins in the SPB are known and many of their sites of phosphorylation have been determined (8). In addition we have identified three proteins involved in the expansion process through a genetic screen (9). In the third aim, the kinetics of expansion and turnover, the role of these three proteins in the expansion process and the role of phospho-regulation of the gamma-tubulin complex will be examined as a model for centrosome maturation. This work will complement the aim in the Winey lab project that will determine the role of the phosphorylation of core proteins in centrosome assembly.

National Institute of Health (NIH)
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University of Colorado at Boulder
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